突变重组人卵透明带蛋白3的生物活性研究

发布时间：2018-04-24 12:33

本文选题：卵透明带 + 巴氏毕赤酵母　；参考：《暨南大学》2007年硕士论文

【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。
[Abstract]:In this study, the recombinant yeast engineering strain of 3(mutant recombinant human zona pellucida protein 3mrhZP3 was used to screen the high expression yeast strain. MrhZP3 was produced by high density fermentation in 2L fermenter. The supernatant of concentrated fermentation was separated and purified by affinity column of nickel ion chelate and the process was optimized. The molecular weight and expression yield of rhZP3 were compared with those of rhZP3 by SDS-PAGE and Western blotting. The acrosome reaction of human spermatozoa and the immunological test of mice were also carried out. The experimental mice were divided into 4 groups. Each group of 10 female mice. Group A was immunized with mrhZP3 protein vaginal suppository. Group B was immunized with mrhZP3 free vaginal suppository. Group C was immunized with mrhZP3 protein. Group D was immunized with blank adjuvant. The animals in each group were immunized 4 times. Serum antibody titers of mice were monitored by ELISA method. The pregnant condition of female mice was counted at the 5th week and the 8th week. The pathological changes of the ovaries of the experimental mice were taken as frozen sections. The results of light microscopy and Western blotting showed that the antigenic activity of mrhZP3. MrhZP3 could induce acrosome reaction of human spermatozoa. No anti- antibody was detected in serum of group A, B and D, respectively, when mrhZP3 and pZP3 were used as coated antigens in serum of mice in group C and group A, respectively. The pregnant rates of group A, B, C and D were 0 ~ 9 / 10 ~ 0 and 8 / 10, respectively. The statistical analysis showed that the difference between group A and group B was statistically significant (P < 0.01). The results showed that mrhZP3 protein had strong antifertility effect. The histopathological results of mouse ovary sections showed that no obvious pathological changes were observed in the ovarian tissues of the experimental mice. The results showed that mrhZP3 had the activity of inducing acrosome reaction and antigenic activity of human spermatozoa similar to that of natural human pellucida, and provided experimental basis for the development of sterility detection reagent and contraceptive vaccine.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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