犬MC4R基因原核表达质粒的构建与表达  

Construction and Expression of Prokaryotic Expression Vector for Canine MC4R Gene

作　　者：贺宝军[1] 巴彩凤[1] 王光川[1] 赵薇[1] 宋慧娟[1] 吕金钢[1] 

机构地区：[1]辽宁医学院实验动物中心,辽宁锦州121001

出　　处：《中国生物制品学杂志》2011年第5期550-553,共4页Chinese Journal of Biologicals

基　　金：辽宁省科技厅基金项目(2009408001-4)

摘　　要：目的构建犬MC4R基因的原核表达质粒,并表达重组蛋白。方法以重组质粒pcDNA3.1-myc-his/A-cMC4R为模板,PCR扩增MC4R基因,将其克隆至原核表达载体pET-30a(+)中,构建重组原核表达质粒pET-30a(+)-MC4R,转化入E.coli Transetta(DE3),IPTG诱导表达,表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒pET-30a(+)-MC4R经双酶切鉴定证明构建正确,DNA测序证实插入序列与GenBank中登录序列的同源性为99%,只有编码区777位碱基T变成了C;表达的重组蛋白相对分子质量约为37 000,诱导4 h表达量最高,占菌体总蛋白的8%;重组蛋白可与抗His单抗特异性结合。结论已成功构建犬MC4R基因原核表达质粒,并表达了重组蛋白,为犬MC4R蛋白多克隆及单克隆抗体的制备提供了抗原。Objective To construct a prokaryotic expression vector for canine MC4R（cMC4R） gene and express recombinant MC4R protein.Methods MC4R gene was amplified by PCR using recombinant plasmid pcDNA3.1-myc-his / A-cMC4R as a template and cloned into prokaryotic expression vector pET-30a（＋）.The constructed recombinant plasmid pET-30a（＋）-MC4R was transformed to E.coli Transetta（DE3） for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Restriction analysis proved that recombinant plasmid pET-30a（＋）-MC4R was constructed correctly.DNA sequencing proved that the homology of inserted sequence to that reported in GenBank was 99%,of which only the base T at site 777 of encoding region changed into C.The relative molecular mass of expressed protein was about 37 000.The expression level reached the maximum after induction for 4 h,which accounted for 8% of total somatic protein.The recombinant protein showed specific binding to anti-His monoclonal antibody.Conclusion The prokaryotic expression vector for cMC4R gene was successfully constructed,and recombinant cMC4R protein was expressed,which provided an antigen for preparation of polyclonal and monoclonal antibodies against cMC4R protein.

关 键 词：受体 黑皮质素 4型 犬科 原核细胞 基因表达 

分 类 号：Q786[生物学—分子生物学]