高表达重组Ret蛋白B16细胞株的建立

ESTABLISHMENT OF B16 CELL LINE WITH HIGH EXPRESSION OF RECOMBINANT RET PROTEIN

目的:探讨建立高表达重组Ret蛋白的B16细胞株的方法,为Ret抗原的免疫原性研究提供靶细胞。方法:通过常规分子克隆的方法,构建Fxyd3-Ret嵌合蛋白的真核表达质粒pIRES-rFxyd3-His-Ret-neo2;将该质粒转染293细胞以验证构建质粒的可行性;将该质粒转染B16细胞,通过筛选获得Fxyd3-Ret蛋白高表达的阳性细胞株。结果:PCR和酶切验证pIRES-rFxyd3-His-Ret-neo2的产物片段大小符合预期,Western blot对转染pIRES-rFxyd3-His-Ret-neo2质粒的细胞进行检测,发现在转染细胞内有目标蛋白高表达。结论:成功构建了pIRES-rFxyd3-His-Ret-neo2真核转染质粒;成功建立了Ret蛋白高表达的B16细胞株。

Objective：To explore the establishment of B16 cell line with high expression of recombinant Ret protein to provide the target cell for the immunogenicity research of Ret antigen.Methods：The euokryotic expression vector pIRES-rFxyd3-His-Ret-neo2 was constructed and the Ret expression of the vector in 293 cell was verified.The pIRES-rFxyd3-His-Ret-neo2 vector was transfected to B16 cell and the clones with high expresion of recombinant Ret protein was selected.Results：The sequence of pIRES-rFxyd3-His-Ret-neo2 was confirmed according to the PCR and restriction enzymic analysis.The transfected cells were proved expressing Ret protein.Conclusion：The results indicate that the eucaryotic expressing vector pIRES-rFxyd3-His-Ret-neo2 was constructed and the B16 cell line with high expression of recombinant Ret was established.