【作者】 郭焱；

【导师】 王忠山；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2006， 博士

【摘要】 透明带是卵细胞表面特有的结构,是精卵结合的重要成分。抗透明带抗体能阻止受精。临床上有相当部分不明原因不孕症妇女通过对抗透明带抗体检测,而找到了不孕的原因。故抗透明带抗体的检测对不孕症原因的判断及治疗有重要意义。由于人卵透明带来源奇缺,猪卵透明带中糖蛋白组分pZP3β是主要的精子受体蛋白之一,抗pZP3β抗体在体外能与人卵透明带发生交叉反应,可抑制人精卵结合,因此,国内外对卵透明带抗体的检测都是用完整猪卵透明带或猪卵透明带抗原全组分进行的,特异性较差。所以,应用基因工程技术生产人ZP3就成为人类获得ZP3的主要手段。有报道表明,出现在卵细胞表面的成熟ZP3分子不存在N端信号肽和C端跨膜区,且全长ZP3由于存在N端的信号肽和C端的跨膜区可影响蛋白的表达。所以,本文采用PCR方法,扩增了删除N端信号肽和C端的跨膜区基因序列的核心片段,将其重组插入融合蛋白原核表达载体pGEX4T-1,构建pGEX-ZP3II,与含有只删除N端信号肽编码区基因序列的pGEX-ZP3I进行比较,实验结果证实pGEX-ZP3II的表达目的蛋白的能力明显高于pGEX-ZP3I。将pGEX-ZP3II转化工程菌E.coliBL21,经优化诱导表达条件,目的蛋白得到大量表达,表达的蛋白经SDS-PAGE分析,主要以包涵体形式存在,经8mM脲素溶解,复性,Glutathion Sepharose 4B亲和层析纯化后,免疫纯系Balb/C小鼠,分离纯化抗血清,经间接酶联免疫吸附试验和间接免疫荧光试验证明,基因重组技术获得的rhuZP3蛋白具有很好的免疫原性和免疫反应性。人ZP3融合蛋白原核表达载体的成功构建和表达,为建立一种特异性强的临床诊断免疫不孕的方法提供了可靠的技术路线。卵透明带糖蛋白ZP3是许多哺乳类动物的精子受体。利用ZP3作抗原可以通过免疫应答抑制精卵结合、阻止受精,从而有望发展成为避孕疫苗。更多还原

【Abstract】 The zona pellucida (ZP), an extracellular glycoprotein coat surroundingmammalian eggs, plays a vital role in initial recognition and binding events ofsperm to the oocyte in a relatively species-specific manner. The human zonapellucida is composed of three glycoproteins, ZP1, ZP2, and ZP3. Considerableevidence suggests that ZP3 serves as the putative primary sperm receptor. SinceZP antibodies can prevent sperm from recognizing oocyte and reduce fertility,therefore, detection of ZP antibodies is very important in clinical diagnosis ofinfertility. The experimental studies show that the availability of large numbersof pig ovaries from abbatoire has made pig ZP glycoproteins promisingcandidates in the development of a contraceptive vaccine. However, theavailability of purified ZP glycoproteins without any other protein contaminantfrom ovaries will be limited and may not a feasible proposition as animmunogen for a contraceptive vaccine. The expression of ZP3 by recombinantDNA technology could help in overcoming this problem. In this experimentalstudy, we have undertaken to express the fusion protein ZP3 in E. coli.Furthermore, the ability of recombinant ZP3 to generate antibodies against themwas also investigated.As mention above, mammalian oocyte is enveloped by a unique, cellular,translucent matrix termed zona pellucida (ZP). Because ZP plays a vital role infertilization, it makes ZP potential candidate antigens for immunocontraception.In 1970’s WHO proposed to develop immunocontraception vaccine that elicit animmune response capable of inhibiting more efficiently the human sperm-egginteraction. Based on previous studies, a modified ZP3 DNA fragment shouldelicit B cell responses only which exclude N-terminal peptide and C-terminaltrans-membrane domain. Thus a eukaryotic expression vector (pIRES1 neo) wasselected for construction of huZP3 expression vector, which acts as DNAvaccine (pIRESl-ZP3). The transformation of pIRESl-ZP3 into CHO cell wascarried out by liposome. ZP3 mRNA could be detected by RT-PCR. Afterimmunization of Balb/C mice using pIRESl-ZP3, antibodies direct againsthuZP3 in serum of mice could be examined by ELISA. In conclusion, theconstruction of huZP3 expression vectors in both prokaryotic and eukaryoticsystem make it possible that anti-fertility effect of active immunization withrecombinant huZP3 will come into reality in the future. In this study, theprotocol given describes the production of immunologically active recombinanthuZP3 using E. coli and CHO cell line as host cell, respectively.更多还原