本文關(guān)鍵詞： 精子特異性乳酸脫氫酶(LDH-C4) 抗LDH-C4抗體 ELISA 免疫不育癥　出處：《福建醫(yī)科大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:(1)原核表達(dá)制備重組人精子特異性乳酸脫氫酶(rhLDH-C4)抗原。(2)以rhLDH-C4為包被抗原,建立人血清抗LDH-C4抗體(IgG型)檢測的間接ELISA方法,為人血清抗精子抗體(ASA)的檢測及免疫不育癥的診斷提供新的實(shí)驗(yàn)方法。(3)探討人血清抗LDH-C4抗體IgG與不育癥的關(guān)系。 方法:(1)應(yīng)用本室構(gòu)建的pET-28a(+)-hLDH-C重組質(zhì)粒,轉(zhuǎn)化大腸桿菌Ecol.BL21(DE3)plysS;經(jīng)菌液PCR及酶切電泳鑒定后,用IPTG誘導(dǎo)rhLDH-C4蛋白的可溶性表達(dá),經(jīng)Ni+-NTA親和層析法一步純化,制備rhLDH-C4抗原;SDS-PAGE鑒定重組蛋白的純度;乳酸脫氫酶同工酶活性染色檢測重組蛋白的酶活性。(2)應(yīng)用Western blotting從不育患者血清標(biāo)本中篩選出一例抗LDH-C4抗體陽性的血清標(biāo)本作為陽性對照,以25例處女血清標(biāo)本OD測定值確定ELISA的判定臨界值(cut-off),建立間接ELISA方法,并優(yōu)化其它檢測參數(shù)。(3)應(yīng)用該ELISA方法,分別檢測74例正常男女(生育組)和177例不明原因不育男女(不育組)血清標(biāo)本中的抗LDH-C4抗體(IgG型)的水平。 結(jié)果:(1)1000ml菌液經(jīng)誘導(dǎo)純化后,得rhLDH-C4蛋白約1.5mg;純化的rhLDH-C4在SDS-PAGE電泳中僅顯示一條區(qū)帶;乳酸脫氫酶同工酶活性染色顯示rhLDH-C4活性區(qū)帶位于LDH-4附近。(2)ELISA檢測參數(shù)的確定:rhLDH-C4抗原最適包被濃度為1μg/ml,判定臨界值(cut-off)為0.120。(3)血清標(biāo)本ELISA檢測結(jié)果:不育組陽性率顯著高于正常生育組(30.51%對9.46%,P0.005);女性不育組陽性率略高于男性不育組,但兩組之間差別無顯著意義(31.46%對29.55%,P0.05)。 結(jié)論:(1)成功地制備了高純度的rhLDH-C4抗原,該蛋白具有類似天然乳酸脫氫酶同工酶的酶活性。(2)首次建立了人血清抗LDH-C4抗體(IgG型)檢測的間接ELISA方法,該方法具有高度的敏感性及特異性。(3)人血清抗LDH-C4抗體IgG與不明原因不育癥關(guān)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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392;R711.6

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1 陳平;重組人精子特異性乳酸脫氫酶抗原的制備及其在不育癥自身抗體檢測中的應(yīng)用[D];福建醫(yī)科大學(xué);2006年

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