摘要
目的 构建携带人胰岛素原突变体的逆转录病毒载体及稳定的包装细胞系。方法 采用PCR重叠延伸定点突变技术 ,将普通细胞内Furin蛋白酶的识别和切割位点Arg X Lys Arg 样序列 ,引入人胰岛素原C肽两端 ,同时加上信号肽、Kozak序列和酶切位点并克隆至逆转录病毒载体 pLXSN中 ,测序正确后转染包装细胞PA317,经G4 18筛选后挑选稳定生产病毒的细胞株 ,用NIH3T3细胞测定病毒滴度。结果 测序证明胰岛素原cDNA的 5个预定位点成功突变 ,其上游加上了信号肽和增强表达的Kozak序列 ;G4 18筛选重组pL mPIN SN转染PA317细胞抗性克隆 ,其病毒滴度为 2 .6× 10 5CFU/ml,而且基因组PCR和病毒液的RT PCR都显示有 2 86bp的条带 ,提示构建携带人胰岛素原突变体的逆转录病毒载体以及稳定的包装细胞系成功。结论 成功构建了携带人胰岛素原突变体的逆转录病毒载体及包装细胞系 。
Objective To construct a recombinant defective vector carrying mutant of human proinsulin and a stable virus-producing packaging cell line, and then to identify them. Methods Human proinsulin gene was mutated by the polymerase chain reaction (PCR) for the site-directed mutagenesis method. Two furin recognized sites of Arg-X-Lys-Arg-motifs were introduced into human proinsulin. Signal peptide, Kozak sequence and restriction enzyme cutting site were added and cloned into pLXSN vector and verified by sequencing analysis. Then this recombinantplasmid was transferred into packaging cell PA317. After screening with G418 (400 mg/L) for 8 weeks, the survived PA317 cells secreting virus stably were selected and enriched to assay virus titer using NIH3T3 cell. Results Five siteswere mutated simultaneously and added signal peptide and Kozak sequence enhancing expression with the upper stream. G418 screen the recombinant pL-mPIN-SN transferring PA317cell. The virus titer was 2.6×105 CFU/ml. Genomic PCR and RT-PCR of viral fluid appeared 286 bp strap. These results showed that recombinantplasmid pL-mPIN-SN, and stable virus-producing cell lines are constructed. Conclusion A mutant human proinsulin with signal peptide retroviral vector and stable virus-producing cell lines are successfully constructed, and it provides a good basis for further research on gene therapy of diabetes.
出处
《山西医科大学学报》
CAS
2004年第2期97-100,共4页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目 ( 3 0 0 70 3 5 6)
