heterotrimeric G proteins regulate nitrogen-use efficiency in rice

heterotrimeric G proteins regulate nitrogen-use efficiency in rice
heterotrimeric G proteins regulate nitrogen-use efficiency in rice

The drive toward more sustainable agriculture has raised the profile of crop plant nutrient-use efficiency. Here we show that a major rice nitrogen-use efficiency quantitative trait locus (qNGR9) is synonymous with the previously identified gene DEP1 (DENSE AND ERECT PANICLES 1). The different DEP1 alleles confer different nitrogen responses, and genetic diversity analysis suggests that DEP1 has been subjected to artificial selection during Oryza sativa spp. japonica rice domestication. The plants carrying the dominant dep1-1 allele exhibit nitrogen-insensitive vegetative growth coupled with increased nitrogen uptake and assimilation, resulting in improved harvest index and grain yield at moderate levels of nitrogen fertilization. The DEP1 protein interacts in vivo with both the G a (RGA1) and G b (RGB1) subunits, and reduced RGA1 or enhanced RGB1 activity inhibits nitrogen responses. We conclude that the plant G protein complex regulates nitrogen signaling and modulation of heterotrimeric G protein activity provides a strategy for environmentally sustainable increases in rice grain yield.Our ability to feed the world’s current population is, in part, a result of the Green Revolution, which was based on the adoption of semi-dwarf cereals that had an increased harvest index; however, the increase in grain yields required substantial increases in nitrogen fertilization

levels 1,2. Unfortunately, these increases in nitrogen fertilizer applications also resulted in what are now well-documented deleterious impacts on the environment 3. Therefore, the continued growth of the human population and the imminent threat of global climate change represent a major challenge for increasing grain yields without simultaneously exacerbating the degradation of the natural environment 4. Thus, there is an urgent need to develop crops that show improved nitrogen-use efficiency 5. However, current understanding of the genetic basis of rice nitrogen-use efficiency remains at the level of the identification of a number of quantitative trait loci (QTLs), with no understanding of the nature of the genes presumed to underlie them 6–9.

Rice varieties exhibit substantial genetic variation with respect to plant height and tiller number at different nitrogen fertilization levels (Supplementary Table 1). For example, the vegetative growth of O. sativa spp. indica variety Nanjing6 (NJ6) is highly responsive to nitrogen, with plant height and tiller number being strongly correlated with nitrogen input level (Fig. 1a and Supplementary Table 2). Conversely, the vegetative growth of the japonica variety Qianzhonglang2 (QZL2) is largely unresponsive to nitrogen fertili-zation level, with plants grown with low nitrogen input having plant height and tiller numbers that are essentially the same as those of plants grown with high nitrogen input (Fig. 1b and Supplementary Table 2). These differences are associated with the consistently supe-rior harvest index (the ratio between grain weight and above-ground biomass at maturity) of QZL2 (Supplementary Table 2). Among a set of 226 recombinant inbred lines (RILs) bred from a QZL2 × NJ6 intercross hybrid progenitor, one line (RIL-D22) exhibited marked nitrogen responses with respect to plant height and tiller number (Fig. 1c –e ) and another (RIL-D04) behaved in a nitrogen-unresponsive manner that was similar to QZL2 (Fig. 1f and Supplementary Table 2). Genetic analysis of a recurrent backcross (BC 2F 2) population having RIL-D22 as the donor parent and QZL2 as the recurrent parent identi-fied qNGR9 as a major QTL for nitrogen-mediated growth responses and mapped this QTL to chromosome 9 (Fig. 1g ). By positional cloning and genetic complementation, we found that qNGR9 is synonymous with DEP1 (DENSE AND ERECT PANICLE 1), a gene that has been shown previously to regulate rice panicle archi-tecture 10–12 (Supplementary Note ).

A variant dep1 allele found in the Italian rice cultivar Balilla con-fers an increased number of grains per panicle (and a consequent increase in grain yield that is characteristic of many japonica rice varieties)10. We developed near-isogenic lines (NILs) carrying DEP1 (from NJ6, called NIL-DEP1) and dep1-1 (from QZL2, called NIL–dep1-1) (Supplementary Fig. 1) and found that although the NIL-DEP1 line exhibited a vegetative growth response to nitrogen

Heterotrimeric G proteins regulate nitrogen-use efficiency in rice

Hongying Sun 1,6, Qian Qian 2,6, Kun Wu 1,6, Jijing Luo 3,6, Shuansuo Wang 1,6, Chengwei Zhang 1, Yanfei Ma 1, Qian Liu 1, Xianzhong Huang 1, Qingbo Yuan 1, Ruixi Han 1, Meng Zhao 1,4, Guojun Dong 2, Longbiao Guo 2, Xudong Zhu 2, Zhiheng Gou 5, Wen Wang 5, Yuejin Wu 4, Hongxuan Lin 3 & Xiangdong Fu 1

1The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, National

Centre for Plant Gene Research, Beijing, China. 2The State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, China. 3The State Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 4Institute of Technical Biology and Agriculture Engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China. 5The State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China. 6These authors contributed equally to this work. Correspondence should be addressed to X.F . (xdfu@https://www.360docs.net/doc/481265898.html, ).

Received 31 October 2012; accepted 19 March 2014; published online 28 April 2014; doi:10.1038/ng.2958

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similar to that of RIL-D22, the vegetative growth of the NIL–dep1-1 line that resembled that of RIL-D04 was not responsive to nitrogen (Fig. 1 and Supplementary Fig. 2). In initial studies of the rela-tionship between DEP1 function and nitrogen response in NIL-DEP1, we found that DEP1 transcript abundance was positively induced by the level of nitrogen supplied (Fig. 2a ). We also found that expression of dep1-1 was affected by nitrogen input level (Supplementary Fig. 3). In further experiments, we compared the nitrogen-response phenotypes conferred by DEP1, dep1-1 and dep1-32, the latter of which is a recessive loss-of-function allele that was identified from a rice dense and erect panicle1-like mutant (Fig. 2b and Supplementary Note ). The NIL–dep1-32 line derived from the backcross between the dep1-32 mutant and NIL-DEP1 (the recurrent parent) had shorter internodes and wider, shorter leaves than NIL-DEP1 (Supplementary Fig. 4). Whereas NIL-DEP1 exhibited nitrogen-responsive vegetative growth, NIL–dep1-32 did not (Fig. 2c ). Rice plants have evolved a number of strategies to sus-tain their growth under limiting nitrogen conditions (for example, an increased root-to-shoot ratio)13. The roots of NIL-DEP1 seed-lings grew longer under lower nitrogen conditions, whereas shoot biomass accumulation was suppressed (Supplementary Fig. 5). We found that the root-to-shoot ratio of NIL–dep1-32 plants was unaffected by the reduction in nitrogen availability (Supplementary Fig. 5). In contrast, the transgenic NIL-DEP1 plants that constitutively overexpressed DEP1 were more sensitive to nitrogenous fertilizer than nontransgenic control NIL-DEP1 plants (Fig. 2d –f ). Thus, the DEP1 protein is required for the vegetative growth response to soil nitrogen, and lack of DEP1 (as conferred by dep1-32) abolishes the nitrogen response.

When compared to indica varieties, vegetative growth with respect to plant height and tillering capacity of the japonica varieties was less sensitive to soil nitrogen (Supplementary Table 1). We then asked whether the variant alleles of DEP1 were involved in the different nitrogen responses. We generated NILs that used the follow-ing accessions as donors: Oryza rufipogon (DEP1W ), the japonica type cv. Nipponbare (DEP1J ) or the indica type cv. Zhenshan97 (DEP1I ). NIL-DEP1W roots grew robustly when exposed to nitrogen deficiency, but shoot growth was strongly suppressed; the root-to-shoot ratio of the NIL-DEP1J seedlings was lower than those of both NIL-DEP1W and NIL-DEP1I seedlings (Fig. 2g ). Under the higher nitrogen regime, tiller numbers of NIL-DEP1W were markedly superior to those of NIL-DEP1J (Fig. 2h ). A nucleotide diversity study showed no sig-nificant excess of low-frequency haplotypes among indica cultivars (Tajima’s D ?0.48065, P > 0.10) but a substantial difference in the japonica varieties (Tajima’s D ?2.39135, P < 0.01) as compared to the wild accessions 14,15 (Supplementary Fig. 6). Moreover, three SNPs in the ORF region clearly differentiate O. rufipogon and the japonica cultivar (Supplementary Fig. 6). This result shows that DEP1 has been subjected to artificial selection during japonica rice evolution.Nitrogen fertilization promotes plant growth by enhancing cell proliferation, and we found that different DEP1 alleles differen-tially affect nitrogen-responsive cell proliferation and organ size at

d

ifferent developmental stages (Supplementary Note ). In particular, we found that the yield-enhancing properties of dep1-1 are associated with repression of longitudinal cell division and plant height during vegetative growth (Supplementary Figs. 7 and 8) and with promo-tion of cell proliferation and panicle branching during reproductive growth 10. We next determined the extent to which DEP1, dep1-1 and dep1-32 confer differential effects on grain yield and nitrogen

m etabolism. The rice root system in a paddy field exists in an anaero-bic environment and as a result is forced to utilize ammonium rather than nitrate as an inorganic source of nitrogen 16. Under low nitrogen conditions, transcript levels of the genes involved in nitrogen metabo-lism were upregulated (Supplementary Note ), but transcript levels of key genes associated with ammonium uptake and assimilation (such as OsAMT1;1, OsGS1;2 and OsNADH-GOGAT1) in NIL–dep1-1 were higher than those set in NIL-DEP1 (Supplementary Figs. 9–11). We found that NIL–dep1-1 plants had higher glutamine synthase activity and accumulated more internal nitrogen than NIL-DEP1 plants, whether the supply of nitrogen was limiting or ample (Fig. 2i ,j and Supplementary Fig. 12). These results suggest that DEP1 also regulates nitrogen uptake and metabolism.

