【摘要】 构建了含不同长度的人α-肿瘤坏死因子(hTNF-a)cDNA的两种重组痘苗病毒。hTNF-α cDNA插入痘苗病毒(天坛株)的tk区,在11k启动子的控制下,表达量可达1.6×10~7U/L,如果缩短hTNF-α cDNA起始密码子ATG与启动子之间距离并去除3′端非编码区,可使hTNF-α的表达量提高3倍。表达产物的细胞毒活性可被抗hTNF-α单克隆抗体所中和,免疫沉淀产物的SDS-PAGE有一特异性17kD的蛋白条带;同时,在细胞毒活性相同的前提下,哺乳动物细胞表达的hTNF-α的抗病毒活性高于大肠杆菌表达的hTNF-α。更多还原

【Abstract】 Two recombinant vaccinia viruses named RVJTNF and RVJTNFPCR were constructed by inserting the modified and unmodified hTNF-α cDNA, driven by the promoter of 11kD protein of vaccinia virus, into the tk region of Tian Tan strain of vaccinia virus respectively. The modified hTNF-α cDNA was obtained by deleting the 5’ and 3’ noncoding reions using PCR technique. Southern blot hybridization showed that hTNF-α cDNA were inserted into the tk region of recombinant vaccinia virus correctly. Expression of TNF protein was assayed by cytotoxic activity for actinomycin D-treated L929 cell. Cultures infected by recombinant vaccinia virus secreted as muca as 1.6×104 units/ml of TNF of culture medium. Synthesis of TNF protein was confirmed by neutra-lizing cytotoxic activity of infected culture supernatant with a monoc-lonal antibody to TNF-α and by immunoprecipitation of 17kD protein from infected culture supernatant. Compared with the unmodified hTN F-α cDNA, the expression Ievel of modified hTNF-α cDNA increased 3 folds approximately. The antiviral activity of TNF-α expressed in mam-malian celis was higher than that of TNF-a expressed in E.coli.更多还原