【摘要】 目的:构建重组人粒细胞-巨噬细胞集落刺激因子乳酸链球菌表达载体,为进一步研究人粒细胞-巨噬细胞集落刺激因子在乳链菌的表达及其治疗价值奠定基础。方法:实验于2005-04/2006-03在南方医科大学南方医院消化病研究所完成。①载体pNCSF的构建:将质粒集落刺激因子及含有P59启动子、USP45蛋白信号肽的pNBC1000质粒分别加入BamH Ⅰ和Pst Ⅰ进行双酶切,并用Apa Ⅰ、Sac Ⅰ进行双酶切鉴定,重组质粒命名为pNCSF。②SDGFP的TA克隆及载体pNCSFGFP的构建:将经过优化适合在乳链菌表达的人粒细胞-巨噬细胞集落刺激因子基因克隆于含有P59启动子、USP45蛋白信号肽的pNBC1000载体,得到重组质粒pNCSF;同时设计上下游引物经PCR扩增增强荧光表达蛋白(EGFP),TA克隆后经测序验证,再连接于pNCSF获得重组质粒pNCSFGFP。③载体pTRCSF、pTRCSFGFP的建立:将获得的pNCSF和pNCSFGFP进一步克隆于穿梭载体pTR1001c,以获得人粒细胞-巨噬细胞集落刺激因子乳链菌表达载体pTRCSF及pTRCSFGFP。结果:①载体pNCSF构建结果:酶切鉴定产物经1.0%的琼脂糖凝胶电泳后,发现有(含启动子P59、信号肽USP45、人粒细胞-巨噬细胞集落刺激因子)720bp的目的片段。②SDGFP的TA克隆及载体pNCSFEGFP的构建结果:SDGFP阳性克隆产物经EcoRⅠ酶切鉴定得到775bp目的片段。pNCSFEGFP酶切鉴定产物经1.0%的琼脂糖凝胶电泳后,发现有(含启动子P59、信号肽USP45、人粒细胞-巨噬细胞集落刺激因子、SDGFP)1495bp的目的片段。③穿梭质粒pTRCSF、pTRCSFGFP酶切鉴定结果:经Xba Ⅰ、Sac Ⅰ进行双酶切鉴定,分别得到约717bp、1492bp大小目的片段。结论:获得了人粒细胞-巨噬细胞集落刺激因子乳链菌表达载体pTRCSF及pTRCSFGFP,并经酶切鉴定和测序证实。更多还原

【Abstract】 AIM: To construct the expressive vector of human granulococyte-macrophage colony stimulating factor (hGM-CSF) in lactococcus lactis so as to lay the foundation for our studies on expression of hGM-CSF in lactococcus lactis and its therapeutic effects.METHODS: This experiment was performed from April 2005 to March 2006 in Institute of Digestive Diseases, Nanfang Hospital,Southern Medical University. ①construction of vector p NCSF: To double restriction enzyme excise plasmid hGM-CSF and plasmid p NBC1000 which contained P59 promoter and USP45 signal peptide and USP45 terminator with BamH I and Pst I, and identified by Apa I and Sac I. The recombinant plasmid was named as p NCSF. ②TA-colony of SDGFP and the construction of vector p NCSFGFP: The optimized hGM-CSF which preferred to express in lactococcus lactis was to cloned to the vector pNBC1000 which contained P59 promoter and USP45 signal peptide and USP45 terminator to obtain pNCSF. Meanwhile, the up-primer and down-primer were designed to amplify EGFP by PCR and the positive SDGFP plasmid was obtained by TA-clone and verified by DNA sequence. Then, the positive SDGFP plasmid was cloned to pNCSF to get plasmid vector pNCSFGFP. ③constructions of vectors p TRCSF and p TRCSFGFP: The obtained plasmids p NCSF and p NCSFGFP were cloned to shuttle vector pTR1001c to acquire the expressive vectors of recombinant hGM-CSF p TRCSF and p TRCSFGFP. RESULTS: ①construction of vector p NCSF: The destination segment about 720 bp which contained P59 promoter, USP45 signal peptide and hGM-CSF was found in the products of p NCSF plasmid after restriction enzyme excision by 1.0% gelose gel electrophoresis. ②construction results of TA-colony of SDGFP and the construction of vector p NCSFGFP: The destination segment about 775 bp was found in the products of SDGFP positive colonies after restriction enzyme excision by EcoR I. There was the destination segment about 1 495 bp which contained P59 promoter, USP45 signal peptide, hGM-CSF and SDGFP in the products of p NCSFGFP plasmid after restriction enzyme excision by 1.0% gelose gel electrophoresis. ③construction results of the shuttle plasmids of p TRCSF and p TRCSFGFP: The two destination segments about 717 bp and 1 492 bp had been found after double restriction enzyme excision by Xba I and SacI.CONCLUSION: The pTRCSF and pTRCSFGFP, the expressive vector of hGM-CSF in lactococcus lactis had been obtained, which had been identified by DNA sequencing and restrictive enzyme excision.更多还原