论文部分内容阅读
目的利用腺病毒的细菌重组系统表达同时具有免疫趋化及血管抑制活性的趋化性细胞因子Crg-2重组蛋白。方法首先在大肠杆菌BJ5183中将穿梭质粒pShuttle-cmv/crg-2与AdE1区基因缺失的骨架质粒pAdEasy-1进行同源重组,筛选后脂质体法转染入具有AdE1区组成性表达的293包装细胞中进行病毒包装、扩增;Westernblot检测病毒感染293细胞的蛋白表达;趋化实验检测感染细胞上清对激活T淋巴细胞的趋化活性。结果穿梭质粒pShuttle-cmv/crg-2与骨架质粒pAdEasy-1重组后,经酶切及PCR鉴定获得重组腺病毒基因组质粒pAd/crg-2;病毒Ad/crg-2经包装并扩增后,用组织培养感染半数剂量法(TCID50)法测定病毒滴度达4×109TCID50/L,感染细胞经Westernblot检测有一接近Mr10000蛋白条带,分泌上清对激活的脾淋巴细胞有明显的趋化作用。结论采用细菌重组法成功获得重组腺病毒Ad/crg-2,可高效表达趋化性细胞因子Crg-2。
Objective To express recombinant chemokine Crg-2, which possesses both chemotaxis and angiogenesis inhibitory activity, using adenovirus bacterial recombination system. METHODS: The shuttle plasmid pShuttle-cmv / crg-2 was inserted into the shuttle plasmid pAdEasy-1 in E. coli BJ5183 for homologous recombination with pAdEasy-1. The recombinant plasmid was transfected into 293 cells with constitutive expression of AdE1 The virus was packaged and amplified in packaging cells. The protein expression of 293 cells infected by virus was detected by Western blot. The chemotactic activity of infected cells supernatant on activated T lymphocytes was detected by chemotaxis assay. Results After recombination with the shuttle vector pShuttle-cmv / crg-2 and the backbone plasmid pAdEasy-1, the recombinant adenovirus plasmid pAd / crg-2 was obtained by restriction endonuclease digestion and PCR. After packaging and amplification, The titer of virus was determined to be 4 × 109 TCID50 / L by TCID50 method. The infected cells detected by Western blot showed a band close to Mr10000. The secreted supernatant had obvious chemotactic effect on activated splenic lymphocytes. Conclusion The recombinant adenovirus Ad / crg-2 was successfully obtained by bacterial recombination method, which can efficiently express chemokine Crg-2.