Under limiting nitrogen conditions, performance with respect to tiller number per plant and grain number per panicle in NIL–dep1-1 was superior to that of both NIL–dep1-32 and NIL-DEP1 (Supplementary Fig. 13). Field-grown NIL–dep1-1 outperformed the other genotypes with respect to both harvest index and grain yield (Fig. 2k and Supplementary Fig. 14a ). For example, at the standard nitrogen fertilization level (180 kg per ha), NIL–dep1-1 exhibited a 14% advantage with respect to grain productivity, reaching a level of yield that would have required an extra 60 kg per ha of nitrogen for NIL-DEP1 to match (Fig. 2k ). The semi-dwarf gene, semi-dwarf1 (sd1), is responsible for the Green Revolution rice ideotype 17,18, which has dominated indica breeding programs over the past 50 years 2. Under a standard management practice, the grain yield of NIL–dep1-1 was ~32% superior to that of NIL-sd1 (Fig. 2k ), and the harvest index

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Figure 1 The effect of nitrogen fertilization levels on rice plant architecture. (a ) Nanjing6 (NJ6). (b ) Qiangzhonglang2 (QZL2). (c ) RIL-D22. (d ) RIL-D04. (e ) The effect of nitrogen fertilization on plant height in RIL-D22 plants. Error bars (d ,e ), s.e.m. (f ) The effect of nitrogen fertilization on tiller number in RIL-D22 plants. (g ) qNGR9 was mapped to the interval between RM3700 and RM257 on chromosome 9 using a BC 2F 2 population derived from the backcross between RIL-D22 and QZL2. LOD, likelihood of odds; PH, plant height. (h ) The contrasting heights of NIL–dep1-1 and NIL-DEP1 plants. Scale bars, 20 cm.

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l e t t e r s of NIL–dep1-1 was ~14% higher (Supplementary Fig. 14b ). Notably, NIL–dep1-1 plants retained their productivity advantage even under conditions of low nitrogen availability (Fig. 2k ). Thus, the dep1-1 allele appears to make a contribution to rice yield specifically by increasing nitrogen-use efficiency.

Heterotrimeric G proteins are multisubunit, integral membrane signal-transduction complexes that mediate intracellular responses to external stimuli in diverse eukaryotic organisms 19. G proteins typically consist of α, β and γ subunits 20,21, and the DEP1 protein has been suggested to be a plant-specific G protein γ subunit 22–24 (Fig. 3a ). We found that the GGL (G protein γ-like) domain of DEP1 interacts with the rice β subunit RGB1 in yeast two-hybrid assays (Fig. 3b ,c ). We detected RGB1-GFP , DEP1-GFP 10 and dep1-1–GFP fusion proteins both on the plasma membrane and within the nucleus of transgenic rice root cells (Fig. 3d and Supplementary Fig. 15). Furthermore, bimolecular fluorescence complementation (BiFC) analysis showed that the GGL domain of DEP1 interacts strongly with RGB1 on the plasma membrane and within the nucleus (Fig. 3e ).

We also found that both downregulation 25 and upregulation of

RGB1 conferred a dwarf phenotype similar to that of NIL–dep1-32 (Fig. 3d ,f ). In addition, overexpression of the genes encoding two canonical rice γ subunits (RGG1 and RGG2)12,20 as well as another atypical γ subunit (GS3, another homolog of DEP1)10,19 also caused dwarfism (Supplementary Fig. 15), whereas there was no obvious phenotype associated with constitutive overexpression of DEP1 (refs. 10–12) (Supplementary Fig. 15).

G βγ acts as a functional monomer, and G β-mediated processes require a γ subunit 26–28. However, the dwarfed phenotype of NIL–dep1-1 plants could not be rescued by the upregulation or downregu-lation of RGB1 (Fig. 3f ). We next examined the genetic interaction between DEP1 and RGA1 (encoding a rice G α subunit) by contrast-ing allelic combinations assembled in a near isogenic background. Whereas the d1-1 allele confers lack of RGA1 and a resultant severe dwarf phenotype 29,30, the semi-dwarf phenotype of the Chinese

cultivar Xueheaizao (XHAZ) is conferred by the d1XHAZ allele and

is due to a low

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Supplemental nitrogen fertilization (kg/ha)Figure 2 DEP1 is required for a nitrogen response. (a ) Expression of DEP1 is induced by the availability of nitrogen. Transcript abundance relative to the level of 2-month-old NIL-DEP1 plants grown without nitrogen fertilization (set to one). Data are shown as the mean ± s.e.m. (n = 3). (b ) A missense polymorphism (c.277G>T) creating a premature stop codon differentiates the cv. Nipponbare DEP1 sequence from that of the dep1-32 mutant. (c ) The phenotype of NIL plants carrying DEP1 and dep1-32 alleles treated with 30 kg/ha (low N)

or 300 kg/ha (high N) nitrogen. Scale bar, 15 cm. (d ) Seedling response to the supply of mineral nitrogen. Seedlings were exposed for to a hydroponic solution containing various concentrations of NH 4NO 3 for 14 d. The concentration of 1.4 mM NH 4NO 3 is set to 1N. Scale bar, 2.5 cm. (e ) The effect of nitrogen fertilizer rates

on shoot length. The rice plants were exposed for to a hydroponic solution containing various concentrations of NH 4NO 3 for 28 d. Data are shown as the mean ± s.e.m. (n = 6). A Student’s t -test was used to generate the P values. (f ) Comparison of the relative ratios of root length between transgenic and nontransgenic NIL-DEP1 plants. Seedlings were exposed for to a hydroponic solution containing various concentrations of NH 4NO 3 for

14 d. The root length of seedlings treated with 1.4 mM NH 4NO 3 was set to one. Data are shown as the mean ± s.e.m. (n = 6). (g ) Seedling response to nitrogen input level. Seedlings were exposed to either 0.035 mM (low N) or 1.4 mM NH 4NO 3 (high N) for 14 d. Scale bar, 4 cm. (h ) The phenotypes of 2-month-old NIL-DEP1W and NIL-DEP1J plants exposed to either 0.035 mM (low N) or 1.4 mM NH 4NO 3 (high N). Scale bar, 15 cm. (i ) The nitrogen content of the aerial parts of mature plants grown under various nitrogen fertilizer regimes. Data are shown as the mean ± s.e.m. (n = 6). (j ) Glutamine synthetase (GS) activity in flag leaf extracts (leaves were sampled before grain filling). Data are shown as the mean ± s.e.m. (n = 6). The presence of the same lowercase letter above the histogram bars in i and j denotes nonsignificant differences across the two panels (P > 0.05). (k ) Grain yield of field-grown NIL plants in response to nitrogenous fertilizer application. Data are shown as the mean ± s.e.m. estimated from six plots (each plot comprised 288 plants) per line per fertilizer.

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sequence polymorphism in its promoter (Supplementary Fig. 16). Conversely, higher expression of RGA1 conferred no detect-able phenotype (Supplementary Fig. 15). We determined the effects of D1, d1-1 and d1XHAZ on nitrogen response and found that although NIL-DEP1-D1 plants responded to nitrogen, both NIL-DEP1–d1-1 and NIL-DEP1-d1XHAZ plants were relatively insensi-tive to nitrogen fertilization (Fig. 4a ). We also

found that NIL–dep1-1–D1 and NIL-DEP1-d1XHAZ plants were taller than NIL–dep1-1–d1XHAZ plants, and NIL–dep1-1–d1-1 plants were severely dwarfed (just like the d1-1 mutant itself) (Fig. 4b ). These results suggest that DEP1 and RGA1 both act in the same signal-ing pathway for a normal nitrogen growth response. Notably, BiFC analysis showed that both DEP1 and dep1-1 interact with RGA1, and

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binding between DEP1 and RGA1. nYFP-tagged DEP1, dep1-1 or deleted versions of DEP1 (as described in Fig. 3b ) were co-transformed with cYFP-RGA1 into tobacco leaves. Fluorescence complementation of

split YFP was determined by confocal microscopy. The rice OsSPL16

transcription factor 15 was used as a negative control. Scale bars, 50 μm.

(d ) The reduced nitrogen response of the transgenic plants overexpressing RGB1. Scale bar, 15 cm. (e ) Comparison of plant height between the transgenic and nontransgenic NIL-DEP1 plants treated with 30 kg/ha (low N) or 300 kg/ha (high N) nitrogen. Data are shown as the mean ± s.e.m. (n = 12). A Student’s t -test was used to generate the P values.

