發(fā)布時間：2018-04-24 12:33

  本文選題：卵透明帶 + 巴氏畢赤酵母�。� 參考：《暨南大學(xué)》2007年碩士論文

【摘要】： 本研究采用突變重組人卵透明帶蛋白3(mutant recombinant human zona pellucida protein 3，mrhZP3)酵母工程菌株，經(jīng)小規(guī)模甲醇誘導(dǎo)表達，篩選高表達酵母菌株。在2L發(fā)酵罐中進行高密度發(fā)酵生產(chǎn)mrhZP3。分離濃縮發(fā)酵上清液，通過螯合鎳離子的親和柱純化mrhZP3并進行工藝優(yōu)化。通過SDS-PAGE和Western blotting鑒定，，與rhZP3的分子量和表達產(chǎn)量進行比較，并進行誘導(dǎo)人精子頂體反應(yīng)實驗和小鼠免疫實驗。實驗小鼠分4組，每組10只雌性小鼠。A組采用mrhZP3蛋白陰道栓劑進行免疫，B組使用不含mrhZP3蛋白的陰道栓劑，C組注射mrhZP3蛋白進行免疫，D組注射空白佐劑做為對照。每組動物接受4次免疫(第0、1、2、3周)。采用ELISA法監(jiān)測小鼠血清抗體滴度變化。第5周合籠，第8周統(tǒng)計雌鼠懷孕情況，取實驗小鼠雙側(cè)卵巢做病理冰凍切片，光鏡觀察卵巢病理學(xué)變化。Western blotting結(jié)果顯示mrhZP3具備抗原活性。mrhZP3可誘導(dǎo)人精子發(fā)生頂體反應(yīng)。分別采用mrhZP3和pZP3作為包被抗原，C組小鼠血清中均檢測到明顯的抗ZP3 IgG抗體，A組、B組和D組小鼠血清中未檢測出抗ZP3抗體。統(tǒng)計受孕率，A組、B組、C組和D組小鼠的受孕率分別為0、9／10、0及8／10，統(tǒng)計學(xué)檢驗分析表明A組與B組，C組和D組間的差異有統(tǒng)計學(xué)意義(P＜0.01)，結(jié)果顯示mrhZP3蛋白具備較強的抗生育效能。小鼠卵巢切片的組織病理學(xué)觀察結(jié)果表明，實驗小鼠的卵巢組織均未觀察到明顯的病理變化。本研究結(jié)果初步表明mrhZP3具有與天然人卵透明帶相似的誘發(fā)人精子頂體反應(yīng)活性和抗原活性，并為發(fā)展不孕不育檢測試劑和避孕疫苗提供了實驗依據(jù)。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R341

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