边缘无浆体MSP1α与MSP1β基因的克隆表达及其对牛红细胞的黏附特性  

Cloning and expression of MSP1α and MSP1β proteins of Anaplasma marginale and their adherence to bovine red cells

作　　者：倪宏波[1] 钱爱东[2] 姜海芳[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]吉林农业大学动物科技学院,吉林长春130118

出　　处：《中国生物制品学杂志》2013年第9期1242-1246,1250,共6页Chinese Journal of Biologicals

基　　金：黑龙江省教育厅骨干教师项目(No.2010td05)

摘　　要：目的原核表达边缘无浆体(Anaplasma marginale,A.marginale)MSP1α和MSP1β蛋白,并检测其对牛红细胞的黏附作用。方法通过PCR法扩增MSP1α和MSP1β基因,插入表达载体pGEX-6p-1中,转化感受态大肠埃希菌(E.coli)BL21(DE3),IPTG诱导表达,表达的重组MSP1α和MSP1β蛋白经透析法纯化后,分别免疫新西兰白兔和商品蛋鸡,制备免疫兔血清及抗MSP1α和MSP1βIgY抗体,Western blot法分析其免疫原性,间接免疫荧光法(immunofluorescence assay,IFA)检测其在大肠埃希菌外膜上的表达,血凝试验(hemagglutination test,HA)和血凝抑制试验(hemagglutination inhibition test,HI)检测其对牛红细胞的黏附作用。结果重组表达质粒pGEX-6p-1-MSP1α和pGEX-6p-1-MSP1β经PCR、双酶切及测序证实构建正确;表达的重组MSP1α和MSP1β蛋白相对分子质量分别约为58 000和107 000,主要以包涵体形式存在,表达量分别占菌体总蛋白的31%和14%;纯化的重组MSP1α和MSP1β蛋白纯度分别可达93%和91%,可与免疫兔血清发生反应;抗MSP1α和MSP1βIgY抗体可分别与表达MSP1α和MSP1β蛋白的重组菌发生反应;重组MSP1α和MSP1β蛋白对牛红细胞具有明显的黏附作用,吸附率可达56.4%和52.7%。结论MSP1α和MSP1β基因可在大肠埃希菌外膜上大量表达,并对牛红细胞具有明显的吸附作用,为进一步揭示牛边缘无浆体病的作用机理奠定了基础。Objective To express the MSP1α and MSP1β proteins of Anaplasma marginale and determine their adher- ence to bovine red cells. Methods MSP1α and MSP1β genes were amplified by PCR and inserted into expression vector pGEX-6p-1. The constructed recombinant plasmids pGEX-6p-1-MSP1α and pGEX-6p-1-MSP1β were transformed to com- petent E. coli BL21 （DE3） for expression under induction of IPTG. The expressed recombinant MSP1α and MSP1α pro- teins were purified by dialysis and immunized to New Zealan rabbits and commercial laying hens, by which rabbit immune sera as well as IgYs against MSP1α and MSP1β were prepared, then analyzed for immunogenieity by Western blot, for ex- pression on outer membrane of E. coli by IFA, and for adherence to bovine red cells by HI test. Results PCR, restric- tion analysis and sequencing proved that recombinant plasmids pGEX-6p-I-MSP1α and pGEX-6p-I-MSP1α were con- structed correctly. The expressed recombinant MSP1α and MSPI1α proteins, with relative molecular masses of about 58 000 and 107 000, mainly existed in a form of inclusion body, and contained about 31% and 14% of total somatic pro- teins, respectively. The purified MSP1α and MSP1β proteins reached purities of 93% and 91%, and showed specific reac- tions with rabbit immune sera. The IgYs against MSP1α and MSP1β showed reactions with recombinant E. coli expressing MSPlct and MSP1β proteins respectively. Recombinant MSP1α and MSP1β proteins showed significantly adherence to

关 键 词：边缘无浆体 MSP1α蛋白 MSP1β蛋白 原核细胞 基因表达 黏附 

分 类 号：S852.61[农业科学—基础兽医学] Q785[农业科学—兽医学]