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Figure 3 The interaction between DEP1 and RGB1. (a ) A phylogenetic analysis of the GGL domain. The conserved residues are indicated by black boxes. DPLIP is a conserved DPLL/I motif. (b ,c ) Yeast two-hybrid assays. Schematic representation of the truncated DEP1 proteins used for the yeast two-hybrid assay. The functional domains comprise GGL, vWFC (von Willebrand factor type C) and TNFR (tumor necrosis factor receptor)/NGFR (nerve growth factor receptor). In the assay, RGB1 is used as bait (GAL4-BD, BD) and DEP1 is used to as prey (GAL4-AD, AD). ?N and ?C represent several N- and C-terminally truncated DEP1 deletion variants. (d ) Localization of the RGB1-GFP fusion protein in the root of transgenic cv. Nipponbare plants. The root tips, scale bar, 50 μm; paddy-grown rice plants, scale bar,

15 cm. (e ) A BiFC analysis in which nYFP-tagged DEP1, dep1-1 or deletions of DEP1

(as described in b ) was co-transformed into tobacco leaf along with cYFP-RGB1. The rice OsSPL16 transcription factor 15 was used as negative control. Scale bars, 50 μm. (f ) Morphology of transgenic NIL–dep1-1 plants in which RGB1 was either suppressed or overexpressed. Scale bar, 20 cm.

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the vWFC (von Willebrand factor type C) domain of DEP1 might participate in this association (Fig. 4c ).

We also found that a functional nucleotide polymorphism (encod-ing p.Cys105Tyr) of DEP1 in the japonica cultivars was correlated with the high-affinity interaction between RGB1 and the GGL domain of DEP1 (Supplementary Fig. 17), which is consistent with a reduced response to nitrogen (Fig. 2g ,h ). Furthermore, the transgenic NIL-DEP1 plants overexpressing RGB1 exhibited nitrogen-insensitive vegetative growth (Supplementary Note ), including plant height, tillering number and root-to-shoot ratio (Fig. 4d ,e and Supplementary Fig. 18). These results show that enhanced RGB1 activity or increas-ing strength of the interaction between DEP1 and RGB1 inhibits nitrogen responses. The findings on nitrogen-heterotrimeric G pro-tein signaling unravel a missing link in the nutrient regulation of plant growth and development, and the modulation of heterotrimeric G protein activity also provides a potential new strategy for environ-mentally sustainable improvement of grain yield in rice by increasing nitrogen-use efficiency.

METHoDs

Methods and any associated references are available in the online version of the paper .

Note: Any Supplementary Information and Source Data files are available in the online version of the paper .

ACKnoWLeDGMentS

We thank N.P . Harberd for the critical comments on the manuscript and

P . Schulze-Lefert, H. Liao and Y. Tong for advice. This research was supported by grants from the 973 Program from National Basic Research Program of China (2011CB915403, 2011CB100302 and 2012AA10A301) and the National Natural Science Foundation (31130070 and 91335207) to X.F.

AUtHoR ContRIBUtIonS

Q.Q., G.D. and L.G. performed assays of nitrogen-use efficiency. S.W ., Q.Y. and X.H. developed the RIL populations. X.H. and K.W . conducted the genetic analysis. J.L. and K.W . were responsible for the positional cloning. K.W ., J.L. and H.L.

developed the NILs. X.Z. and Y.M. identified the mutants. R.H. and Q.L. performed DNA sequence analysis. H.S. and X.H. carried out the yeast two-hybrid and BiFC experiments. H.S. and M.Z. characterized the phenotype of transgenic plants. C.Z., S.W ., K.W . and Y.W . performed field experiments. Z.G. and W .W . were responsible for diversity analysis. X.F. designed the experiments and wrote the manuscript.

CoMPetInG FInAnCIAL InteReStS

The authors declare no competing financial interests.

Reprints and permissions information is available online at https://www.360docs.net/doc/481265898.html,/reprints/index.html .

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rice, contains a defective gibberellin 20-oxidase gene. Proc. Natl. Acad. Sci. USA 99, 9043–9048 (2002).

19. New, D.C. & Wong, J.T. The evidence for G-protein–coupled receptors and

heterotrimeric G proteins in protozoa and ancestral metazoa. Biol. Signals Recept. 7, 98–108 (1998).

20. Perfus-Barbeoch, L., Jones, A.M. & Assmann, S.M. Plant heterotrimeric G protein

function: insights from Arabidopsis and rice mutants. Curr. Opin. Plant Biol. 7, 719–731 (2004).

21. Jones, J.C. et al. The crystal structure of a self-activating G protein α subunit

reveals its distinct mechanism of signal initiation. Sci. Signal. 4, ra8 (2011).22. Mao, H. et al. Linking differential domain functions of GS3 protein to natural

variation of grain size in rice. Proc. Natl. Acad. Sci. USA 107, 19579–19584 (2010).

23. Chakravorty, D. et al. An atypical heterotrimeric G-protein γ-subunit is involved in

guard cell K +-channel regulation and morphological development in Arabidopsis thaliana . Plant J. 67, 840–851 (2011).

24. Li, S. et al. The plant-specific G protein γ subunit AGG3 influences organ size and

shape in Arabidopsis thaliana . New Phytol. 194, 690–703 (2012).

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gene, RGB1, causes dwarfism and browning of internodes and lamina joint regions. Plant J. 67, 907–916 (2011).

26. Ford, C.E. et al. Molecular basis for interaction of G protein βγ subunits with

effectors. Science 280, 1271–1274 (1998).

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Arabidopsis . Science 292, 2066–2069 (2001).

28. Trusov, Y. et al. Heterotrimeric G protein γ subunits provides functional

selectivity in G βγ dimer signaling in Arabidopsis . Plant Cell 19, 1235–1250 (2007).

29. Fujisawa, Y. et al. Suppression of the heterotrimeric G protein causes abnormal

morphology, including dwarfism, in rice. Proc. Natl. Acad. Sci. USA 96, 7575–7580 (1999).

30. Ashikari, M., Wu, J., Yano, M., Sasaki, T. & Yoshimura, A. Rice gibberellin-insensitive dwarf mutant gene Dwarf1 encodes the α-subunit of GTP-binding protein. Proc. Natl. Acad. Sci. USA 96, 10284–10289 (1999).

l e t t e r s

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oNLINE METHoDs

Plant materials and growth conditions. A set of 226 RILs was bred from the cross between NJ6 and QZL2. Details regarding other rice accessions used are given in Supplementary Table 1. The dep1-32 mutant was selected among an ethylmethane sulfonate mutagenized population of cv. Minghui63. NILs carrying various variant alleles of DEP1, D1 and SD1 were constructed by crossing the relevant donor seven times with the recurrent parent (WYJ7). Field-grown NIL plants were raised in a rice paddy at an interplant spacing of 20 cm during the standard growing season at the National Rice Research Institute stations in Hangzhou and Hainan. The primer sequences for the genotyping assays are provided in Supplementary Table 3.

Transgene constructs. Full-length cDNAs for RGA1, RGB1, RGG1 and RGG2 were amplified from NIL-DEP1 plants and were then inserted into the pAct-in ?HA-nos vector 10. The DEP1 and GS3 coding sequences were amplified and subcloned into the pActin ?MYC-nos vector. The 367-bp portion of RGB1 cDNA was used to develop the pActin ?RNAi-RGB1 construct, which provided the transgene used for RNAi-based downregulation of RGB1. For the p35S ?RGB1-GFP fusion construct, the RGB1 coding sequence was amplified and subcloned into the p35S ?GFP-nos vector 15. Transgenic rice plants were gen-erated by Agrobacterium -mediated transformation as described elsewhere 31. The sequences of all PCR primers used are provided in Supplementary Table 4.Transcript analysis. Total RNA was extracted using the TRIzol reagent (Invitrogen) and reverse transcribed using an M-MLV Reverse Transcriptase kit (Promega). Real-time RT-PCR analysis was performed as described else-where 32. Each experiment was performed at least three times with three independent RNA preparations, and rice actin3 was used as a reference. The relevant PCR primer sequences are given in Supplementary Table 5.Yeast two-hybrid assay. Yeast two-hybrid assays were performed as described elsewhere 15. The RGB1 coding sequence was inserted into pGBKT7 (Clontech), and the full-length DEP1 coding sequence as well as its N-terminal and C-terminal truncated deletions were inserted into pGADT7 (Clontech). The vectors were then transformed into yeast strain AH109. The sequences of the primers used are provided in Supplementary Table 4.

BiFC analysis. Intact DEP1 and its truncated deletions were amplified and inserted into pSPYNE-35S, and RGA1 and RGB1 cDNAs were subcloned into pSPYCE-35S. BiFC experiments based on a tobacco transient expression system were performed as described elsewhere 33.

Microscopy. GFP fluorescence was observed in the root tips of relevant trans-genic rice plants by excitation at 488 nm with an argon laser. The emission signal was captured in the wavelength range 500–600 nm by laser confocal microscopy as described elsewhere 34.

Measurement of glutamine synthase activity. Fresh plant material (~1 g) was ground in 400 μL of 50 mM Tris-HCl (pH 8.0) containing 1 mM MgCl 2, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% polyvinylpyrrolidone (PVP) and 10 mM β-mercaptoethanol. The homogenate was centrifuged at 15,000 r.p.m. for 20 min at 4 °C. A 10 μL aliquot of the supernatant was added to 50 μL of assay mixture (0.6 mL of 0.25 M imidazole-HCl buffer, 0.4 mL of 0.3 M sodium hydrogen glutamate, 0.4 mL of 30 mM ATP-Na and 0.2 mL of 0.5 M MgSO 4) and held for 15 min at 37 °C. The reaction was then stopped by the addition of 200 μL of a 1:1:1 solution of 10% FeCl 3, 24% trichloroacetic acid and 50% HCl. The reactions were centrifuged at 3,500 r.p.m. for 15 min, and the resulting supernatant was assayed spectrophotometrically at 550 nm. Albumin bovine V (Sigma) was used as a standard.

