维氏气单胞菌脂蛋白的原核表达及其抗血清的抗菌作用研究  

Prokaryotic expression of lipoprotein in Aeromonas veronii and study on antimicrobial effect of its antiserum

作　　者：宋格格 岳涛涛 盛天鸽 张俊辉[1] 王文东[1] 杨振国[1] 卢强[1] SONG Ge-ge;YUE Tao-tao;SHENG Tian-ge;ZHANG Jun-hui;WANG Wen-dong;YANG Zhen-guo;LU Qiang(Institute of Zoonosis,Jilin University,Changchun 130062,China;College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学人兽共患病研究所,吉林长春130062 [2]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医科学》2021年第7期891-897,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31672686)。

摘　　要：为了探究维氏气单胞菌毒力因子脂蛋白的生物学功能,对脂蛋白基因进行原核表达,对表达产物进行鉴定并制备多克隆抗体研究其抗菌效应。通过PCR扩增脂蛋白基因,连接至pET-32a(+)载体,构建重组质粒pET-32a(+)-Lpp。将重组质粒pET-32a(+)-Lpp转化入感受态细胞BL21(DE3),用IPTG诱导表达。利用His标签镍离子蛋白纯化柱对表达产物进行纯化,并采用SDS-PAGE和Western-blot鉴定重组蛋白的表达产物。用纯化后的重组脂蛋白免疫新西兰大白兔制备多克隆抗体,多抗经皮下接种小鼠研究其抗菌效应。结果显示,经酶切鉴定和序列测定发现重组质粒pET-32a(+)-Lpp构建成功。通过SDS-PAGE检测发现,重组蛋白LPP在原核表达系统中以可溶性形式表达,分子质量约为69 ku。Western-blot结果显示,纯化后的重组蛋白LPP为单一条带。制备的多克隆抗体的效价为102 400,该多抗可提高荷菌小鼠的生存率,减少脾荷菌量。因此,本实验成功构建了重组质粒pET-32a(+)-Lpp,表达并纯化了重组蛋白LPP,制备的多抗具有抗菌作用,为该蛋白生物学功能的研究及疫苗的制备奠定了基础。In order to investigate the biological function of lipoprotein of Aeromonas veronii,prokaryotic expression of lipoprotein genes of A.veronii was carried out,and the expression products were identified and the polyclonal antibodies were prepared to study its antimicrobial effect.The lipoprotein gene was amplified by PCR and linked to the pET-32a(+) vector to construct the recombinant plasmid pET-32a(+)-Lpp.The recombinant plasmid pET-32a(+)-Lpp was transformed into competent cell BL21(DE3) and induced by IPTG.The expression products were purified by His-tag nickel ion protein purification column,and the expression of the recombinant protein was identified by SDS-PAGE and Western-blot.The purified recombinant lipoprotein was used to immunize New Zealand white rabbits to prepare polyclonal antibodies,which were used to subcutaneously inoculate mice to study their antibacterial effects.The results showed that the recombinant plasmid pET-32a(+)-Lpp was successfully constructed after restriction digestion and sequencing.SDS-PAGE detected that the recombinant protein LPP was expressed in a soluble form in the prokaryotic expression system,with a molecular weight of about 69 ku.Western-blot showed that the purified recombinant protein LPP was a single band.And the prepared polyclonal antibody had a titer of 102 400,which could improve the survival rate of bacteria-bearing mice and reduce the bacterial load in the spleen.Therefore,this study successfully construct the recombinant plasmid pET-32a(+)-Lpp,and the recombinant protein LPP was expressed and purified. The prepared polyclonal antibody has the antibacterial effect,which laid a foundation for the study of the biological function of the protein and the research of vaccines.

关 键 词：维氏气单胞菌 脂蛋白LPP 原核表达 抗菌作用 

分 类 号：S852.615[农业科学—基础兽医学]