汉滩病毒G1S0.7嵌合基因重组腺病毒的构建及鉴定

Construction and identification of recombinant adenovirus containing chimeric gene G1S0.7 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G1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G1S0.7嵌合基因的转移载体pShuttle-G1S0.7，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.coli JM109，获得重组腺病毒Adeno-G1S0.7 DNA，转染HFX293细胞得到重组腺病毒。进一步对重组腺病毒的滴度和表达产物进行鉴定。结果构建的含G1S0.7嵌合基因的重组腺病毒，滴度可达10^13～10^15 PFU／L；该重组腺病毒感染Vero-E6细胞后，表达出可被抗汉滩病毒核蛋白及糖蛋白G1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G1生物学活性的融合蛋白，为进一步研究其免疫学特性奠定了基础。

Objective To express the chimeric gene containing the G1 fragment of M segment and the 0.7 kb fragment of S segment from Hantaan virus in adenovirus expression system. Methods The recombinant adenovirus transfer vector pShuttle-G1S0.7 was constructed. Then the chimeric gene was inserted into adenovims DNA. After transfection of HEK293 cells, the recombinant adenovims was obtained and its fiter was determined. The expressed product was detected by indirect immunofluorescence assay （IFA）. Results The recombinant adenovirus containing the chimeric gene G1S0.7 was constructed successfully and its fiter was about 10^13 - 10^15 PFU/L. After infection with the recombinant adenovims, Vero-E6 cells could express fusion protein, which could be recognized by both the hantaan virus NP-specific mAb and glycoprotein G1-specific mAb. Conclusion G1S0.7 with biological activity was expressed in adenovims expression system successfully, which lays a foundation for further research on its immunological characteristic.