31. Hiei, Y., Ohta, S., Komari, T. & Kumashiro, T. Efficient transformation of rice

(Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271–282 (1994).

32. Jiang, C. et al. Root architecture and anthocyanin accumulation of phosphate

starvation responses are modulated by the GA-DELLA signalling pathway in Arabidopsis . Plant Physiol. 145, 1460–1470 (2007).

33. Walter, M. et al. Visualization of protein interactions in living plant

cells using bimolecular fluorescence complementation. Plant J. 40, 428–438 (2004).

34. Fu, X. & Harberd, N.P . Auxin promotes Arabidopsis root growth by modulating

gibberellin response. Nature 421, 740–743 (2003).

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体液免疫球蛋白测定

体液免疫球蛋白测定 第一节血清IgG、IgA、IgM测定 血清免疫球蛋白IgG、IgA、IgM定量测定方法一般有单向环状免疫扩散法、火箭免疫电泳法、ELlSA、免疫比浊法、放射免疫分析法等。临床常用单向环状免疫扩散法和免疫比浊法来测定血清免疫球蛋白含量。 一、血清IgG、IgA、lgM测定 (一)单向环状免疫扩散法 该法的原理是将抗血清均匀地分散于琼脂或琼脂糖凝胶内,胶板上打孔,孔内注入抗原或待测血清,抗原在含有抗血清的胶内呈放射状(环状)扩散,在抗原抗体达到一定比例时形成可见的沉淀环。在一定条件下,抗原含量越高,沉淀环越大。 (二)免疫比浊法 该法具有检测范围宽、测定结果准确、精密度高、检测时间短(一般在几分钟内即可完成测试)、敏感度高、稳定性好等优点。 二、血清IgG、IgA、IgM测定的临床意义 (一)年龄 新生儿可由母体获得通过胎盘转移来的IgG,故血液中含量较高,接近成人水平。 (二)免疫球蛋白IgG、IgA、IgM均升高 慢性肝脏疾病如慢性活动性肝炎、原发性胆汁性肝硬化、隐匿性肝硬化患者血清中可见3种Ig均升高。慢性细菌感染如慢性支气管炎、肺结核,血IgG可升高。宫内感染时脐血或出生后的新生儿血清中IgM含量可增高。SLE患者以IgG、IgA升高较多见。类风湿关节炎患者以IgM增高为主。 (三)单一免疫球蛋白升高 主要是指患者血清中某一类免疫球蛋白含量显著增多,大多在30g/L以上,这种异常增多的免疫球蛋白其理化性质十分一致,称为单克隆蛋白(MP)即M蛋白。此类异常增多的免疫球蛋白多无免疫活性,故又称副蛋白。由它所致的疾病称为免疫增殖病如多发性骨髓瘤、巨球蛋白血症、恶性淋巴瘤、重链病、轻链病等。 (四)免疫球蛋白降低 先天性低Ig血症,主要见于体液免疫缺陷病和联合免疫缺陷病。IgA缺乏患者,易发生反复呼吸道感染。IgG缺乏患者,易发生化脓性感染。IgM缺乏患者,易发生革兰阴性细菌败血症。 第二节血清IgD和IgE测定 正常人血清中IgD含量很低,仅占血清免疫球蛋白总量的0.2%。膜结合型IgD(mIgD)构成BCR,是B 细胞分化发育成熟的标志。未成熟的B细胞仅表达mIgM,成熟B细胞可同时表达mIgM和mIgD。活化的B 细胞或记忆B细胞其表面的mIgD逐渐消失。 IgE是正常人血清中含量最少的免疫球蛋白,要由黏膜下淋巴组织中的浆细胞分泌。其重要特征为糖含量高达12%。IgE为亲细胞抗体,可引起Ⅰ型超敏反应。IgE可能与机体抗寄生虫免疫有关。 第1页

免疫比浊法检测免疫球蛋白

免疫比浊法检测免疫球蛋白 一、实验目的 利用免疫比浊法绘制标准曲线,并检测样品中免疫球蛋白的浓度。(本小组检测的为IgG样品) 二、实验原理 1.抗原抗体反应(Antigen-antibody reaction):抗原与其刺激机体产生的相应抗体在体内或体外发生特异性结合的反应。反应特点有:特异性、比例性、可逆性、敏感性。影响因素有:电解质、温度、酸碱度。 2.免疫比浊法:合适比例的抗原抗体形成的免疫复合物,在PEG作用下形成微粒,使样品浊度发生变化。当一束光线通过溶液受到光散射和光吸收两个因素的影响而使光的强度减弱,根据光的强度改变可测得微粒浓度。 分类:①透射比浊法(Transmission tubidimetry)当一定波长光线通过浊度发生变化的反应混合物时,由于被不溶性免疫复合物吸收而减弱,故在一定范围内吸光度与免疫复合物量呈正相关。当抗体浓度固定(过量),样品的浊度与其中所含抗原量成正比。(特点)透射比浊操作简便,适用于普通的自动生化分析仪和普通的分光光度计,几乎所有的实验室均能开展。不足的是灵敏度和精密度均不够理想,所需的抗血清量大,检测的时间较长。②散射比浊法(Nephelometry)光线通过检测溶液时,被其中所含的抗原抗体复合物折射而部分偏转,产生散射光,其强度与复合物的数量和散射夹角成正比,与光的波长成反比。(特点)优点是灵敏度、精密度均较高,检测快速。其缺点是需特定的分析仪器,试剂价格高。 本实验采用透射法。 3.聚乙二醇PEG的作用:在免疫反应中,为增强抗原抗体反应常使用增聚剂,3~4%的聚乙二醇,可破坏抗原抗体的水化层,促进抗原抗体靠近反应,但如浓度不适合,会影响其它溶质或产生非特异性聚集影响结果。 三、实验材料 免疫球蛋白A,G(IgA,IgG)测定试剂(试剂1[PEG],试剂2[羊抗人IgA, IgG])(1管/每组)免疫球蛋白A, G(IgA,IgG)校准品,蒸馏水,血清样本(1管) 微量加样枪、ep管(1.5mL离心管) 酶标仪、水浴箱 四、实验步骤 1.在7个EP管中各加250μL IgG试剂1(PEG)。 2.7管分别加入蒸馏水、校准品原液、1:2校准品、1:4校准品、1:8校准品、1:16校准品、样本各2μL。 3.混匀后37℃水浴5min。 4.7管各加入85μL IgG试剂2(羊抗人IgG)。 5.混匀后37℃水浴10min。 6.分别吸取200μL至96孔酶标板中,用酶标仪在700nm处读取OD值。 五、实验结果与数据处理 2.标准曲线

免疫球蛋白的结构

第一节免疫球蛋白的结构(The Structure of Immunoglobulin) B淋巴细胞在抗原刺激下增殖分化为浆细胞,产生能与相应抗原发生特异性结合的免疫蛋白,这类免疫球蛋白被称为抗体(antibody, Ab)。 1937年,Tiselius用电泳方法将血清蛋白分为白蛋白、α1、α2、β及γ球蛋白等组分,其后又证明抗体的活性部分是在γ球蛋白部分。因此,相当长一段时间内,抗体又被称为γ球蛋白(丙种球蛋白)。 实际上,抗体的活性除γ球蛋白外,还存在于α和β球蛋白处。1968年和1972年的两次国际会议上,将具有抗体活性或化学结构与抗体相似的球蛋白统一命名为免疫球蛋白(immunoglobulin,Ig)。 Ig是化学结构的概念,它包括正常的抗体球蛋白和一些未证实抗体活性的免疫球蛋白,如骨髓瘤病人血清中的M蛋白及尿中的本周氏(Bence Jones, BJ)蛋白等。 免疫球蛋白可分为分泌型(secreted Ig,SIg)和膜型(membrane Ig, mIg)。前者主要存在于血清及其他体液或外分泌液中,具有抗体的各种功能;后者是B细胞表面的抗原识别受体。 ☆☆相关素材☆☆ 图片正常人血清电泳分离图 一免疫球蛋白的基本结构 The basical structure of immunoglobulin 免疫球蛋白分子是由两条相同的重链(heavy chain,H链)和两条相同的轻链(light chain,L链)通过链间二硫键连接而成的四肽链结构。 X射线晶体结构分析发现,IgG分子由3个相同大小的节段组成,位于上端的两个臂由易弯曲的铰链区(hinge region)连接到主干上形成一个"Y"形分子,称为Ig分子的单体,是构成免疫球蛋白分子的基本单位。

血清免疫球蛋白测定.docx

血清免疫球蛋白测定 B-淋巴细胞受抗原刺激后,引起一系列细胞形态与生化特性变化,转化为淋巴母细胞并增生繁殖,最后演化为浆细胞,合成免疫球蛋白。免疫球蛋白普遍存在于生物体的血液、体液、外分泌液及某些细胞(如淋巴细胞)的细胞膜上。免疫球蛋白是一组具有抗体特性的球蛋白,其分子量很大,含有1000个以上的氨基酸分子,均由其共同抗原的两个相同的轻链(L链)和具有特异性抗原的两个不同的重链(H链)组成。 1分类 根据重链的氨基酸组成不同,免疫球蛋白可分为分五类:免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)和免疫球蛋白E(IgE)。 1.1IgG IgG是人体的主要免疫球蛋白,也是唯一能通达胎盘的免疫球蛋白。根据IgG的重链氨基酸顺序的差别,可分为IgG1,IgG2,IgG3,IgG4各亚类。IgG是主要抗感染抗体,对各种细菌、病DU有抗体活性,并有激活补体的作用。 1.2IgA IgA是分泌液中主要抗体,可分为IgA1和IgA2两个亚类。存在于血清中的称血清型IgA,存在于泪液、乳汁、胃肠道、呼吸道和泌尿生殖道分泌液中的称分泌型IgA,对局部粘膜有抗菌和抗病DU作用。

