摘要
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。
AIM： To express an immunochemotactic and angiostatic anti-cancer molecule, CXC chemokine Crg-2 by E. coil recombination system of adenovirus. METHODS： By E. coli recombination system of adenovirus, the shuttle vector pShuttle-cmv/crg-2 was co-transfected into BJ5183 E. coli with adenovirus backbone plasmid pAdEasy-1. The resulting recombinants were transfected into 293 packaging cells and adenovirus was packaged and amplified. The molecular weight and bioacUvity of recombinant protein were determined by Western blot and chemotaxis assay. RESULTS： Recombinant adenovirus genome backbone bearing crg-2 gene, pAd/crg-2, was successfully obtained as evidenced by endonucleases digestion and PCR. Adenovi- rus was packaged and amplified to a titration of 4 × 10^9 TCID50/L. The cultured supernatant 293 cells were infected by adenovirus contented a protein of nearly 10000 and presented a significantly chemotatic effect towards activated spleen lymphoblasts. CONCLUSION. Recombinant adenovirus Ad/crg-2 obtained by E. coli recombinant system of adenovirus can efficiently express bioactive Crg-2 chemokine.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第3期283-285,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30400016)
第四军医大学后备人才基金资助(No.4138A4324)
