突变重组人卵透明带蛋白3的生物活性研究
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摘要
本研究采用突变重组人卵透明带蛋白3(mutant recombinant human zona pellucida protein 3，mrhZP3)酵母工程菌株，经小规模甲醇诱导表达，筛选高表达酵母菌株。在2L发酵罐中进行高密度发酵生产mrhZP3。分离浓缩发酵上清液，通过螯合镍离子的亲和柱纯化mrhZP3并进行工艺优化。通过SDS-PAGE和Western blotting鉴定，与rhZP3的分子量和表达产量进行比较，并进行诱导人精子顶体反应实验和小鼠免疫实验。实验小鼠分4组，每组10只雌性小鼠。A组采用mrhZP3蛋白阴道栓剂进行免疫，B组使用不含mrhZP3蛋白的阴道栓剂，C组注射mrhZP3蛋白进行免疫，D组注射空白佐剂做为对照。每组动物接受4次免疫(第0、1、2、3周)。采用ELISA法监测小鼠血清抗体滴度变化。第5周合笼，第8周统计雌鼠怀孕情况，取实验小鼠双侧卵巢做病理冰冻切片，光镜观察卵巢病理学变化。Western blotting结果显示mrhZP3具备抗原活性。mrhZP3可诱导人精子发生顶体反应。分别采用mrhZP3和pZP3作为包被抗原，C组小鼠血清中均检测到明显的抗ZP3 IgG抗体，A组、B组和D组小鼠血清中未检测出抗ZP3抗体。统计受孕率，A组、B组、C组和D组小鼠的受孕率分别为0、9／10、0及8／10，统计学检验分析表明A组与B组，C组和D组间的差异有统计学意义(P＜0.01)，结果显示mrhZP3蛋白具备较强的抗生育效能。小鼠卵巢切片的组织病理学观察结果表明，实验小鼠的卵巢组织均未观察到明显的病理变化。本研究结果初步表明mrhZP3具有与天然人卵透明带相似的诱发人精子顶体反应活性和抗原活性，并为发展不孕不育检测试剂和避孕疫苗提供了实验依据。
In the present study, the Pichia pastoris strains which protease cleavage sites of hZP3 hadbeen replaced for producing the mutant recombinant human zona pellucida protein 3(mrhZP3)were adopted. Induced by methanol, mrhZP3 were expressed in shake flasks. High-efficiencystrains were chose to produce mrhZP3 in high-density fermentation. The fermentativesupernatants were separated and concentrated. Under the optimized conditions, mrhZP3 werepurified by Ni~(2+) metal chelating chromatography. The purified mrhZP3 were identified bySDS-PAGE and Western blotting. Then mrhZP3 were compared with rhZP3 in molecularweight and yield. Human sperm acrosome reaction was induced by mrhZP3. Female mice wereimmunized with mrhZP3. The mice were divided into four groups at random, each group 10mice. Group A were immunized with mrhZP3 suppository by vagina, the suppository withoutmrhZP3 were administered to group B. Group C were immunized with mrhZP3 by muscleinjection, and group D were injected with adjuvant only. All the groups were administered onweek 0, 1, 2, and 3. Serum samples of the immunized mice were collected on week 1, 2, 3, and5. The antibodies against ZP3 were detected by ELISA. The female mice were mated on week 5and killed on week 8 for checking pregnancy. Ovarian frozen sections were prepared formicroscopical pathological observation. The antigenic activity of mrhZP3 was confirmed byWestern blotting. The human sperm acrosome reaction was induced with mrhZP3. WithmrhZP3 and pZP3 as coating antigens respectively, the antibodies could be detected in group C,while none were detected in the sera of group A, B and D. Pregnancy rate of group A was 0, Bwas 9/10, C was 0 and D was 8/10. There were significant statistic differences between group Aand B, between group C and D (P＜0.01). No obvious pathological changes were observed inthe mice ovarian frozen sections. The results implied mrhZP3 are similar to natural human ZPin inducing sperm acrosome reaction and mrhZP3 possess antigenic activity, mrhZP3 has thepotential to develop diagnostic kits for AR and effective contraceptive vaccine.
