摘要
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
Objective:To provide the target antigen for the development of a ZP vaccine by using a prokaryotic expression system. Methods:To obtain nonfusion expression, the fragment (249 bp) from ATG to Apa Isite of pZP3α cDNA, which was deleted the 5'terminus noncoding region, was amplified by PCR. The amplified sequenced EcoRI Apa α fragment was ligased with the 3'terminus Apa I IαSal I fragment of pZP3α cDNA from pZ58 plasmid, and then the reconstructed pZP3α with a complete open reading frame was inserted in frame downstream of the P RP L promoter in a pBV221 vector. Results:After transformation of E.coli BL21 (DE3) and thermoinduction, the pZP3α protein with 61 kD was expressed at the level of more than 10% of the total cellular proteins and the specific protein band on SDSPAGE gel could be recognized by antiporcine ZP IgGs in Western blotting. Conclusion: The availability of native and nonglycosylated pZP3α protein will help in the development and evaluation of a contraceptive vaccine based on pZP3α protein.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2000年第6期303-306,共4页
Chinese Journal of Immunology
基金
国家计划生育药具重点实验室资助
