摘要
将鸡痘病毒 2 82 E4株 TK基因和 1 .5kb B片段 2个非必需区克隆后 ,分别在 TK基因 Nco 位点和 B片段 Eco R 位点插入复合启动子 (由牛痘病毒 A型包涵体启动子和 2 0个串联的痘苗病毒 ( VV)突变型 p7.5早期启动子串联组成 ) ,然后在复合启动子上游插入单一启动子 (人工合成的由 1 6个串联的 VV突变型p7.5早期启动子组成 )调控的 Lac Z基因或 GFP基因作为报告基因 ,构建成分别以 TK基因或 B片段为侧翼、以复合启动子调控目的基因的 2个以 Lac Z为报告基因的表达载体 p UTA-2 -1 6Lac Z和 p UBA-7-1 6Lac Z,2个以 GFP为报告基因的表达载体 p UTA-2 -F71 6和 p UBA-7-F71 6。在 TK基因 Nco 位点和 B片段 Eco R 位点插入单一启动子和单一启动子调控的 Lac Z基因 ,得到 2个分别以 TK基因或 B片段为侧翼、以单一启动子调控目的基因、以 Lac Z为报告基因的表达载体 p UT1 61 6Lac Z和 p UB1 61 6Lac Z。将构建的 6个表达载体分别转染 FPV HIPRA株感染的鸡胚成纤维 ( CEF)细胞 ,仅在转染以 TK基因为侧翼、以 Lac Z为报告基因的 2个表达载体 p UTA-2 -1 6Lac Z和 p UT1 61 6Lac Z收获的病毒中 ,在 X-gal存在下 ,筛选出了表达 Lac Z的蓝色重组病毒蚀斑 ,而转染以 B片段为侧翼的表达载体在同样条件下未筛选到蓝色病?
Four FPV transfer vectors for producing recombinant FPV(rFPV )were constructed by firstly inserting a hybrid promoter(the A type inclusion body promoter of cowpox virus followed by 20 tandemly repeated mutant p7.5 early promoters of vaccinia virus), into the Nco I site of TK gene and the EcoR I site of 1.5 kb fragment B of strain 282E4 respectively. The LacZ gene and the GFP gene regulated by P16 (16 tandemly repeated mutant p7.5 early promoters of vaccinia virus) respectively, were then inserted into the Hind III site upstream of the hybrid promoter, but with opposite oritentation to it. 2 of the vectors, pUTA 2 16LacZ and pUTA 2 F716, the foreign genes were designed to be flanked by TK sequence, the reporter was designed as GFP(pUTA 2 F716) or lacZ (pUTA 2 16LacZ), the other 2 vectors, pUBA 7 F716 and pUBA 7 16LacZ, the foreign genes were designed to be flanked by the sequence of fragment B.Another 2 transfer vectors, pUT1616LacZ and pUB1616LacZ, with the designation of foreign gene to be regulated by P16, were also constructed by insertion P16 with LacZ gene regulated by another P16, into the Nco I site of TK gene and the EcoRⅠ site of fragment B respectively.For generating recombinant FPV by homology recombination, the 6 constructed transfer vectors were transfected into CEF cells pre infected with FPV strain HIPRA respectively. Blue FPV plaques were observed only from those viruses obtained by co transfecting with pUTA 2 16LacZ and pUT1616LacZ in the presence of X gal. No GFP expressing virus plaques was observed under fluorescent microscope at wave length of 509nm. By inserting the F gene of NDV strain Miyadera into the Sma I site downstream of the hybrid promoter in pUTA 2 16LacZ, an expression plasmid pUTA 2 16LacZF was constructed. By transfecting the plasmid into CEF cells pre infected with FPV strain HIPRA, a rFPV expressing the F protein of NDV was obtained. Moreover, the F protein was expressed in a cleavaged form.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2000年第5期423-428,共6页
Chinese Journal of Veterinary Science
基金
国家"8 6 3"计划项目 !(10 1-0 5 -0 3-1)
关键词
鸡痘病毒疫苗
载体构建
新城疫病毒
F蛋白
表达
fowlpox virus
vector construction
Newcastle disease virus
F protein
expression
