摘要
目的 构建谷胱甘肽转硫酶 TLR4融合蛋白表达载体 ,并研究其编码的蛋白质在大肠杆菌中的表达。方法 利用PCR从含TLR4全长cDNA序列的质粒中扩增其胞外段 ,约 180 0bp ,然后将其克隆入pMD18 T载体中。测序确证后重组入谷胱甘肽转硫酶融合表达载体pGEX 4T 1中 ,并用IPTG诱导其在大肠杆菌中表达。结果 PCR扩增的TLR4胞外区基因序列与GenBank数据库中的序列一致。重组质粒经酶切鉴定及测序证实 ,TLR4基因已正确插入到pGEX 4T 1中。重组表达质粒转化感受态大肠杆菌HB10 1后用IPTG诱导 ,获得Mr92 0 0 0的GST TLR4融合蛋白 ;37℃ ,0 .2mmol LIPTG诱导 4h ,SDS PAGE电泳后 ,经薄层凝胶扫描分析该融合蛋白占菌体蛋白总量 34.2 5 % ,部分以包涵体存在 ,可溶性蛋白约占 2 7.84 % ;亲和层析纯化后纯度大于 90 %。Western blot证实GST TLR4融合蛋白能与TLR4单抗特异性结合。结论 成功地构建融合蛋白表达载体pGEX TLR4 ,并在大肠杆菌中获得有效表达 ,获得了纯度大于 90 %的可溶性GST TLR4融合蛋白 。
Objective To construct expression vector of glutathione s-transferase-TLR4 fusion protein and express it in Escherichia coli.Methods TLR4 extra-cellular fragment was directly amplified from pCMV-TLR4 plasmid DNA by PCR ,then inserted into vector pMD18-T. After sequencing, the TLR4 gene was inserted into pGEX-4T-1, the recombinant expression plasmid pGEX-TLR4 was identified by restriction endonuclease digestion and sequencing. pGEX-TLR4 expressed proteins in E.coli HB101 via induction of IPTG.Results Sequencing showed that sequence of human TLR4 gene was identical with that of TLR4 cDNA recorded in the GenBank. The analysis for recombinant plasmid DNA digested by BamHⅠ and SalⅠ demonstrated that the TLR4 gene was inserted exactly into pGEX-4T-1. The recombinant plasmid pGEX-TLR4 was transferred into competent HB101 strain. The GST-TLR4 fusion protein was expressed in the bacteria under induction of IPTG. After induction, a new protein band of M r 92 000 appeared on SDS-PAGE. The recombinant protein of M r 92 000 amounted to 34.25% of the total bacteria protein after inducting with IPTG for 4 hours at 37 ℃. The fusion protein existed in the pattern of inclusion body, the soluble fusion protein was about 27.84%. After affinity purification the purity of GST-TLR4 was over 90%.Western-blot analysis indicated that the recombinant protein could react specifically with anti-TLR4 antibody.Conclusion Expression vector of GST-TLR4 fusion protein has been constructed successfully and expressed effectively in Escherichia coli, the purity of GST-TLR4 fusion protein was over 90%. These results make it possible to study further on its biological function and to obtain the TLR4 monoclonal antibody.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2003年第4期274-277,共4页
Immunological Journal
基金
国家重点基础研究发展规划项目 (G19990 5 4 2 0 3)
国家自然科学基金 (30 170 96 8)资助项目
