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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
引用本文:张芳琳,刘勇,于澜,胡刚,吴兴安,史梦远,白文涛,王海涛,徐志凯.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[J].第四军医大学学报,2004,25(12):1057-1060.
作者姓名:张芳琳  刘勇  于澜  胡刚  吴兴安  史梦远  白文涛  王海涛  徐志凯
作者单位:第四军医大学基础部微生物学教研室,陕西,西安,710033
基金项目:国家自然科学基金资助项目 (30 0 70 686,30 1 74847),军队“十五”科研规划青年基金 (0 1Q1 1 5),国家教育部骨干教师资助计划资助项目
摘    要: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.coli JM109并用PCR方法进行筛选和鉴定,获得重组腺病毒Adeno-G2S0.7DNA,转染HEK293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10^13~10^15pfu/L;该重组腺病毒感染HEK293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,为进一步研究其免疫学特性奠定了基础.

关 键 词:汉坦病毒  基因表达  腺病毒科  重组  遗传
文章编号:1000-2790(2004)12-1057-04
修稿时间:2004年1月15日

Construction and identification of recombinant adenovirus containing chimeric gene G2S0.7 of Hantaan virus
Abstract: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.
Keywords:Hantaan virus  gene expression  adenoviridae  recombination  genetic
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