含编码人精子膜多肽cDNA和乙肝表面抗原基因的重组痘苗病毒

合成编码一种人精子膜蛋白YWK-Ⅱ胞外区的一段多肽片段的双链寡核苷酸链,HSD-2a.将HSD-2a和3’端乙肝表面抗原主S基因连接并与pUC18质粒重组.经鉴定的重组质粒用BamH Ⅰ和EcoRl酶切后,可分离纯化得到HSD-2a和HBsAg的主S基因连接片段,将该连接片段插入痘苗病毒通用表达载体pGJP-5的痘苗病毒启动子P_(7.5)下游,构建成重组质粒pGJP-HSD/HBs.将该重组质粒传染已感染了天坛痘苗病毒的猴肾细胞CV-1,pGJP-5中含有的TK基因与出发株病毒基因组的TK基因发生体内同源重组,构成重组痘苗病毒vv-HSD/HBs.HuTK~-细胞经vv-HSD/HBs感染后,在5-溴脱氧尿苷存在下进行空斑分析,筛选出TK~-重组病毒.应用ELISA方法对已感染了重组病毒的HuTK~-细胞培养上清和细胞裂解液测定,表明有HSD-2a编码的多肽片段的表达.Western-blot法分析呈现28和30Kd两条显色带.重组痘苗病毒vv-HSD/HBs有可能作为抗生育疫苗用途.

The Expression of Sperm Membrane Peptide- Hepatitis B Surface Antigen Fusion Protein with Recombinant Vaccinia Virus

A synthetic oligonucleotide, HSD - 2a, encoding a peptide segment of the extracellular domain of a human sperm membrane protein, YWK - I , was fused with hepatitis B surface antigen gene (HBs gene). The fused gene was then cloned into pUC18 plasmid. The fused gene was prepared from the recombinant pUC18 plasmid by BamH I and EcoR I digestion, and then cloned into the transfer vector pGJP - 5 under the control of P7.5 promoter, designated as pGJP -HSD/HBs. CV - 1 cells were co - transfected with vaccinia virus (Tian Tan strain) and pGJP-HSD/HBs and homologous recombination occurred between the vaccinia virus TK gene of the plasmid flanking the foreign gene and the same sequences within the virus genome. TK~ phenotype recombinant wirus, vv -HSD/HBs, was selected from transfected HuTK~ cells by plaque purification technique. The expression of HSD - 2a in spent medium and cellular protein of HuTK~ cells infected with vv -HSD/HBs was determined by ELISA and Western -blot analysis using anti - YWK - I antiserum. The present study indicates that the vv - HSD/HBs shows promise as and antifertility vaccine.