摘要
本文构建了分别含K88,K99和LT-B亚单位抗原基因的重组痘苗病毒,并观察了这3种基因的表达情况。首先将K88亚单位基因克隆入痘苗转移或体PGJP-5中,得到嵌合质粒P_5-K88,将K99和LT-B亚单位基因分别克隆至痘苗转移载体PJ16中,得到重组质粒PJK-9和PLT-B。再经细胞体内同源重组技术,将以上3种基因分别插入到痘苗病毒天坛株的TK基因中,经5-BUdR的选择压力在人TK^--143细胞中挑选TK^-表型病毒,分别以相应的^(32)P标记的基因片段为探针进行杂交筛选重组病毒株。我们得到7株含K99亚单位基因的重组病毒V-K99(Ⅰ～Ⅷ),5株含K88亚单位基因的重组病毒V-K88(Ⅰ～Ⅴ);1株含LT-B亚单位基因的重组病毒V-LTB,ELISA检测结果表明,在以上3种重组病毒感染的人TK^--143细胞培养液和细胞裂解物中均未测出相应的基因表达产物。以上结果表明这3种基因可能不易在痘苗系统内有效表达。
This article described the construction of recombinant vaccinia viruses containing K88, K99 and LT-B subunit genes and their expression in vaccinia virus expression system. The chimeric plasmid P_5-K88 was obtained by cloning K88 subunit gene into vaccinia transfer vector PGJP-5 and the chimeric plasmids PJK-9, PLT-B were constructed by insertion K99 and LT-B subunit genes into transfer vector PJ16 respectively. These three kinds of genes were individually inserted into TK gene of vaccinia virus genome by the homologous recombinant technique in vivo. The TK-phenotype recombinant viruses were screened in the presence of 5-BUdR on human TK^--143 cells and identified by Dot-blot hybridization. 7 strains of recombinant viruses with K99 subunit gene, named V-K99 (Ⅰ-Ⅶ), 5 strains of recombinant viruses with K88 subunit gene, named V-K88 (Ⅰ-Ⅴ), and 1 strain of recombinant virus with LT-B subunit gene, designated V-LTB were obtained. ELISA results showed that no corresponding gene expression production was detected in both the supernatants and cell lysates of human TK^--143 cells infected by these three kinds of recombinant vaccinia viruses. The above results also showed that these three kinds of genes were not probably expressed in vaccinia virus expression system.
出处
《军事医学科学院院刊》
CSCD
北大核心
1991年第3期186-191,共6页
Bulletin of the Academy of Military Medical Sciences
基金
国家自然科学基金资助项目
