猪生长激素基因在昆虫细胞中的分泌表达

Secretion Expression of Porcine Growth Hormone Gene in Insect Cells

将PCR扩增得到的 pGH基因插入到带有 polh启动子和 gp6 7强信号肽序列的杆状病毒转移载体pAcGP6 7 A中 ,构建重组质粒 pGP6 7pGH ,并与线性化致死缺失型苜蓿丫纹夜蛾核多角体病毒 (AcMNPV OCC-)基因组DNA共转染Sf9细胞 ,构建出重组病毒AcMNPV pGP6 7 pGH OCC-。感染重组病毒的Hi5细胞的表达产物的SDS PAGE和Westernblot结果表明 ,细胞可溶蛋白和培养液上清中均有一条分子量约为 2 2kDa的猪生长激素特异性反应带 ,且培养液上清中的目的蛋白分子量比天然pGH略大一些。薄层扫描仪扫描估测可知 ,重组pGH分别占细胞可溶蛋白的 8 98%和培养液上清总蛋白的 3 6 1%。将表达 96h的培养液上清浓缩液进行N 糖基化分析 ,结果显示重组 pGH无N 糖基化加工修饰。

The porcine growth hormone gene was obtained by PCR and inserted into the baculovirus expression vector of pAcGP67-A which contains polh promoter and gp67 signal sequence.The recombinant plasmid of pGP67-pGH was cotransfected Hi5 cells with the linearized parental virus Ac MNPV-OCC - DNA to produce the recombinant virus Ac MNPV-pGP67-pGH-OCC -.SDS-PAGE and Western blot analysis showed that pGH gene products were observed,those in the supernatant with a little larger molecular weight size than the natural pGH's. The expressed pGH accumulated up to about 3.61% of the supernatant proteins and 8.98% of the soluble cellular proteins respectively as estimated by CS-930 scanning of the SDS-PAGE.The N-glycosylation analysis showed the recombinant pGH had no N-glycosylation.