用Gateway技术克隆并表达汉滩病毒G1蛋白ITAM样基序

Cloning and expressions of G1-ITAM-like sequences in Hantaan virus by gateway technology

目的:克隆并表达汉滩病毒(HTNV)囊膜糖蛋白(G1)胞质区尾段ITAM样基序及其突变体.方法:采用RT-PCR方法,扩增HTNVG1蛋白ITAM样基序及其中2个保守酪氨酸残基突变基序编码基因,应用Gateway技术将目的基因克隆至pDESTTM15表达载体,在BL21-AI中经L-阿拉伯糖诱导表达,免疫印迹确证融合蛋白的表达.结果:成功地构建了2个含汉滩病毒G1胞质区尾段ITAM样基序编码序列表达载体pDEST-ITAM-L,pDEST-ITAM和1个含保守酪氨酸残基突变的G1ITAM样基序编码序列表达载体pDEST-ITAM-YY-FF(Y615F,Y628F),并在大肠埃希杆菌中得到高效表达,免疫印迹证实目的蛋白与GST融合表达.结论:获得了3种与谷胱甘肽-S-转移酶(GST)融合的G1ITAM样基序及其突变基序的重组蛋白,为进一步探讨HTNVG1蛋白ITAM样基序在HFRS免疫信号传递中的作用奠定了基础.

AIM： To clone and express wild-type GI-ITAM-like sequence-containing constructs and tyrosine-mutated GI-ITAM- like sequence constructs of Hantaan virus. METHODS： G1- ITAM-like sequence-encoding genes of Hantaan virus were ampli- fied from cDNA of the virus strain 84FLi using RT-PCR. Muta- genesis of the ITAM-like sequence was obtained by artificial syn- thesis. The desired blunt-end PCR products were TOPO^○R cloned into a Gateway^○R entry vector named pENTRrM/D-TOPO. The recombination reaction were performed using Gateway^○R LR Clona- seTM II enzyme mix to transfer the desired genes into the Gateway^○R destination vector pDESTTM 15. After three expression clones were generated, the recombinant proteins were expressed in BI21-AI^TM strain of E. coli under the induction of L-arabinose. The expres- sions of recombinant proteins were confirmed by Western Blot assay. RESULTS ： The expression vectors, pDEST-ITAM-L （ aa569-aa642 ）, pDEST-ITAM （ aa602-aa634 ） and pDEST- ITAM-YY-FF （Y615F, Y628F）, were successfully constructed and highly effective expression of recombinant proteins in E. coli were measured by SDS-PAGE. Western Blot assay revealed that recombinant proteins were tagged by GST. CONCLUSION： Recombinant fusion proteins containing wild-type and tyrosine mutated GI-ITAM-like sequences were obtained. This work lays a basis for the further study of the function of Hantaan virus G1- ITAM-like sequence.