携带Herceptin与MICA胞外结构域融合基因的重组腺病毒的构建及鉴定

Construction and Identification of a Recombinant Adenovirus Vector Carrying a Fusion Gene Composed of Heceptin and the ECD of MICA

通过构建能表达抗体Herceptin与MICA胞外结构域的融合蛋白的重组腺病毒,为进一步联合NK细胞的治疗研究奠定基础。采用Overlap-PCR的方法获得Herceptin与MICA胞外结构域的融合基因,并将融合基因连接到载体pDC339上。接着再利用酶切及PCR方法鉴定载体及重组病毒基因的正确性,并通过ELISA和West-ern Blot检测融合蛋白的表达情况,最后经间接免疫荧光分析(IFA)检测融合蛋白的结合特性。构建的载体酶切后经电泳鉴定证实构建成功,重组病毒DNA的PCR显示病毒携带了Herceptin的轻链基因、重链基因及MICA胞外结构域的基因;ELISA和Western Blot证实重组腺病毒成功地表达出了目的融合蛋白;IFA实验表明融合蛋白能与SK-OV-3细胞表面的Her-2受体特异性地结合。最后成功地构建了能表达具有正常结合特性的抗体Herceptin与MICA胞外结构域的融合蛋白的重组腺病毒,为之后与NK细胞的联合治疗研究奠定基础。

To construct a recombinant adenovirus which can express a fusion protein composed of Herceptin and the extracellular domain（ECD） of major histocompatibility class I related chain A（MICA）.The authors fuse the ECD sequence of gene MICA to the Herceptin through Overlap-PCR,and then connect the fusion gene to the adenovirus vector pDC339.The recombinant vector is identified by restriction enzyme digestion of DNA and PCR.Enzyme-linked immunosorbent assay（ELISA） and Western Blot are applied to analyze the fusion protein expressed in HEK 293 cells;the binding characteristics of the fusion protein is checked by indirect immunofluorescence assay（IFA）.Restriction enzyme digestion of DNA demonstrates that the vector is successfully constructed;PCR identification indicates the existence of fusion gene in the recombinant adenovirus;ELISA and Western Blot show that the recombinant adenovirus can normally express the fusion protein;IFA confirms that the fusion can specially bind to surface HER-2 receptor on SK-OV-3 cells.The authors successfully construct a recombinant adenovirus which can express a fusion protein with normal binding capacity,this contributes to the future research of therapy with natural killer（NK） cells.