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目的构建含HIV-1tat基因重组腺病毒,观察在不同细胞中外源蛋白Tat的表达,作为DC抗HIV疫苗的基础。方法通过PCR扩增,获得HXB2tat的cDNA片段,定向克隆入腺病毒转移载体pTrack-CMV,线性化后转化含有腺病毒骨架pAd-easy-1的大肠杆菌BJ5183,获得同源重组的质粒prAd-tat,PacⅠ酶切纯化后转染293细胞,包装成具有感染力的复制缺陷型重组腺病毒vAd-tat。结果经PCR、酶切及DNA序列测定,插入片段大小、方向正确,获得具有感染力的含有HIV-1tat基因的重组腺病毒;通过Western blot方法检测,重组腺病毒在293细胞中表达出Mr为15000的蛋白。结论成功构建了含有HIV-1tat基因的腺病毒,并观察到该基因在细胞中的表达。
Objective To construct a recombinant adenovirus containing HIV-1tat gene and observe the expression of the foreign protein Tat in different cells as the basis of DC anti-HIV vaccine. Methods The cDNA fragment of HXB2tat was obtained by PCR amplification and cloned into the adenovirus vector pTrack-CMV. After linearization, the recombinant plasmid was transformed into E. coli BJ5183 containing adenovirus backbone pAd-easy-1 to obtain homologous recombined plasmid prAd-tat The recombinant plasmids were transfected into 293 cells by PacⅠ digestion and purified and packaged into infectious replication-deficient recombinant adenovirus vAd-tat. Results Recombinant adenovirus containing HIV-1 at gene was obtained by PCR, restriction enzyme digestion and DNA sequencing. The recombinant adenovirus was transfected into 293 cells by Western blot, 15000 protein. Conclusion The adenovirus containing HIV-1 tat gene was successfully constructed and the expression of this gene in cells was observed.