携带IL-2和NK4双基因真核共表达载体的减毒沙门菌株的构建

Construction of an Attenuated Salmonella Typhimurium Containing IL-2 and NK4 Co-expressed Eukaryotic Vector

目的构建携带IL-2/NK4双基因真核表达载体的减毒沙门菌菌株。方法利用分子克隆技术将IL-2及NK4基因分别插入PIRES-SEQ质粒IRES序列的上下游中,构建为双基因真核共表达载体pCMV-IL-2-IRES-NK4,将该质粒利用脂质体转染入细胞,采用ELISA及PCR法检测pCMV-IL-2-IRES-NK4转染细胞后IL-2/NK4基因及蛋白表达情况。将所构建质粒以电转化法转化减毒沙门菌菌株Ty21a,从而得到能够表达IL-2/NK4双基因的重组减毒沙门菌菌株。结果 ELISA及PCR法检测该质粒可在细胞中以较高的效率稳定表达IL-2及NK4基因和蛋白;同时表达IL-2/NK4双基因的减毒沙门菌菌株TPIN构建成功。结论本研究成功构建了携带IL-2/NK4双基因的减毒沙门菌菌株。

Objective To construct an attenuated Salmonella typhimurium strain （TPIN） containing IL-2 and NK4 co-expressed eukaryotic vector. Methods A Eukaryotic expression vector （pCMV-IL-2-IRES-NK4） was construc-ted which contained IL-2 and NK4 genes by genetic engineering technology. The vector was transfected into cells using li-pofectamin and detected the expression levels of IL-2 and NK4 by enzyme linked immunosorbent assay （ELISA） and pol-ymerase chain reaction （PCR） methods. And then, the vector pCMV-IL-2-IRES-NK4 was conducted into an attenuated Salmonella typhimurium Ty21a by electrotransformation to build TPIN. Results The IL-2 / NK4 co-express vector steadi-ly expressed the IL-2 and NK4 genes and albumen with high levels by ELISA and PCR detection; the attenuated Salmo-nella typhimurium DNA vaccine with IL-2 / NK4 genes （TPIN） was constructed successfully. Conclusion An attenuated Salmonella typhimurium strain carrying IL-2 / NK4 genes has been successfully constructed.