重组腺病毒载体AdOX40Ig的构建、表达及相关体外实验

Construction and in vitro research of the recombinant adenovirus vector AdOX40Ig

目的:构建并鉴定含人OX40-IgG1融合基因的重组腺病毒载体,并感染人肝HL-7702细胞,筛选出最佳感染剂量并检测OX40Ig的表达情况,为进一步用于器官移植后的抗排斥治疗奠定基础。方法:根据基因库公布的人OX40和hIgG1 Fc片断序列设计特定引物,通过聚合酶链反应扩增并鉴定后,酶切插入穿梭质粒pAdTrack-CMV中。随后与pAdeasy-1腺病毒质粒以氯化钙法共转化BJ5183感受态细胞,重组质粒经鉴定正确后,大量扩增为腺病毒载体。再经293A细胞包装、扩增并纯化后感染HL-7702肝细胞,筛选出最佳感染剂量,并采用间接免疫荧光法检测其中外源性OX40Ig基因的表达,采用ELISA法测定肝HL-7702细胞上清中OX40Ig蛋白的含量。结果:转移质粒pTOX40Ig经酶切及测序鉴定正确。重组腺病毒质粒经酶切鉴定正确,并成功转染了293A细胞。得到的病毒液经纯化扩增后进一步感染肝HL-7702细胞,经筛选,10 MOI为最佳感染剂量,并且可以检测到OX40Ig目的基因及其蛋白的表达。结论:成功构建了重组腺病毒载体AdOX40Ig,并且可在肝细胞HL-7702中表达相应的目的基因及蛋白。

Objective： To construct and identify recombinant adenovirus vector containing the human OX40-IgG1 fusion gene,then to infect the HL-7702 liver cells to filter out the best infective dose and detect expression of OX40Ig,which lay a foundation for the immune therapy inhibitory after organ transplantation.Methods： Specific OX40 and hIgG1 Fc primers were designed according to GenBank,and subcloned into the pAdTrack-CMV shuttle plasmid after PCR and identification.Retransformed into BJ5183 competent cells with pAdeasy-1 by calcium chloride method.The recombinant plasmid was detected by PCR and DNA sequence analysis and was transfected into 293A cells.The recombinant adenovirus infected HL-7702 liver cells and filtered out the best infective dose.OX40Ig gene expression was detected by indirect immunofluorescence assay.OX40Ig protein expression was detected by ELISA.Results：pTOX40Ig and pAdOX40Ig were constructed successfully according to restriction endonuclease analysis.293A cells were transfected,and then the purified recombinant adenovirus infected HL-7702 liver cells.10 MOI was the best infective dose.OX40Ig effective expression was detected in HL-7702 liver cells.Conclusion： The recombinant adenovirus vector AdOX40Ig carrying the human OX40-IgG1 fusion gene was successfully constructed,and OX40Ig effective expression was found in HL-7702 liver cells in vitro.