摘要
在前期研究克隆得到黄颡鱼(Pelteobagrus fulvidraco)全长cDNA的基础上,为进一步研究黄颡鱼免疫球蛋白M(IgM)重链的生物学功能,设计特异性引物PCR扩增获得了编码成熟免疫球蛋白M重链基因的编码序列。将该基因编码片段连接到原核表达载体pQE30中,构建黄颡鱼IgM重链的重组表达质粒IgM-pQE30。该重组质粒经酶切和测序鉴定后,转入表达宿主大肠杆菌M15中诱导表达,在30℃下,经0.2 mmol/L IPTG诱导表达8 h,可获得大量以包涵体形式存在的黄颡鱼IgM重链蛋白,经SDS-PAGE电泳和Western blotting分析表明,新增的62 kDa蛋白条带与预期值相符,且能与鼠源抗6×His的单克隆抗体特异性结合,证明黄颡鱼IgM重链基因获得了高效原核表达。为进一步纯化该蛋白制备特异抗体,研究其生物学功能奠定了基础。
Yellow catfish（Pelteobagrus fulvidraco） is an important cultured catfish in China,and suffering from various diseases.Immunoglobulin M（IgM） is an important molecule against pathogens in fish immune response.In order to elucidate its expression pattern and mechanism during the period of immunological reaction for prevention of fish diseases,it is necessary to obtain enough IgM protein for preparation of anti-IgM specific antibodies.Therefore,the recombinant expression plasmid（IgM-pQE30） of yellow catfish was constructed with the IgM heavy chain coding sequence fragment cloned from the catfish linking the prokaryotic expression vector pQE30 after complete digestion of sequencing plasmid（IgM heavy chain/pMD-18T） and pQE30 by two endonucleases Sph 1 and Kpn 1.Then the recombinant plasmid was transferred into the host bacterium Escherichia coli M15,and induced to express fusion protein,which was confirmed by means of SDS-PAGE and Western blotting.A 62 kDa protein was greatly induced in unsoluble status in M15,and confirmed to be the recombinant protein（target protein） using monoclonal antibody against 6xHis tag in Western blotting.The recombinant protein was expressed in the greatest quantity at 30 0C for the induction of 8 hours with 0.2 mmol/L IPTG.The IgM heavy chain fusion protein can be greatly expressed in the recombinant plasmid expression system under the most suitable induction conditions mentioned above.This lays a foundation for further researches on the IgM of yellow catfish.
出处
《生态科学》
CSCD
北大核心
2013年第1期44-50,共7页
Ecological Science
基金
国家自然科学基金项目(40576056
40976066
31170474)
中央高校基本科研业务费专项资金资助(21612111)
关键词
免疫球蛋白M
重链
重组质粒
原核表达
黄颡鱼
immunoglobulin M
heavy chain
recombinant plasmid
prokaryotic expression
Pelteobagrus fulvidraco