1.3IgM IgM可分为IgM1和IgM2两个亚类。是抗原刺激最先产生的免疫球蛋白,有较强的固定补体、溶解细菌及细胞的能力。IgM是抗革兰氏阴性菌和异体红细胞的主要抗体,也是存在于B淋巴细胞表面的主要免疫球蛋白。 1.4IgD 其作用与过敏反应有关。据报道,IgD可对某些抗原起反应,如青霉素、胰岛素、核抗原、甲状腺抗原等。它存在于B淋巴细胞表面,构成早期的膜受体,具有识别抗原,制动B淋巴细胞的分化作用。 1.5IgE 与I型变态反应有关,具有亲细胞特性。IgE通过Fc段与肥大细胞、嗜碱粒细胞上的Fc受体结合,故属亲同种细胞性抗体。当机体再次接触同种抗原后,过敏原即与结合在细胞表面的IgE作用,使细胞释放介质,引起平滑肌痉挛,血管通透性增加等过敏反应,还具有抗寄生虫感染作用。 2正常参考值 IgG:8~16g/L,IgA:1~4g/L,IgM:0.5~1.9g/L,IgD:0.01~0.4g/L,IgE:0.001~0.009g/L。 3临床意义 3.1血清免疫球蛋白降低 血清免疫球蛋白水平取决于免疫球蛋白合成和分解代谢的速率以及由体内丢失的程度。血清免疫球蛋白降低可由于合成不足,丢失

免疫球蛋白IgE

免疫球蛋白I g E This manuscript was revised on November 28, 2020

免疫球蛋白IgE 免疫球蛋白lgE(人体的一种抗体),存在于血中。是正常人血清中含量最少Ig,可以引起I型超敏反应 球蛋白偏高,与慢性肝病,机体免疫系统有关。人血浆内的免疫球蛋白大多数存在于丙种球蛋白(γ-球蛋白)中。可分为五类,即免疫球蛋白G (IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)和免疫球蛋白E(IgE)。其中IgG是最主要的免疫球蛋白,约占人血浆丙种球蛋白的70%,分子量约15万,含糖2~3%。IgG分子由4条肽链组成。其中分子量为2.5万的肽链,称轻链,分子量为5万的肽链,称重链。轻链与重链之间通过二硫键(—S—S—)相连接。免疫球蛋白是机体受抗原(如病原体)刺激后产生的,其主要作用是与抗原起免疫反应,生成抗原-抗体复合物,从而阻断病原体对机体的危害,使病原体失去致病作用。另一方面,免疫球蛋白有时也有致病作用。在慢性乙肝患者,长期白、球比例倒置,警惕有肝硬化迹象免疫球蛋白Ig的医学定义是具有免疫功能或结构与抗体相似的球蛋白,其基本结构是:两条相同的重链和两条相同的轻链由链间二硫键连接而成。重链根据恒定区中氨基酸种类和排列顺序不同分为5种:α,γ,δ,ε,μ。分别对应5种含相应重链的免疫球蛋白:IgA,IgG,IgD,IgE,IgM。 血清免疫球蛋白E(IgE)又称为反应素,是血清中含量最少的一类免疫球蛋白,只占血清总量的0.001%。IgE测定用国际单位IU或ng表示, 1IU=2.4ng,相当于WHO标准冻干血清制剂0.00928内所含的IgE量。 IgE是正常人血清中含量最少的Ig,正常浓度是5×10的-5次方 mg/ml。正常人群IgE水平受环境、种族、遗传、年龄、检测方法及取样标准等因素的影响,各家各医院的正常值相差甚远。它是一种亲细胞抗体,与具有吞噬作用的肥大细胞,嗜中性粒细胞(可以吞噬被细菌,病毒等有害病原体感染的细胞)有很高的亲和力。因此可以介导Ⅰ型超敏反应,即我们平时所说的过敏反应。此外,IgE可能与抗寄生虫感染有关。 一般大于100-333IU/ml(ku/L)即大于250-800ng/ml(μg/L)即表现为增高,主要见于过敏性体质及过敏性疾病,如哮喘、过敏性鼻炎、荨麻疹等。

血清免疫球蛋白测定(一)

血清免疫球蛋白测定(一) B-淋巴细胞受抗原刺激后,引起一系列细胞形态与生化特性变化,转化为淋巴母细胞并增生繁殖,最后演化为浆细胞,合成免疫球蛋白。免疫球蛋白普遍存在于生物体的血液、体液、外分泌液及某些细胞(如淋巴细胞)的细胞膜上。免疫球蛋白是一组具有抗体特性的球蛋白,其分子量很大,含有1000个以上的氨基酸分子,均由其共同抗原的两个相同的轻链(L链)和具有特异性抗原的两个不同的重链(H链)组成。 1分类 根据重链的氨基酸组成不同,免疫球蛋白可分为分五类:免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)和免疫球蛋白E(IgE)。 1.1IgG IgG是人体的主要免疫球蛋白,也是唯一能通达胎盘的免疫球蛋白。根据IgG的重链氨基酸顺序的差别,可分为IgG1,IgG2,IgG3,IgG4各亚类。IgG是主要抗感染抗体,对各种细菌、病毒有抗体活性,并有激活补体的作用。 1.2IgA IgA是分泌液中主要抗体,可分为IgA1和IgA2两个亚类。存在于血清中的称血清型IgA,存在于泪液、乳汁、胃肠道、呼吸道和泌尿生殖道分泌液中的称分泌型IgA,对局部粘膜有抗菌和抗病毒作用。 1.3IgM IgM可分为IgM1和IgM2两个亚类。是抗原刺激最先产生的免疫球蛋白,有较强的固定补体、溶解细菌及细胞的能力。IgM是抗革兰氏阴性菌和异体红细胞的主要抗体,也是存在于B淋巴细胞表面的主要免疫球蛋白。 1.4IgD 其作用与过敏反应有关。据报道,IgD可对某些抗原起反应,如青霉素、胰岛素、核抗原、甲状腺抗原等。它存在于B淋巴细胞表面,构成早期的膜受体,具有识别抗原,制动B淋巴细胞的分化作用。 1.5IgE 与I型变态反应有关,具有亲细胞特性。IgE通过Fc段与肥大细胞、嗜碱粒细胞上的Fc受体结合,故属亲同种细胞性抗体。当机体再次接触同种抗原后,过敏原即与结合在细胞表面的IgE作用,使细胞释放介质,引起平滑肌痉挛,血管通透性增加等过敏反应,还具有抗寄生虫感染作用。 2正常参考值 IgG:8~16g/L,IgA:1~4g/L,IgM:0.5~1.9g/L,IgD:0.01~0.4g/L,IgE:0.001~0.009g/L。3临床意义 3.1血清免疫球蛋白降低 血清免疫球蛋白水平取决于免疫球蛋白合成和分解代谢的速率以及由体内丢失的程度。血清免疫球蛋白降低可由于合成不足,丢失增多和分解加快引起。合成不足:血清免疫球蛋白降低可由于原发性或继发性合成缺陷所致。原发性免疫缺陷病有性联先天性丙种球蛋白缺乏症、婴儿暂时性丙种球蛋白低下症、伴IgM升高的性联低丙种球蛋白血症、选择性IgA缺乏症、选择性IgM缺乏症、选择性IgG亚类缺乏症等。继发性免疫缺陷病有慢性淋巴细胞性白血病、多发性骨髓瘤、巨球蛋白血症和恶性肿瘤等。丢失增多:从胃肠道丢失的见于溃疡性结肠炎、消化道癌肿、吸收不良综合征、淋巴瘤、肠淋巴管扩张症。从皮肤丢失的有急性灼伤、特异反应性皮炎。从浆膜腔丢失的有胸膜炎、腹膜炎、反复抽胸腹水。从肾脏丢失的有肾小球肾炎(微小病变型、膜性、增殖性)、肾静脉血栓形成、SLE和淋巴瘤。主要是由于蛋白从尿中丢失或毒素抑制免疫球蛋白合成,后者首先影响IgM,其次影响IgA,然后再影响IgG。分

