融合基因GM-CSF-BZLF1重组腺病毒表达载体的构建

CONSTRUCTION OF RECOMBINANT ADENOVIRUS EXPRESSION VECTOR ENCODING GM-CSF-BZLF1 FUSION GENE

目的构建粒细胞-巨噬细胞集落刺激因子(GM-CSF)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-CSF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-CSF-BZLF1。将融合基因GM-CSF-BZLF1定向亚克隆至pAdTrack-CMV质粒,在原核细胞E.coliBJ5183中完成穿梭质粒与骨架质粒pAdEasy-1的同源重组,构建融合基因GM-CSF-BZLF1真核表达载体pAd-GM-CSF-BZLF1。将真核表达载体pAd-GM-CSF-BZLF1转染293细胞,获得复制缺陷型重组腺病毒vAd-GM-CSF-BZLF1。RT-PCR鉴定感染重组腺病毒的293细胞中GM-CSF-BZLF1基因的表达。结果 GM-CSF-BZLF1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-GM-CSF-BZLF1的293细胞中检测到融合基因GM-CSF-BZLF1的转录表达。结论成功地构建了融合基因GM-CSF-BZLF1重组腺病毒表达载体,为进一步探讨GM-CSF-BZLF1的功能提供了理论基础和实验依据。

Objective To construct human granulocyte-macrophage colony-stimulating factor（GM-CSF）gene and 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.