In the present study, the Pichia pastoris strains which protease cleavage sites of hZP3 hadbeen replaced for producing the mutant recombinant human zona pellucida protein 3(mrhZP3)were adopted. Induced by methanol, mrhZP3 were expressed in shake flasks. High-efficiencystrains were chose to produce mrhZP3 in high-density fermentation. The fermentativesupernatants were separated and concentrated. Under the optimized conditions, mrhZP3 werepurified by Ni~(2+) metal chelating chromatography. The purified mrhZP3 were identified bySDS-PAGE and Western blotting. Then mrhZP3 were compared with rhZP3 in molecularweight and yield. Human sperm acrosome reaction was induced by mrhZP3. Female mice wereimmunized with mrhZP3. The mice were divided into four groups at random, each group 10mice. Group A were immunized with mrhZP3 suppository by vagina, the suppository withoutmrhZP3 were administered to group B. Group C were immunized with mrhZP3 by muscleinjection, and group D were injected with adjuvant only. All the groups were administered onweek 0, 1, 2, and 3. Serum samples of the immunized mice were collected on week 1, 2, 3, and5. The antibodies against ZP3 were detected by ELISA. The female mice were mated on week 5and killed on week 8 for checking pregnancy. Ovarian frozen sections were prepared formicroscopical pathological observation. The antigenic activity of mrhZP3 was confirmed byWestern blotting. The human sperm acrosome reaction was induced with mrhZP3. WithmrhZP3 and pZP3 as coating antigens respectively, the antibodies could be detected in group C,while none were detected in the sera of group A, B and D. Pregnancy rate of group A was 0, Bwas 9/10, C was 0 and D was 8/10. There were significant statistic differences between group Aand B, between group C and D (P＜0.01). No obvious pathological changes were observed inthe mice ovarian frozen sections. The results implied mrhZP3 are similar to natural human ZPin inducing sperm acrosome reaction and mrhZP3 possess antigenic activity, mrhZP3 has thepotential to develop diagnostic kits for AR and effective contraceptive vaccine.
引文
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[3] Martini S. Animal models in human reproduction [M]. New York: Raven Press.1980, 299-302.
[4] Paterson M, Koothan P T, Morris K D, O'Byrne K T, Braude P, Williams A, Aitken R J. Analysis of the contraceptive potential of antibodies against native and deglycosylated porcine ZP3 in vivo and in vitro [J]. Biology of reproduction. 1992, 46(4): 523-534.
[5] Dunbar B S, Avery S, Lee V, Prasad S, Schwahn D, Schwoebel E, Skinner S, Wilkins B. The mammalian zona pellucida: Its biochemistry, immunochemistry, molecular biology, and developmental expression [J]. Reproduction, Fertility and Development. 1994, 6(3): 331-347.
[6] 潘勇顾，左嘉客．局部免疫和避孕疫苗[J]．生殖与避孕．1999，19(6)：328-334．
[7] Vandevoort C A, Schwoebel E D, Dunbar B S. Immunization of monkeys with recombinant complimentary deoxyribonucleic acid expressed zona pellucida proteins [J]. Fertility and sterility. 1995, 64(6): 838-847.
[8] 潘善培，谢琪璇，陈海玲，刘学高，陈乾生，马昭林，梁兴林．用猪卵透明带55 kDa糖蛋白抗原(ZP3)免疫恒河猴的初步研究[J]．暨南大学学报．1995，16(1)：902-971．
[9] Sun W, Lou Y H, Dean J, Tung K S. A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida [J]. Biology of reproduction. 1999, 60(4): 900-907.
[10] Lou Y H, Coonrod S, Kramer W, Tung K S K. Destruction of zona pellucida of ovulated oocytes by anti-ZP3 antibodies: a potential mechanism of ZP-based contraceptive vaccine [J]. Journal of Reproductive Immunology. 1997, 34(1): 29-35.
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