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目的:探索将huZP3a22~348蛋白编码基因拆分成两段,分别表达的可行性,并研究表达产物的免疫原性。方法:用PCR技术, 将去N-端信号肽和C-端跨膜区的huZP322~348蛋白编码基因拆成大小相近的两段, 以完整阅读框的形式分别重组插入热诱导型pBV221的多克隆区。结果:大肠杆菌分别特异地表达了可单独或复合应用的huZP3a22~176和huZP3b177~348,在经过抗人ZP3不同抗原区合成肽抗体的蛋白印迹鉴定后, 用改良的制备性PAGE方法分离纯化这两种表达产物。同时用兔抗猪ZP IgGs蛋白印迹试验表明, huZP3a和pZP3b的几个共同线性抗原表位都存在于其肽链的前半区域。结论:通过基因重组技术可获得足够量的huZP3a和huZP3b,这为开展huZP3a和huZP3b的免疫原性以及huZP3诱发人精子顶体胞吐的功能域等研究奠定了基础。
OBJECTIVE: To explore the feasibility of splitting huZP3a22 ~ 348 protein coding genes into two segments and expressing them separately, and to study the immunogenicity of the expressed products. Methods: The huZP322 ~ 348 protein coding genes of N-terminal signal peptide and C-terminal transmembrane region were separated into two segments with similar size by PCR. The multiple copies of heat-inducible pBV221 Clone area. Results: Escherichia coli specifically expressed huZP3a22 ~ 176 and huZP3b177 ~ 348, which could be used alone or in combination. After identification by Western blotting of the synthetic peptide antibodies against different antigenic regions of ZP3, E. coli were isolated and purified by a modified preparative PAGE method Two expression products. At the same time, the rabbit anti-porcine ZP IgGs Western blotting experiments showed that several common linear epitopes of huZP3a and pZP3b existed in the first half of the peptide chain. Conclusion: HuZP3a and huZP3b can be obtained in sufficient quantities by gene recombination technology, which lays the foundation for the research on the immunogenicity of huZP3a and huZP3b and the function domain of huZP3 induced human sperm acrosome exocytosis.