血清免疫球蛋白G的分离纯化及鉴定

实验八血清免疫球蛋白G的分离纯化及鉴定 为了初步掌握蛋白质的分离纯化技术,选择了从家畜血清中分离纯化免疫球蛋白G (Immunoglobulin G,简称IgG)的实验。血清中的蛋白质有数十种之多,IgG是球蛋白的一种。 分离纯化蛋白质的方法是利用不同蛋白质的某些物理、化学性质(如在一定条件下带电的情况、分子量、溶解度等)的不同而建立起来的,其中有盐析、离子交换、凝胶过滤、亲和层析、制备电泳和超速离心等。在分离纯化时,要根据情况选用几种方法互相配合才能达到分离纯化一种蛋白质的目的。 本实验采用硫酸铵盐析、DEAE-纤维素离子交换及凝胶过滤等方法,提取家畜血清中IgG。其原理及操作如下: ㈠硫酸铵盐析 1.试剂 饱和硫酸铵溶液pH7.0:称取(NH4)2SO4760g,加蒸馏水至1000ml,加热至5℃,使绝大部分硫酸铵溶解,置室温过夜,取上清液,用氢氧化铵调pH至7.0。 (内含0.15mol/L NaCl,简称PBS):取0.2mol/L Na2HPO4磷酸缓冲溶液(0.01mol/L,pH7.0) 溶液30.5ml,0.2mol/L NaH2PO4溶液19.4ml,加NaCl8.5g,加蒸馏水至1000ml。 磷酸缓冲溶液(0.0175mol/L,pH6.7)(不含NaCl,简称PB):取0.2mol/L Na2HPO4溶液43.5ml,0.2mol/L NaH2PO4溶液56.6ml混合,用蒸馏水稀释至1000ml。 2.操作①取家畜血清5ml,加磷酸缓冲溶液(0.1mol/L,pH7.0)5ml,混匀,滴加(边摇边搅拌)饱和硫酸铵4ml(此时溶液硫酸铵饱和度约为20%)。静置20min,以3000r/min 离心15min,沉淀为纤维蛋白(弃去),上清液中含清蛋白、球蛋白。②取上清液,再加饱和硫酸铵溶液6ml(方法同前),此时溶液硫酸铵饱和度为50%,静置20min,以3000r/min 离心15min,上清液中含清蛋白,沉淀为球蛋白。③倾去上清液,将沉淀溶于5ml磷酸缓冲液(0.01mol/L pH7.0),再加饱和硫酸铵 3.2ml,此时溶液硫酸铵的饱和度约为33%,静置20min,以3000r/min离心15min,除去上清液,沉淀即为γ-球蛋白。 ㈡凝胶过滤脱盐 1.试剂①Sephadex G-25:用前以蒸馏水浸泡5h或在沸水浴中溶胀2h;②磷酸盐缓冲液(pH6.7,0.0175mol/L):见前;③磷酸盐缓冲液(pH7.0,0.01mil/L)见前;④奈氏试剂; ⑤20%磺酰水杨酸。 2.操作①取层析柱一根(1.5cm×20cm),垂直于固定架上,夹住流出口。加10ml磷酸缓冲液(0.0175mol/L, pH6.7)于柱中。将已溶胀好的Sephadex G-25倾倒去水,加入磷酸盐缓冲液并搅拌成悬浮液,慢慢装入柱中,打开流出口,继续加入Sephadex G-25使自然沉降至15cm高,关闭出口。②打开出口,使柱中多余的磷酸缓冲液流出凝胶柱床面(切勿低于柱床面)。用吸管吸取盐析所得的蛋白质溶液2ml,沿管壁加入凝胶面上,打开流出口,让样品进入凝胶柱,再用滴管小心加入磷酸盐缓冲液至离凝胶面2~3cm高,编号,按顺序放在试管架上。③在收集的同时,检查蛋白质是否流出。于每管中取出1滴放在黑色比色板孔中(按编号顺序),再分别加入1滴20%磺酰水杨酸,如出现白色沉淀即表示蛋白质已流出凝胶柱,如此直检查到蛋白质全流出为止。与此同时,再从含蛋白质的管中取出1滴于白色比色板中,加入奈氏试剂1滴,如蛋白管中不出现棕色,即表示蛋白质中的硫酸铵已除去,合并无硫酸铵的蛋白质管,待用。 ㈢DEAE-纤维素纯化免疫球蛋白G 1.试剂①DEAE-纤维素:DE-11、DE-22、DE-32、DE-52均可;②0.5mol/L HCl溶液;③0.5mol/L NaOH溶液;④pH6.7,0.0175mol/L磷酸盐缓冲液。 2.操作

测定血清中免疫球蛋白的临床意义

测定血清中免疫球蛋白的临床意义 王 丹1,姜 芳2 (11哈尔滨医科大学第一临床医学院,黑龙江哈尔滨150001;21哈尔滨电机厂医院,黑龙江哈尔滨150040) 摘要:机体患某种疾病时,可有1种或几种免疫球蛋白明显升高或减低。因此,检测血清中免疫球蛋白的含量对于某些疾病的诊断和指导治疗有实际临床意义。该文简单介绍引起高免疫球蛋白血症和低免疫球蛋白血症的主要疾病。 关键词:免疫球蛋白;高免疫球蛋白血症;低免疫球蛋白血症中图分类号:R39217 文献标识码:A 文章编号:1004-5775(2002)03-0167-02 Clinical Significance of Determination on Immunoglobulin in Serum W ANG Dan ,J I ANG Fang (The First Affiliated Hospital of Harbin Medical University Harbin 150001China ) Abstract :There might be one or several kinds of immunoglobulin increased or decreased when body su ffered s ome diseases.There might be clin 2ical significance in diagnosis and indication to detect the content of immunoglobulin in serum for s ome diseases.This paper had introduced about s ome diseases that could cause hyperimmunoglobulinia or hypoimmunoglobulinia briefly.K ey w ords :Immunoglobulin ;Hyperimmunoglobulinia ;Hypoimmunoglobulinia 免疫球蛋白(Ig )是抗体的表现形式和物质基础,是具有抗体活性和结构相似的血清球蛋白。 免疫球蛋白的基本结构是4条肽链,2轻2重,肽链之间由二硫键相联结。任何一种免疫球蛋白,只有一类别的重链、轻链相同。根据重链的不同,免疫球蛋白分为IgG 、IgA 、IgM 、IgD 和IgE 5种。免疫球蛋白由浆细胞、前浆细胞或淋巴细胞产生,在血浆和血管外体液中的分布大致相等。循环中的免疫球蛋白,每日约更换1/4。健康成人每日合成免疫球蛋白2~5g ,但在发生感染时,合成可增加7倍。正常成人血清中各种免疫球蛋的含量和某些特性见表1。在机体患某种疾病时,可有1种或几种免疫球蛋白明显升高或减低。因此,检测血清中免疫球蛋白的含量对于某些疾病的诊断和指导治疗有实际临床意义。检测IgG 、IgA 、IgM 所用的方法,根据检样不同有所不同。血液中含量测定既可用单向环状免疫扩散法,也可用免疫比浊法。前法简单经济,但工作量大,耗时长;后法精密度较高,可自动化,需时短,但需要昂贵的自动化仪器。 作者简介:王丹(1978-),女,汉族,检验士。 〔16〕姜林娣,梅振武,王吉耀1甲氨喋呤治疗类风湿关节炎临床疗效评价〔J 〕1中华风湿病学杂志,1998,2(4):204~2071 〔17〕胡晋江,吕随峰,朱全刚,等1来氟米特治疗类风湿关节炎研究进展〔J 〕1中国新药杂志,2001,10(4):294~3001 〔18〕Sm olen JS ,K alden JR ,Scott D L ,et al .E fficacy and safely of leflunomide com pared with placebo and sulphasalazine in active rheumctoid arthritis :a double -blind ,randomised ,muliticentre trial 〔J 〕.Lancet ,1999,353:259~266. 〔19〕Leirisalo -Repo M.Therapeutic aspects of spondylo -arthropathies -a.review 〔J 〕.Scand -Rheumatol ,1998,27(5):323~328. 〔20〕S ieper J.Basic therapeutics in the management of Various forms of arturitis 〔J 〕.Orthopade ,1998,27(8):576~580. 〔21〕G eher P ,G omr B.Repeated cyclosporing therapy of periph 2eral arthritis ass ociated with ankylosing 〔J 〕.med -sci -m onit ,2001,7(1):105~107. 〔22〕Durez P ,H orsmans Y.Dramatic response after an intra 2venous loading dose of azathioprine in one case of severe and refracto 2ry ankglosing sponclylitis 〔J 〕.Rheumatology ,2000,39(2):182~184. 〔23〕Lorenz H M.T NF inhibitors in the treatment of arthritis 〔J 〕.Curr -opin -Investig -Drugs ,2000,1(2):188~193. 〔24〕Maim R ,S tclair EW ,Breedvdd F ,et al .In fliximab (chimeric anti -tum our necrosis factor m onoclonal an tibody )versus placebo in rheumatoid arthritis patients.recrosis concomitant methotrexate :a ranaomised phise Ⅲtrial 〔J 〕.Lancet ,1999,354:1932~1939. 〔25〕Brandt J ,Haibel H ,C omelg D ,et al .success ful treatment of active ankylosing S pondylitis with the anti -tum or necrosis factor alpha m onoclonal antibody in fliximab 〔J 〕.Arthritis -Rheum ,2000,43(6):1346~1352. 〔26〕Weinblatt ME ,kremer JM ,Bankhurst AR ,et al .A trial of etanercept.a recombinant tum our necrosis factor receptor :Fc fusion protein ,in patients with rheumatoid arthritis receiving methotrexate 〔J 〕.N Engl J mecl ,1999,340:253~259. 〔27〕郭玉海,曹贻训1强直性脊柱炎研究进展〔J 〕1山东中医杂志,2000,4:251~2531 〔28〕唐传芝,李林1强直性脊柱炎中医治疗进展〔J 〕1中医药信息,2001,1:20~231 〔29〕林昌松,续青1中医治疗强直性脊柱炎经验〔J 〕1河北中医,2001,2:115~1161 〔30〕林昌松,刘晓玲,关彤,等1陈纪教授治疗强直性脊柱炎经验〔J 〕1新中医,2001,2:10~111 〔31〕陈林囡,郭邵浩1雷公藤合独活寄生汤治疗强直性脊柱炎临床观察〔J 〕1南京中医药大学学报,1998,14(5):2741 〔32〕戈吉庆,樊宇富,钱小奇1强直性脊柱炎中医辨证论治研究进展〔J 〕1江苏中医,2000,12:57~581 (编辑:谢忠艳) (收稿日期:2001-01-24)

