FasL非增殖腺病毒载体的构建及其在肾细胞中的表达

目的构建携带FasL的非增殖腺病毒载体,并研究其生物学特性。方法将人FasL全长cDNA克隆到非增殖腺病毒载体pSuCMV中,获得pSuCMVFasL重组子,经293包装细胞包装,获得氨苄青霉素抗性克隆,命名为pSuCMVFasL。体外感染人近端肾小管上皮细胞株HK-2细胞72h后,Western blot检测其在HK-2细胞中的表达。结果获得携带FasL非增殖腺病毒,滴度为7.3×109pfu/ml;Western blot法检测在其感染的HK-2细胞中能检测到重组的蛋白表达。结论将FasL基因导入非增殖腺病毒载体是一种行之有效的基因转染途径,制备的非增殖腺病毒在体外能有效感染HK-2细胞,为进行体内移植免疫耐受的基因治疗研究奠定基础。

Construction of Replication-defective Adenovirus Vector Containing Human Fas Ligand Gene and Its Expression

Objective To construct replication-defective adenovirus vector containing human FasL and study its biological function. Methods The human Fas ligand complementary DNA was subcloned to the replication-defective adenovirus vector pSuCMV to obtain pSuCMV-FasL recombinant with direct inserting then packed with 293 amphotropic packaging cells,antiAMP clones were acquired and named as pSuCMVFasL. After infecting HK-2 cells with pSuCMV-FasL for 72 h,the expression of FasL was detected by Western blot. Results The titer of virus was 7.3×10^9 pfu/ml.Soluble fusion protein FasL was expressed in HK-2 cells infected with pSuCMVFasL. Conclusion Introduction of FasL gene to replication-defective adenovirus vector is an effevtive way. pSuCMVFasL prepared in this study can efficiently transfer exogenous gene into HK-2 cells.It may be used as a novel immunosuppressive agent for gene therapy in organ transplantation.