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

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作  者:牟丹蕾[1] 姜泓[1] 王英鹏[1] 李光玉[1] 潘蕾[1] 李新红[1] 王临旭[1] 张岩[1] 黄长形[1] 白雪帆[1] 

机构地区:[1]第四军医大学唐都医院全军感染病诊疗中心,陕西西安710038

出  处:《第四军医大学学报》2007年第15期1345-1348,共4页Journal of the Fourth Military Medical University

基  金:国家自然科学基金(30471540)

摘  要: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.

关 键 词:汉坦病毒 囊膜糖蛋白类 免疫受体酪氨酸活化基序 

分 类 号:R512.8[医药卫生—内科学]

 

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