人nm23-H1基因重组腺病毒载体的构建与鉴定

Construction and identification of recombinant adenovirus vector containing human nm23-H1 gene

目的:构建表达人nm23-H1基因的重组腺病毒载体,并在人胚肾293细胞中进行鉴定。方法:以重组质粒pcDNA3.1-nm23-H 1中提取的nm23-H 1基因为模板,采用PCR法扩增基因片段。将扩增的基因片段插入穿梭质粒pSh uttle-CMV,并转化大肠肝菌DH 5α。筛选重组质粒pSh uttle-CMV-nm23-H 1,电转化已转化了pAdEasy-1的BJ5183细胞。筛选重组腺病毒质粒pAdEasy-nm23-H 1,用Lipofectamine转染293细胞,制备携带人全长nm23-H 1基因的重组复制缺陷型腺病毒Ad-nm23-H 1。大量扩增Ad-nm23-H 1病毒颗粒,并测定病毒滴度。采用RT-PCR法检测nm23-H1基因在293细胞中的转录。结果:经PCR、酶切鉴定及DNA测序结果证实pSh uttleCMV-nm23-H 1及pAdEasy-nm23-H 1质粒正确构建,纯化后的Ad-nm23-H 1病毒颗粒感染性滴度为109.5(3.4×109)CCID50/ml,经RT-PCR扩增出的基因片段,与目的片段大小一致。结论:已成功构建了表达nm23-H1重组腺病毒载体,为肿瘤的基因治疗提供了依据。

Objective： To construct a recombinant adenovirus vector containing human nm23-H1 gene and identify it in human embryo kidney （HEK） 293 cell. Methods： nm23-H1 gene extracted from recombinant plasmid pcDNA3.1-nm23-H1 was served as a template and amplified by PCR.. Amplified gene fragment was inserted into shuttle plasmid pShuttle-CMV and E. coli DH5a was transformed. Recombinant plasmid pShuttle-CMV-nm23-H1 was screened and competent BJ5183 cells previously transformed with pAdEasy-1 were transformed. Recombinant plasmid pAdEasy-nm23-H1 was screened and transfected to 293 cells in mediation of Lipofectamine to prepare replication-deficient adenovirus Ad-nm23-H1 carrying full length of nm23-H1 gene. Ad-nm23-H1 virus particles were amplified and the tite was determined. Transcription of nm23-H1 gene in 293 cells was determined by RT-PCP. Results： The pShuttle-CMV-nm23-H1 and the pAdEasy-nm23-H1 had been successfully constructed proved by PCR analysis, enzyme digestion and DNA sequence analysis. The virus particle titer of purified Ad-nm23-H 1 was 1095（3.4 ~ 109） CCIDsdml. The gene fragment was amplified by RT-PCP from transfected 293 cells, which was consistent with that expected. Conclusion： Recombinant adenovirus Ad-nm23-H1 for expression of nm23-HI was successfully constructed which provided a basis for gene therapy of tumors.