重组人脂联素基因在减毒沙门氏菌中的表达
Construction and expression of attenuated salmonella typhimurium carrying human adiponectin gene eukaryotic expression vector
周静 1周洲 1吴莹 1向廷秀 1姜政 1王丕龙1
作者信息
- 1. 重庆医科大学附属第一医院消化内科,重庆,400016
- 折叠
摘要
目的:构建携带人脂联素(AdipoQ)基因真核表达载体并在减毒沙门氏菌中表达,为AdipoQ对非酒精性脂肪性肝病(NAFLD)的基因治疗提供依据.方法:从人脂肪组织中提取总RNA,通过实时荧光定量PCR(rRT-PCR)方法获得Adi-poQ基因并将其克隆到真核表达载体pEGFP-NI上,构建pEG-FP-N1-AdipoQ重组载体.重组质粒经鉴定后再电转入减毒沙门氏菌SL7207中表达.结果:克隆的人AdipoQ基因744 bp测序结果显示:1个碱基发生突变,654位:A→T,突变率为0.1%,氨基酸Glu→Asp.经SDS-PAGE,Western Blot检测融合蛋白Mr约为55×10~3(绿色荧光蛋白Mr约为27×10~3),能够被AdipoQ抗体识别.结论:成功构建携带AdipoQ基因真核表达载体的减毒沙门氏菌株,AdipoQ基因能够在减毒沙门氏菌中表达并与绿色荧光蛋白融合.为进一步研究其在NAFLD中的作用机制奠定了基础.
Abstract
AIM: To construct and express an attenuated Salmonella typhimurium strain contraining human adiponectin gene eukaryotic expression vector, in order to lay the foundation for the genetic therapy of nonalcoholic fatty liver disease in future. METHODS: Total mRNA was extracted from human fat tissue and the adiponectin cDNA was obtained by RT-PCR, and then cloned into eukaryotic expression vector pEGFP-N1. The recombinant vector was identified, and transformed into attenuated Salmonella typhimurium strain SL7207, and the recombinant strain SL7207/pEGFP-NI-AdipoQ was screened by green fluorescent microscope and Western Blot. RESULTS: Enzyme digestion analysis and sequencing showed that the target genes was found to be 744 bp, and had been inserted into eukaryotic expression vector, but as compared with gene reported by GenBank, 0. 1% of the gene mutation and O. 4% of amino acid residues change, respectively: 654 bp: A→T, amino acids: Glu→Asp. SDS-PAGE demonstrated that pEGFP-N1-AdipoQ was expressed in SL7207 strain, and the relatived molecular mass of the GFP-AdipoQ fusion protein was about 55×10~3, while GFP protein expressed by pE(;FP-NI was about 27×10~3. The Western Blot analysis indicated that the EGFP-AdipoQ fusion protein could be recognized by anti-AdipoQ antibody, suggesting that this fusion protein had good biological activity. CONCLUSION: The recombinant attenuated Salmonella typhimurium strain contraining plasmid pEGFP-N1- AdipoQ can be successfully constructed and expressed, and can be a basic foundation for the genetic therapy of NFLD in future with adiponectin..
关键词
脂联素/沙门菌属/疫苗/减毒Key words
adiponeetin/Salmonella/vaccines/attenuated引用本文复制引用
基金项目
出版年
2009
NETL
NSTL
维普
万方数据