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目的 :通过原核表达系统获得研制ZP疫苗的靶抗原。方法 :用PCR方法删除猪卵透明带 3α(pZP3α)cDNA 5’端非编码区碱基顺序。在扩增的该基因 5’端小片段与双酶切 3’端大片段在ApaI位点相连后 ,通过双酶切位点将读框完整的目的基因定向插入pBV2 2 1表达质粒PRPL 启动子下游的多克隆区。结果 :此重组表达质粒转化宿主菌后经热诱导培养 ,可在SDS PAGE胶上观察到 6 1kD的特异蛋白表达条带 ,而且它在Westernblot鉴定实验中能被抗pZPIgGs识别。结论 :非糖基化天然pZP3α蛋白的可获得性将有助于研制和评价基于pZP3α蛋白的避孕疫苗
OBJECTIVE: To obtain the target antigen of ZP vaccine by prokaryotic expression system. Methods: The nucleotide sequence of 5 ’noncoding region of porcine zona pellucida 3α (pZP3α) cDNA was deleted by PCR. After amplification of the 5 ’end of this gene and double digestion of the 3’ end of the large fragment at the ApaI site, the double-digested site was used to insert the complete reading frame of the target gene into the pBV2 21 expression plasmid PRPL promoter Downstream polyclonal region. Results: The recombinant plasmid was transformed into host bacteria and then induced by heat induction. The specific band of 61kD protein was observed on SDS PAGE gel, and it could be recognized by anti-pZPIgGs in Western blot. CONCLUSIONS: The availability of the non-glycosylated native pZP3α protein will facilitate the development and evaluation of pZP3α-based contraceptive vaccines