血清免疫球蛋白在乙型肝炎患者中的检验意义分析

血清免疫球蛋白在乙型肝炎患者中的检验意义分析 发表时间:2018-07-13T15:34:15.670Z 来源:《健康世界》2018年10期作者:吴仲锋[导读] 分析探讨血清免疫球蛋白在乙型肝炎患者中的检验意义。 阜阳市人民医院安徽阜阳 236000 摘要:目的分析探讨血清免疫球蛋白在乙型肝炎患者中的检验意义。方法本次选取我院在2016年2月—2017年2月收治的60例乙型肝炎患者作为研究的对象,以病情严重程度分为:轻型慢性乙型肝炎32例(A组)、重型慢性乙型肝炎28例(B组),取同期健康体检者60名作为对照组对象,通过临床检验比较三组相关血清免疫球蛋白指标水平(IgA、IgG、IgM)情况。结果在IgA、IgG、IgM三项血清免疫球蛋白指标水平上,A组、B组与对照组比较,均明显更高,组间数据差异具备统计学意义(P<0.05)。此外,B组和A组比较,在在IgA、IgG、IgM三项血清免疫球蛋白指标水平上均明显更高,组间数据差异具备统计学意义(P<0.05)。结论乙型肝炎患者的血清免疫球蛋白水平和健康人群比较明显更高,且重型慢性乙型肝炎患者的血清免疫球蛋白水平高于轻型慢性乙型肝炎;加强血清免疫球蛋白指标水平检测,能够为乙型肝炎患者的进一步诊疗工作提供必要的科学依据;因此,值得采纳及应用。关键词:血清免疫球蛋白;乙型肝炎;检验意义 [Abstract] objective to explore the significance of serum immunoglobulin in patients with hepatitis b. methods The select our hospital in February 2016 - February 2017,60 patients with hepatitis B were treated as the research object,using disease severity was divided into:light 32 cases of chronic hepatitis B(group A),28 cases severe chronic hepatitis B(group B),take the same physical examination 60 as A control object,through clinical trial compared three groups of related index of serum immunoglobulin levels(IgA,IgG,IgM). Results in IgA,IgG and IgM serum immunoglobulin index levels,the comparison between group A,group B and control group was significantly higher,with statistically significant difference between groups(P < 0.05). In addition,group B and group A were significantly higher in IgA,IgG and IgM serum immunoglobulin index levels,with statistically significant differences between groups(P < 0.05). Conclusion serum immunoglobulin levels in patients with hepatitis b and healthy population is significantly higher,and severe chronic hepatitis b patients serum immunoglobulin levels higher than light chronic hepatitis b;The enhancement of serum immunoglobulin index level detection can provide the necessary scientific basis for the further diagnosis and treatment of hepatitis b patients. Therefore,it is worth adopting and applying. [Key words] serum immunoglobulin;Hepatitis b;Inspection significance 乙型肝炎,是感染科较为常见的一种疾病,主要由乙型肝炎病毒诱发,该类患者主要的临床表现为乏力疲劳、食欲不振、恶心、厌油以及右上腹痛等。为了提高乙型肝炎患者的生存质量,需加强临床诊疗工作的开展[1]。本次将我院在2016年2月—2017年2月收治的60例乙型肝炎患者作为研究的对象,其目的是分析探讨血清免疫球蛋白在乙型肝炎患者中的检验意义,现将研究成果作如下报道: 1.资料及方法 1.1一般资料 本次选取我院在2016年2月—2017年2月收治的60例乙型肝炎患者作为研究的对象,以病情严重程度分为:轻型慢性乙型肝炎32例(A 组)、重型慢性乙型肝炎28例(B组),均符合《慢性乙型肝炎防治指南》[2]中的诊断标准,且均经医学伦理委员会审批通过,将合并其他严重脏器疾病及严重精神障碍者排除在外。其中,A组32例中,男性20例、女性12例;年龄分布在18-76岁,年龄均值为(44.8±1.3)岁。B组28例中,男性18例、女性10例;年龄分布在19-75岁,年龄均值为(44.5±1.4)岁。取同期健康体检者60名作为对照组对象,男性38例、女性22例;年龄分布在19-73岁,年龄均值为(44.3±1.6)岁。结合上述数据可知,60例乙型肝炎和60名健康体检者的一般资料比较无明显差异(P>0.05),有可比价值。 1.2检测方法 (1)首先,进行样本的采集,对所有受检者基于晨间、空腹以及无菌条件下,进行静脉血的采集,约采集5ml;其次,进行离心处理,离心速度为每分钟3000转,将离心时间控制在10分钟,得到上层血清,基于抗凝管当中放置,进一步在-20℃的冰箱当中保持,予备用。 (2)本次选取我院使用的全自动生化分析仪,并配套相关检验试剂。通过免疫比浊法的使用,进一步对血清免疫球蛋白指标水平进行测定,包括:IgA、IgG、IgM。 1.3判定标准 比较A组、B组、对照组的血清免疫球蛋白指标水平(IgA、IgG、IgM)情况。 1.4统计学分析 本次在数据处理方法使用的统计学软件型号为SPSS21.0,计量资料选用()表示,组间涉及的数据比较用t检验;P<0.05表示2组数据有明显差异,有统计学意义。 2.结果 在IgA、IgG、IgM三项血清免疫球蛋白指标水平上,A组、B组与对照组比较,均明显更高,组间数据差异具备统计学意义(P<0.05)。此外,B组和A组比较,在在IgA、IgG、IgM三项血清免疫球蛋白指标水平上均明显更高(P<0.05)。详细数据见下表1:表1 三组血清免疫球蛋白指标水平情况比较(,g/L)

血清免疫球蛋白测定

血清免疫球蛋白测定 【中图分类号】R446.11 【文献标识码】A 【文章编号】1672-5085(2009)20-0263-02 B-淋巴细胞受抗原刺激后,引起一系列细胞形态与生化特性变化,转化为淋巴母细胞并增生 繁殖,最后演化为浆细胞,合成免疫球蛋白。免疫球蛋白普遍存在于生物体的血液、体液、 外分泌液及某些细胞(如淋巴细胞)的细胞膜上。免疫球蛋白是一组具有抗体特性的球蛋白,其分子量很大,含有1000个以上的氨基酸分子,均由其共同抗原的两个相同的轻链(L链) 和具有特异性抗原的两个不同的重链(H链)组成。 1 分类 根据重链的氨基酸组成不同,免疫球蛋白可分为分五类:免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)和免疫球蛋白E(IgE)。 1.1 IgG IgG是人体的主要免疫球蛋白,也是唯一能通达胎盘的免疫球蛋白。根据IgG的重链氨基酸 顺序的差别,可分为IgG1,IgG2,IgG3,IgG4各亚类。IgG是主要抗感染抗体,对各种细菌、病毒有抗体活性,并有激活补体的作用。 1.2 IgA IgA是分泌液中主要抗体,可分为IgA1和IgA2两个亚类。存在于血清中的称血清型IgA,存 在于泪液、乳汁、胃肠道、呼吸道和泌尿生殖道分泌液中的称分泌型IgA,对局部粘膜有抗 菌和抗病毒作用。 1.3 IgM IgM可分为IgM1和IgM2两个亚类。是抗原刺激最先产生的免疫球蛋白,有较强的固定补体、溶解细菌及细胞的能力。IgM是抗革兰氏阴性菌和异体红细胞的主要抗体,也是存在于B淋 巴细胞表面的主要免疫球蛋白。 1.4 IgD 其作用与过敏反应有关。据报道,IgD可对某些抗原起反应,如青霉素、胰岛素、核抗原、 甲状腺抗原等。它存在于B淋巴细胞表面,构成早期的膜受体,具有识别抗原,制动B淋巴 细胞的分化作用。 1.5 IgE 与I型变态反应有关,具有亲细胞特性。IgE通过Fc段与肥大细胞、嗜碱粒细胞上的Fc受体 结合,故属亲同种细胞性抗体。当机体再次接触同种抗原后,过敏原即与结合在细胞表面的IgE作用,使细胞释放介质,引起平滑肌痉挛,血管通透性增加等过敏反应,还具有抗寄生虫感染作用。 2 正常参考值 IgG:8~16g/L,IgA:1~4g/L,IgM:0.5~1.9g/L,IgD:0.01~0.4g/L,IgE:0.001~0.009g/L。 3 临床意义 3.1血清免疫球蛋白降低

