融合基因GMCSFBZLF1重组腺病毒表达载体的构建_医学论文.docVIP

融合基因GM重组腺病毒表达载体的构建_医学论文 融合基因GM重组腺病毒表达载体的构建_医学论文 作者：崔瑛 王云 刘成玉 张东峰 【摘要】 目的 构建粒细胞巨噬细胞集落刺激因子(GM和EB病毒即刻早期基因(BZLF1)融合基因的重组腺病毒表达载体。方法 采用逆转录聚合酶链反应分别获得GM和BZLF1编码序列的cDNA，应用剪接式重叠延伸(SOE)技术将两段基因通过多肽接头(Gly4Ser)3 的DNA 序列进行连接，构建融合基因GM。将融合基因GM定向亚克隆至pAdTrack质粒，在原核细胞E. coli BJ5183中完成穿梭质粒与骨架质粒pAdEasy的同源重组，构建融合基因GM真核表达载体pAd。将真核表达载体pAd转染293细胞，获得复制缺陷型重组腺病毒vAd。RT鉴定感染重组腺病毒的293细胞中GM基因的表达。结果 GM基因插入重组腺病毒表达载体的预期位置，且插入序列完全正确感染重组腺病毒vAd的293细胞中检测到融合基因GM的转录表达。结论 成功地构建了融合基因GM重组腺病毒表达载体，为进一步探讨GM的功能提供了理论基础和实验依据。 【关键词】 疱疹病毒4型，人粒细胞巨噬细胞集落刺激因子基因，即早基因融合遗传载体 [ABSTRACT] Objective To construct human granulocyted EBV encoding immediate early genes (BZLF1) fused to GMCSFBZLF1 gene and the fusion gene recombinant adenovirus expression vector. Methods GMCSF cDNA and BZLF1 cDNA were acquired by RTPCR. The fusion gene GMCSFBZLF1 was constructed through a polypeptide linker (Gly4Ser) 3 by using spliced overlap extension (SOE). The fusion gene GMCSFBZLF1 was subcloned into pAdTrackCMV plasmid. The shuttle plasmid and the backbone plasmid pAdEasy1 were recombinated homologously in E.coli BJ5183 cells, then fusion gene eukaryotic expression vector pAdGMCSFBZLF1 were constructed. pAdGMCSFBZLF1 vector was transfected into 293 cells to obtain recombinant adenovirus vAdGMCSFBZLF1. RTPCR was used to identify the expression of fusion gene GMCSFBZLF1 in 293 cells infected with vAdGMCSFBZLF1. Results The result of DNA sequencing demonstrated that GMCSFBZLF1 gene inserted in the expected site in the recombinant adenovirus expression vector and the insertion sequence was wholly correct. The expression of fusion gene GMCSFBZLF1 could be detected in the 293 cells infected with vAdGMCSFBZLF1. Conclusion The fusion gene GMCSFBZLF1 recombinant adenovirus expression vector has been constructed successfully, which might furnish the rationale and experimental evidence for further study on the function of GMCSFBZLF1 gene. 　　[KEY WORDS] herpesvirus 4, human granulocyte 　　EB病毒(EBV)是嗜B淋巴细胞的疱疹病毒，为致瘤病毒之一。