汉滩病毒76-118株G2S0.7嵌合基因重组腺病毒的构建及表达产物鉴定

Construction and identification of recombinant adenovirus containing chimeric gene G2S0.7 of Hantaan virus

目的 :在本室前期工作的基础上构建汉滩病毒M基因G2片段与S基因 0 .7kb片段嵌合基因的重组腺病毒 .方法 :构建含有汉滩病毒G2S0 .7嵌合基因的转移载体pShuttle G2S0 .7,然后通过特异性的酶切将嵌合基因与腺病毒DNA相连 ,电转化E .coliJM1 0 9并用PCR方法进行筛选和鉴定 ,获得重组腺病毒Adeno G2S0 .7DNA ,转染HEK2 93细胞得到重组腺病毒 .进一步对重组腺病毒滴度和表达产物进行鉴定 .结果 :构建了含G2S0 .7嵌合基因重组腺病毒 ,滴度可达 1 0 1 3 ～ 1 0 1 5pfu/L ;该重组腺病毒感染HEK2 93细胞后 ,表达出可被抗汉滩病毒核蛋白及糖蛋白G2的特异性单抗 (mAb)所识别的融合蛋白 .结论 :利用腺病毒表达系统 ,成功地表达同时具有核蛋白及糖蛋白G2生物学活性的融合蛋白G2S0 .7。

AIM: To express the chimeric gene containing M segment and 0.7 kb fragment of S segment of Hantaan virus in adenovirus expression system. METHODS: The recombinant adenovirus transfer vector pShuttle G2S0.7 was constructed and the chimeric gene was inserted into adenovirus DNA. After transfecting HEK293 cells, the recombinant adenovirus was obtained and the titer of it was determined and the expressed product was detected by ELISA. RESULTS: The recombinant adenovirus containing the chimeric gene G2S0.7 was constructed successfully and the titer of it was about 1013 1015pfu/L. HEK293 cells were then infected by the recombinant adenovirus and fusion protein could be expressed in the infected cells. The expressed protein could be recognized by the Hantaan virus NP specific mAb and glycoprotein G2 specific mAb. CONCLUSION: The successful expression of G2S0.7 with biological activity in adenovirus expression system lays the basis for further research on its immunological characteristics.