重组人精子特异性乳酸脱氢酶抗原的制备及其在不育症自身抗体检测中的应用

发布时间：2018-01-27 01:20

本文关键词： 精子特异性乳酸脱氢酶(LDH-C4) 抗LDH-C4抗体 ELISA 免疫不育症　出处：《福建医科大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的:(1)原核表达制备重组人精子特异性乳酸脱氢酶(rhLDH-C4)抗原。(2)以rhLDH-C4为包被抗原,建立人血清抗LDH-C4抗体(IgG型)检测的间接ELISA方法,为人血清抗精子抗体(ASA)的检测及免疫不育症的诊断提供新的实验方法。(3)探讨人血清抗LDH-C4抗体IgG与不育症的关系。方法:(1)应用本室构建的pET-28a(+)-hLDH-C重组质粒,转化大肠杆菌Ecol.BL21(DE3)plysS;经菌液PCR及酶切电泳鉴定后,用IPTG诱导rhLDH-C4蛋白的可溶性表达,经Ni+-NTA亲和层析法一步纯化,制备rhLDH-C4抗原;SDS-PAGE鉴定重组蛋白的纯度;乳酸脱氢酶同工酶活性染色检测重组蛋白的酶活性。(2)应用Western blotting从不育患者血清标本中筛选出一例抗LDH-C4抗体阳性的血清标本作为阳性对照,以25例处女血清标本OD测定值确定ELISA的判定临界值(cut-off),建立间接ELISA方法,并优化其它检测参数。(3)应用该ELISA方法,分别检测74例正常男女(生育组)和177例不明原因不育男女(不育组)血清标本中的抗LDH-C4抗体(IgG型)的水平。结果:(1)1000ml菌液经诱导纯化后,得rhLDH-C4蛋白约1.5mg;纯化的rhLDH-C4在SDS-PAGE电泳中仅显示一条区带;乳酸脱氢酶同工酶活性染色显示rhLDH-C4活性区带位于LDH-4附近。(2)ELISA检测参数的确定:rhLDH-C4抗原最适包被浓度为1μg/ml,判定临界值(cut-off)为0.120。(3)血清标本ELISA检测结果:不育组阳性率显著高于正常生育组(30.51%对9.46%,P0.005);女性不育组阳性率略高于男性不育组,但两组之间差别无显著意义(31.46%对29.55%,P0.05)。结论:(1)成功地制备了高纯度的rhLDH-C4抗原,该蛋白具有类似天然乳酸脱氢酶同工酶的酶活性。(2)首次建立了人血清抗LDH-C4抗体(IgG型)检测的间接ELISA方法,该方法具有高度的敏感性及特异性。(3)人血清抗LDH-C4抗体IgG与不明原因不育症关系密切。
[Abstract]:Objective to prepare recombinant human spermatozoa specific lactate dehydrogenase rhLDH-C4 antigen by prokaryotic expression. To establish an indirect ELISA method for the detection of anti LDH-C4 antibody in human serum. To explore the relationship between human serum anti sperm antibody (IgG) and infertility, and to provide a new experimental method for the diagnosis of immune infertility. Methods the recombinant plasmid pET-28a (hLDH-C) was constructed and transformed into E. coli Ecol.BL21DE3 plysS. The soluble expression of rhLDH-C4 protein was induced by IPTG, and then purified by Ni NTA affinity chromatography to prepare rhLDH-C4 antigen. The purity of recombinant protein was identified by SDS-PAGE. Detection of enzyme activity of Recombinant protein by lactate dehydrogenase isozyme staining. Western blotting was used to screen a positive serum sample of anti LDH-C4 antibody from the serum samples of infertile patients as a positive control. An indirect ELISA method was established to determine the critical value of ELISA by determining the OD value of 25 virginal serum samples. The ELISA method is applied to optimize other detection parameters. The serum levels of anti LDH-C4 antibody were measured in 74 normal male and female (fertility group) and 177 unexplained male and female infertile (infertile group). Results the rhLDH-C4 protein was about 1.5 mg / ml after induction and purification. The purified rhLDH-C4 showed only one band in SDS-PAGE electrophoresis. The activity of lactate dehydrogenase isoenzyme staining showed that the active zone of rhLDH-C4 was located near LDH-4. The optimal coating concentration of rhLDH-C4 antigen was 1 渭 g / ml. The positive rate of ELISA in the infertile group was significantly higher than that in the normal fertility group (30.51% vs 9.46%). P0.005; The positive rate of female infertility was slightly higher than that of male infertility, but there was no significant difference between the two groups (31.46% vs 29.55%, P 0.05). Conclusion the high purity rhLDH-C4 antigen was successfully prepared. The enzyme activity of this protein is similar to that of natural lactate dehydrogenase isozyme. (2) an indirect ELISA method for the detection of human serum anti LDH-C4 antibody was established for the first time. This method has high sensitivity and specificity. The human serum anti LDH-C4 antibody IgG is closely related to unexplained infertility.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;R711.6

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