血清免疫球蛋白的提取分离、纯化及鉴定

I 血液及组织样品的制备 分析组织中某种物质的含量、探索物质代谢的过程和规律,经常使用动物的肝、肾、脑、粘膜和肌肉等组织,也选用全血、血浆、血清或者无蛋白血滤液等血液样品,有时也采用尿液、胃液等完成各种生化实验。掌握以上各种实验样品的正确处理和制备方法是保证生化实验顺利进行的关键。 一、血液样品 (一)采血 测定用的血液,多由静脉采集。一般在饲喂前空腹采取,因此时血液中化学成分含量比较稳定,采血时所用的针头、注射器,盛血容器要清洁干净;接血时应让血液沿着容器壁慢慢注入,以防溶血和产生泡沫。 (二)血清、全血及血浆的制备 1.血清的制备 血清是全血不加抗凝剂自然凝固后析出的淡黄清亮液体。制备方法是:将刚采集的血液直接注入试管或离心管中。将试管放成斜面,让其自然凝固,一般经3h 血块自然收缩而析出血清;也可将血样放入37 ℃恒温箱内,促使血块收缩,能较快约析出血清。为了缩短时间,也可用离心机分离(未凝或凝固的均可离心),分离出的血青,用吸管吸出置于另一试管中,若不清亮或带有血细胞,应重离心,加盖冷藏备用。 2.全血及血浆的制备 取清洁干燥的试管或其它容器,收集动物的新鲜血液,立即与适量的抗凝剂充分混合,所得到的抗凝血为全血。每毫升血液中加入抗凝剂的种类可以根据实验的需要进行选择,但是用量不宜过大,否则将影响实验的结果。将已抗凝的全二于2,000r / min 离心10min ,沉降血细胞,取上层清液即为血浆。血浆比血清分离得快而且量多:两者的差别,主要是血浆比血清多含一种纤维蛋白原,其它成分基本相同。 3.抗凝剂 凡能够抑制血液凝固反应进行的化合物称为抗凝剂。抗凝剂种类甚多,实验室常用的有如下几种,可根据情况选择使用。 (1)草酸钾(钠)优点是溶解度大,可迅速与血中钙离子结合,形成不溶性草酸钙,使血液不凝固。每毫升血液用1-2mg 即可。 配制方法:配制10%草酸钾水溶液二吸取此液0.1ml 放入一试管中,慢慢转动试管,泛草酸钾尽量铺散在试管壁上,置80 ℃烘箱烤干(若超过150 ℃则分解),管壁即呈一薄层三色粉末,加塞备用。可抗凝血液5ml 。 此抗凝血,常用于非蛋白氮等多种测定项目,但不适用于钾、钙的测定。对乳酸脱氯酸性磷酸酶和淀粉酶具有抑制作用,使用时应注意。 ( 2)草酸钾-氟化钠氟化钠是一种弱抗凝剂。但浓度2mg / ml 时能抑制血液内葡萄糖的分解,因此在测定血糖时常与草酸钾混合使用。 配制方法:草酸钾6g、氟化钠3g,溶于100ml 蒸馏水中。每个试管加入0.25ml,于80℃烘干备用。每管含混合剂22. 5mg,可抗凝5ml 血液。 此抗凝血,因氟化钠抑制睬酶,所以不能用于脉酶法的尿素氮测定;也不能用于淀粉酶及磷酸酶的测定。 (3)乙二胺四乙酸二钠盐(简称EDTANa2 ) EDTANa2 易与钙离子络合而使血液不凝。 有效浓度为0.5mg 可抗凝lml 血液。 配制方法:配成4 % EDTANa :水溶液。每管装0.lml , 80 ℃烘干,可抗凝5ml 血液。此抗凝血液适用于多种生化分析。但不能用于血浆中含氮物质、钙及钠的测定。

免疫球蛋白检查的意义

免疫球蛋白检查的意义 免疫球蛋白是人体血清和体液中具有抗体活性的一类蛋白质。又称丙种球蛋白。具有抗菌、抗病毒作用和加强细胞的吞噬作用,并能在补体的协同下,杀灭或溶解病原微生物。是机体抗御疾病的重要成分。 血清免疫球蛋白可分为五种类型,即IgG、IgM、IgA、IgD、IgE。其正常值由于检查的对象、年龄、地区和方法不同而差异。 各种免疫球蛋白不但量上有区别,而且在功能上也各有特点: IgG:具有抗菌、抗病毒、抗毒素作用的大部分抗体属于IgG。它是唯一能通过胎盘的免疫球蛋白。IgG增高见于IgG型多发性骨髓瘤、系统性红斑狼疮、类风湿性关节炎、慢性活动性肝炎、结核病、黑热病及某些感染性疾病等。降低见于肾病综合征、某些肿瘤、白血病、重链病、轻链病及某些免疫缺陷病。 IgA:IgA具有抗细菌和抗病毒的作用。不能通过胎盘,小儿只能从母乳中得到。 免疫球蛋白A(IgA):参考值:成人0.7~3.9克/升。在人体的血液中IgA分为两型,即血清型和分泌型,前者以单体形式存在,后者是由一种连接的二聚体和分泌片组成,且合成和分泌的部位在肠道、呼吸道、乳腺、唾液腺和泪腺,因此主要存在于

胃肠道、支气管分泌液、初乳、唾液和泪液中,是参与粘膜部位免疫的主要抗体,在局部抗感染中发挥重要作用。婴儿正是通过初乳获得分泌型IgA,达到自然被动免疫的效果。 IgA增高见于IgA型多发性骨髓病、系统性红斑狼疮、类风湿性关节炎、肝硬化、湿疹、血小板减少等疾病。降低见于重链病、轻链病、吸收不良综合征、某些免疫缺陷病、反复呼吸道感染、输血反应、自身免疫性疾病等。 IgM是一种高效能的抗体,杀菌力强,特别是对大肠杆菌等革兰氏阴性菌有效。 免疫球蛋白M(IgM):参考值:成人0.4~3.5克/升。是个体发育过程中最早合成和分泌的抗体,在胎儿发肓晚期即可产生IgM,在新生儿中含量极微,出生逐渐升高,到1/2~1岁时达到成人水平。在脐带血中此抗体含量增加提示胎儿有宫内感染(如风疹病毒、巨细胞病毒等)。天然的血型抗体为IgM,由血型不合的输血所引起的溶血反应亦是由此抗体所致。当机体遇有病原体感染时,IgM是体液免疫应答中产生最早的抗体,在临床上检测此抗体可特异性的早期诊断和治疗疾病。 临床意义:增高见于巨球蛋白血症、自身免疫性疾病(系统性红班狼疮、类风湿关节炎、干燥综合征)、病毒感染、慢性淋巴细胞性白血病和恶性淋巴瘤。降低见于多发性骨髓瘤、烧伤、营养不良和免疫低下疾病。

免疫球蛋白49977

1、简述各类型人免疫球蛋白的特点。 1、1 Ig与Ab的区别 免疫球蛋白(immunoglobulin Ig ):具有抗体活性或化学结构与抗体相似的球蛋白,统称为Ig。抗体(Antibody Ab):B 细胞识别抗原后增殖分化为浆细胞,由浆细胞合成并分泌的一种能与相应抗原特异性结合,具有免疫功能的球蛋白。 1、2 Ig的分类 在同一种属的所有个体内,Ig重链C区所含抗原表位不同,可将免疫球蛋白的重链分为5种(重链AA组成以及排列顺序不同):g、a、m、d、e,与此对应的免疫球蛋白分为5类(class),分别就是IgG、IgA、IgM、IgD、IgE。同一类的免疫球蛋白中,根据其重链抗原性与二硫键的数目与位置的不同,又可分为不同的亚类(subclass)。IgG有IgG1~IgG4四个亚类;IgA有IgA1与IgA2两个亚类;IgD与IgE尚未发现亚类。 1、3 Ig的功能(Ab的生物学活性) ①识别并特异性结合Ag:Ig单体可结合两个抗原表位,为双价 ②激活补体:IgG1-3、IgM 型抗体结合抗原后,通过经典途径活化补体;聚合的IgA、IgG4可经旁路途径活化补体 ③结合细胞表面的Fc受体:调理、促吞噬作用,IgG(IgG1、IgG3);Ab依赖的细胞介导的细胞毒作用,即表达Fc受体的细胞通过识别抗体的Fc段,直接杀伤被抗体包被的靶细胞的作用;IgE能介导Ⅰ型超敏反应;穿过胎盘(IgG)与粘膜(SIgA)。 1、4 五类Ig的特性与功能

一、IgM 1、脾脏中浆细胞合成,分子量最大,五聚体,称巨球蛋白 2、个体发育过程中最早出现的Ig。母体中的IgM不能通过胎盘,如果胎儿或新生儿的血液中发现有IgM,说明已发生过宫内感染。 3、Ag刺激后最先产生的Ab,早期免疫防御;血清中IgM增高,就是感染早期的诊断依据。IgM就是免疫应答中首先分泌的抗体,一经感染快速产生,经过一段时间,IgM抗体量逐渐减少而消失(半衰期短)。它们在与抗原结合后启动补体的级联反应。它们还把入侵者相互连接起来,聚成一堆以便于巨噬细胞的吞噬。IgM 的免疫调理作用比IgG强。 4、B细胞表面的Ag受体之一。 5、天然的ABO血型Ab、类风湿因子等均就是IgM。 二、IgG 1、脾、淋巴结中浆细胞合成,出生后3个月开始合成,含量多,分布广。就是唯一能在母亲妊娠期穿过胎盘保护胎儿的抗体。还从乳腺分泌进入初乳,使新生儿得到保护。母体传递给胎儿的IgG于生后6个月几乎全部消失,而婴儿自身产生IgG从3个月时才逐渐增多,故6个月后易患感染。 2、IgG抗体激活补体,中与多种毒素,调理吞噬、ADCC及结合SPA等作用。发挥主力免疫作用。IgG就是血清主要的抗体成分,约占血清Ig的75%,其中40~50%分布于血清中,其余分布在组织中。 3、感染后产生较晚,但在血清中半衰期达20-23天,体内持续的时间长。 三、IgA 1、按其免疫功能又分为血清型及分泌型两种。

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