9ku颗粒溶素活性肽重组减毒沙门菌的构建与表达

Construction and expression of the recombinant attenuated Salmonella carrying human 9 ku-granulysin gene

目的构建人9ku颗粒溶素(granulysin)的重组减毒沙门菌,并在真核细胞中表达,为下一步利用颗粒溶素基因治疗肿瘤奠定基础。方法以含有颗粒溶素cDNA序列的质粒为模板,通过聚合酶链反应(PCR)扩增获得含分泌肽编码基因的9ku的颗粒溶素基因片段后,定向插入真核表达载体pBudCE4.1中,获得重组表达质粒pBudCE4.1-S9K,通过PCR、双酶切及插入片段序列测定对重组质粒进行鉴定;采用RT-PCR,免疫细胞化学与Dot-ELISA法检测pBudCE4.1-S9K在RAW264.7细胞的瞬时表达与分泌;重组质粒电转化减毒沙门菌后,将重组菌感染巨噬细胞,以RT-PCR检查颗粒溶素的表达。结果成功扩增出含分泌肽编码基因的9ku的颗粒溶素基因片段;经酶切,PCR及测序鉴定证明得到读码框正确的重组质粒;转染细胞证实可分泌表达9ku颗粒溶素;重组减毒沙门菌感染细胞后,检测到颗粒溶素的表达。结论成功构建含9ku颗粒溶素活性肽基因的重组减毒沙门菌,该菌能成功递呈携带基因在感染细胞内表达。

To construct and express the recombinant attenuated Salmonenella carrying human 9 ku-granulysin gene in order to lay a foundation for the study on granulysin as an anti-tumor agent used in tumor gene therapy, the 9 ku-granulysin gene fragment including leader peptide gene was amplified by PCR from the plasmid cDNA, and cloned into eukaryotic expression vector pBudCE4.1 to construct recombinant expression plasmid pBudCE4.1-S9K. This recombinant expression vector was identified by PCR, restriction enzyme digestion and sequencing, and was then transfected to RAW264.7 cells. The 9 ku-granulysin could be expressed and secreted in the transfeeted cells as demonstrated by the detection with RT-PCR,Immunocytochemical staining and dot ELISA assay. In this way, the 9 ku-grunulysin gene fragment coding the secreted peptide was successfully amplified, and the recombinant plasmid with corrected reading frame was obtained after identification with RT-PCR, restriction enzyme digestion and sequencing. The 9 ku-granulysin protein could be expressed and secreted from the transfected cells. And the expression of granulysin could be detected after infection of with recombinant attenuated Salmonella strain.