【作者】 钱东；

【导师】 张富春；

【作者基本信息】 新疆大学 ， 动物学， 2001， 硕士

【摘要】 对许多种类的有害野生动物种群数量的控制已经成为一个非常值得研究的课题。通过免疫避孕的方式来减小种群的数量，已经被提上了议事日程，在本研究中，我们将鼠卵透明带3（mZP3）作为研究对象。由于卵透明带3作为精子的初级受体是鼠卵透明带的一种主要糖蛋白，而且抗ZP3抗体能够阻止精卵结合，因此ZP3被作为一种在免疫避孕领域非常有发展前景的候选免疫原。本论文的研究工作主要包括mZP3 cDNA的克隆、融合蛋白的表达及抗血清的制备。 首先，从Gene Bank^*上检索到ZP3cDNA序列，根据查到序列设计出特异的引物。同时，解剖适龄雌性未孕的昆明白小鼠分离卵巢，从中提取出总RNA，用偶连有OligodT的磁珠纯化出mRNA，在反转录酶的作用下，通过RT-PCR得到mZP3 cDNA，将克隆到的cDNA连接到pUCm-T质粒上，通过蓝白斑筛选和酶切鉴定，确定连接的正确性，最后用DNA测序仪对该序列进行测序（使用引物为T₇和SP6反向引物），确认该克隆出序列同Gene Bank是一致的，长度为1317bp。 然后利用测序结果和原核表达载体pGEX-4T-1的多克隆位点序列及开放阅读框确定共用酶切位点，将pUCm-ZP3上的mZP3基因用XhoI和EcoRI切下，用同样的酶切割pGEX-4T-1，混合后连接，用HindⅢ＆BgⅢ双酶切和PCR的方法配合鉴定重组质粒GST-ZP3构建的正确性，将该质粒转化入大肠杆菌BL21中，用IPTG诱导后，裂解细胞，用SDS-PAGE鉴定诱导效果，发现在72kDa处出现多出的条带，表明有融合蛋白的表达。 大量培养表达融合蛋白的大肠杆菌，诱导后裂解，用偶连有GST的琼脂珠大量纯化融合蛋白，纯化的融合蛋白SDS-PAGE显示为一条带，用提纯的融合蛋白免疫大白兔，经过两次加强免疫后，采得血清，用ELISA检测校价为1:100O，用Western blot检测在72kDa处出现条带，证明制备的纯化的融合蛋白和抗血清是正确的。 克隆出小鼠ZP3的cDNA，并且通过原核表达载体在大肠杆菌中大量表达ZP3基因所编码的活性蛋白，为以后进一步利用ZP3作为靶抗原进行免疫不孕研究打下了基础。更多还原

【Abstract】 Population control has become a major problem in many wildlife species. Fertility control through immunocontraception has been proposed as a method for reducing population size. We selected the murine zona pellucida 3(mZP3) as the main role. Ovarian mZP3 is a major glycoprotein of murine zona pellucida and the primary sperm receptor. Because antibodies raised against mZP3 block sperm-egg interaction, ZP3 has been considered a candidate immunogen in the development of a contraceptive vaccine. In this study, we had cloned the mZP3 cDNA, expressed the fusion protein and produced the rabbit-anti-mZP3 antisera. Firstly, the sequence of murine zona pellucida 3 cDNA was obtained from the Gene Bank. According to the sequence , the special primers were designed. Meanwhile, the ovaries were gotten by dissected the non-pregnant Kunmingbai mouse. The total RNA was abstracted from ovaries and mRNA was purified by agarose linked with OligodT. Then the cDNA of mZP3 was cloned and joined into pUCm-T. The correction of the recombinant vector pUCm-ZP3 was tested by blue and white spot screen and restrictive digestion. Finally the sequence of the cloned mZP3 eDNA was defined by PE 377 DNA Sequencer (the primers were T7 and reverse SP6 ). The results showed that the sequence of the cloned mZP3 was the same with the Gene Bank and its length was 131 7bp. And then the ORF and the common restrictive digestion sites were judged on the basis of the results of sequencing and the restriction map and multiple cloning site of pGEX-4T- 1. The mZP3 gene was cut from the pUCm-ZP3 by XhoI and EcoRI and the same restrictive enzyme was used to cut pGEX-4T-1. Then the mZP3 gene was cloned into the pGEX-4T- 1. The correction of the recombinant vector GST-ZP3 was verified by the digestion of HindJII&BglI and PCR in which primers were special. Then GST-ZP3 was transformed into E. co/i BL2 1. After induced by IPTG, the cells were lysised and the fusion expression was measured by SDS-PAGE. And a 72kDa band was found. It suggested that the fusion protein be expressed in E.coli BL21. 3 ~I~h~i~c The E. coli BL2I which expressed the fusion protein was cultivated in big bulk and induced by IPTG. After the cell was lysised, the fusion protein was purified by agarose linked with GST. The big white rabbit was injected by the purified fusion protein with Feufund complete adjnvant. After boosted in two times, the rabbit-anti-mZP3 antisera was obtained. Because the result of Western blot showed a band in 72kDa., the immunocompetence of the fusion protein was verified. The titer of rabbit-anti-mZP3 antisera was measured by ELISA. The result was 1:1000. The base that mZP3 would be deeply studied as a candidate immunogen in the development of a contraception in mouse was founded by cloning mZP3 eDNA and expressed the active fusion protein which was coded by the cDNA of mZP3.更多还原