基于RNA复制子GHRH基因表达载体的构建及在293细胞中的表达  

The Construction of GHRH Gene Expression Vector Based on RNA Replicon and Its Expression in 293 Cell

作　　者：任晓慧[1] 乔继光 刘松财[1] 张明军[1] 章倩倩[1] 侯峰[1] 吕铁钢[1] 周立光[1] 吴琼[1] 欧阳松应[3] 张永亮[1] 

机构地区：[1]吉林大学畜牧兽医学院 [2]深圳市肉品卫生检验所,深圳518004 [3]中国协和医科大学,北京100730

出　　处：《畜牧兽医学报》2007年第4期395-399,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金资助项目(30270973)

摘　　要：本研究将GHRH基因克隆到塞姆利基森林病毒(SFV)复制子质粒型表达载体pCMV-Rep-lacZ,构建了真核表达载体pCMV-Rep-GHRH。采用脂质体介导将表达质粒pCMV-Rep-GHRH转染293细胞,经RT-PCR、IFA、Tricine-SDS-PAGE和Western Blot检测,证明表达质粒pCMV-Rep-GHRH在293细胞中正确地表达了GH-RH多肽分子。本研究还利用放免法检测质粒载体转染细胞培养上清中GHRH浓度,初步比较了RNA复制子载体pCMV-Rep-GHRH和普通载体(pIRES-GHRH和pcDNA3-GHRH)的表达效率,结果表明,转染48 h,RNA复制子载体表达GHRH的含量显著高于普通载体,分别比pIRES-GHRH和pcDNA3-GHRH高出36.12%(P<0.05)和67.94%(P<0.05)。结论:构建的真核表达载体pCMV-Rep-GHRH能够在真核细胞293中表达GHRH多肽分子,且表达水平显著高于普通载体,为下一步更好地调控动物生长以提高动物生产性能奠定基础。In this study, GHRH gene was cloned into pCMV-Rep-lacZ which digested by BamH I in order to replace the LacZ gene with our gene of interest and dephosphorylated by shrimp alkaline phosphatase,to creat pCMV-Rep-GHRH, pCMV-Rep-GHRH was transfected by lipofectin to prepared 293 cells. Results of RT-PCR, Tricine-SDS-PAGE ,Western Blot and immunofluorescence antihody assay（IFA） showed that the target gene was expressed efficiently in transfected 293 cell. In order to compare the expressed efficacy between RNA replicon vector（pcMV-Rep- GHRH） and ordinary vector（pIRES-GHRH and pcDNA3-GHRH）, we also detected the concentration of GHRH from transfected cell supernatant by RIA in this study. The results showed that the concentration of GHRH from pCMV-Rep-GHRH transfected cell supernatant was higher （P 〈0. 05） signifiantly than that of GHRH from plRES-GHRH and pcDNA3-GHRH transfected cell supernatant respectively. We conclude that GHRH can be expressed efficiently in eukaryotic expression vector pCMV-Rep-GHRH transfected 293 cell. We can also draw another conclusion that the express level of RNA replicon vector was superior to ordinary vector. It may be a potential way to increase animal productivity.

关 键 词：生长激素释放激素 塞姆利基森林病毒 复制子 

分 类 号：Q789[生物学—分子生物学]