摘要
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: It is hard to obtain a large number of human ovaries and to purify each proteinmponent from natural human zona pellucida (huZP) that surrounds the oocytes; the zona pellucida consists ofP1, ZP2 and ZP3 glycoproteins. Thus, it may be a feasible approach to produce each ZP component by geneticgineering for conducting immunological and reproductive biology studies. Methods: Although there have beenveral reports that huZP3 proteins were expressed in eukaryotic and prokaryotic expression systems, expressionvels are low and all are fusion proteins. In this study, we apply a new strategy of expressing two peptides of coreuZP322~ 348. This sequence, representing the huZP3 cDNA, excluding sequences encoding N-terminal signal se-uence and C-terminal transmembrane domain, was cut into two fragments by polymerase chain reaction whichcode huZP3a22 ~ 176 and huZP3b177 ~ 348 peptides, respectively. Afterwards, each amplified fragment with a reading-ame was inserted into a frame downstream of the PRPL promoter in the temperature-inducible pBV221 vector.esults: Two target peptides were expressed in E. coli BL21(DE3)pLysS strain at a higher-level, which can becognized by antisera against synthetic peptide151~ 165 (137~ 150) or 327~ 340 present in huZP3. Finally, expression productsere purified using our improved method of preparative gel polyacrylamide gel electrophoresis easily. In addition,estern blot analysis by anti- pZP IgGs suggests that there might be at least two common antigenic regions present huZP3 and pZP3b proteins because anti- pZP IgGs reacted with the rec-huZP3 and rec-huZP3a more stronglyan AS ZP3-5. Conclusion: The availability of two huZP3 peptides will help in testing the immunogenicity ofuZP3a and huZP3b through animal experiments and in determining if one or both recombinant peptides will initiaterosomal exocytosis in human spermatozoa in vitro.
出处
《生殖与避孕》
CAS
CSCD
北大核心
2004年第3期143-148,共6页
Reproduction and Contraception
基金
上海市人口和计划生育委员会局管基金资助项目(No. 03JG05014)
关键词
人透明带蛋白-3
基因表达
免疫印迹
纯化
human zona pellucida-3
gene expression
western blot analysis
purification
