摘要
目的构建pGEX-6P-3/GAD65原核表达载体,获得大量纯化的大鼠GAD65蛋白。方法通过RT-PCR技术从大鼠脑组织中扩增GAD65cDNA,克隆至PGEM-Teasy载体上并测序,利用引物上的BamHI与EcoRI酶切位点将GAD65基因插入PGEX-6P-3,转化大肠杆菌BL21,限制性内切酶消化鉴定。IPTG诱导重组菌株表达,用SDS-PAGE与Western-blot对表达产物进行鉴定,并用Sepharose4B亲和层析柱对表达产物进行纯化,PreScissionProteas酶对融合蛋白进行酶切。结果扩增的GAD65长度为1758bp,测序结果与已知序列吻合。重组子经酶切鉴定,获得PGEX-6P-3GAD65原核表达菌株,获得分子量约91000的PGEX-6P-3/GAD65融合蛋白,分离纯化出约65000的GAD65蛋白,Western-blot表明重组蛋白能被GAD65单克隆抗体特异结合。结论成功获得GAD65蛋白,为研究GAD65生物学作用奠定了基础。
Objective To construct prokaryotlc expression vector of PGEX-6P-3/GAD65, and obtain bulk of purified GAD65 protein. Method GAD65 cDNA was amplified from brain tissue of rat with RT-PCR, cloned into PGEM-Teasy and sequenced. GAD65 gene was inserted into PGEX-6P-3 through pre-set enzyme digestion sites of BamHI and EcoRI in primers to construct recombinant gene, then the plasmid was transfected into E.coli BL21 and the transformants were identified by restriction enzyme digestion method. The recombinant protein was purified with sepharose 4B affinity column and the fusion protein was cleaved by PrescissionProtesa enzyme, and then identified by SDS-PAGE and Western-blot analysis. Result The length of amplified GAD65 fragment was 1758 base pairs, and the sequence of the gene is the same as the reported sequence. The expression plasmid, PGEX-6P-3 /GAD65, was constructed. Recombinant GAD65 fusion protein was expressed in E.coli with a M.W. of 91kDa. The fusion protein was puirifed and cleaved to generate the GAD65 with a M.W.of 65kDa. Results of Western-blot indicate the recombination protein can be idio-conjugated with GAD65 monoclonal antibody. Conclusion The GAD65 gene was cloned and recombinant GAD65 was produced and purified from E.coli. It is an important reagent for the studies of the biological function of GAD65.
出处
《热带医学杂志》
CAS
2006年第6期675-678,共4页
Journal of Tropical Medicine
基金
广东省自然科学基金(No.04009589)
广州医学院项目基金(No.303005273)
