表达红斑丹毒丝菌SpaA和CbpB蛋白的重组枯草芽孢杆菌的构建及其在小鼠上的免疫原性评价  

Construction of a recombinant Bacillus subtilis strain expressing SpaA and CbpB of Erysipelothrix rhusiopathiae and evaluation of the strain immunogenicity in a mouse model

作　　者：程中林 黄浩[1] 曹思艺 石华辉 高继业 李继祥[1] CHENG Zhonglin;HUANG Hao;CAO Siyi;SHI Huahui;GAO Jiye;LI Jixiang(College of Veterinary Medicine,Southwest University,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆402460

出　　处：《生物工程学报》2024年第12期4521-4532,共12页Chinese Journal of Biotechnology

基　　金：重庆市技术创新与应用发展专项(cstc2021jscx-tpyzxX0012)。

摘　　要：为了构建表达红斑丹毒丝菌SpaA和CbpB蛋白的口服重组枯草芽孢杆菌活载体候选疫苗菌株,经融合PCR及无缝克隆构建了重组整合质粒pDG1730-CBJA,使用自然法转化枯草芽孢杆菌KC菌株,通过壮观霉素抗性平板筛选、淀粉降解实验及PCR检测获得重组菌KC-spaA-cbpB。用免疫印迹及间接免疫荧光实验检测重组菌表达的SpaA和CbpB蛋白,并测定了重组菌在小鼠体内的遗传稳定性。构建壮观霉素抗性基因敲除质粒pMAD-△spe^(r),转化KC-spaA-cbpB,筛选、鉴定获得KC-spaA-cbpB::△spe^(r),并进行了小鼠的口服免疫实验。实验结果显示,重组菌KC-spaA-cbpB在菌体表面表达SpaA蛋白,在芽孢表面表达CbpB蛋白,在小鼠体内稳定。壮观霉素抗性基因敲除菌KC-spaA-cbpB::△spe^(r)能表达SpaA和CbpB蛋白,其芽孢经灌胃免疫小鼠42d后,小鼠血清中SpaA和CbpB蛋白的特异性抗体IgG和粪便中特异性sIgA均极显著高于阴性对照(P<0.01),攻毒保护率为67.5%。本研究成功构建了表达红斑丹毒丝菌SpaA和CbpB蛋白的重组枯草芽孢杆菌,为研究猪丹毒口服活载体疫苗奠定了基础。To construct a recombinant Bacillus subtilis strain expressing SpaA and CbpB of Erysipelothrix rhusiopathiae for oral administration,we constructed the recombinant plasmid pDG1730-CBJA by fusion PCR and seamless cloning.The plasmid was introduced into B.subtilis KC strain by natural transformation,and the recombinant strain KC-spaA-cbpB was screened out on the plate containing spectinomycin(spe^(r))and confirmed by PCR and starch degradation test.The SpaA and CbpB expressed by KC-spaA-cbpB were detected by Western blotting and indirect immunofluorescence assay,and the genetic stability of the recombinant strain in mice was determined.The plasmid pMAD-△spe^(r) with knockout of spe^(r) was constructed and transformed into KC-spaA-cbpB.The spe^(r)-deleted mutant strain KC-spaA-cbpB::△spe^(r) was screened and identified,and its immunogenicity in a mouse model was evaluated by oral immunization.The results showed that the recombinant strain KC-spaA-cbpB was stable in mice,expressing SpaA on the cell surface and CbpB on the spore surface.KC-spaA-cbpB::△spe^(r) expressed SpaA and CbpB.The mice vaccinated with the spores of KC-spaA-cbpB::△spe^(r) had higher levels of SpaA and CbpB-specific IgG in the serum that those vaccinated with the wild-type spores 42 days after vaccination by gavage(P<0.01).The protective rate of mice immunized with the recombinant spores was 67.5%.The results indicated that a recombinant B.subtilis strain expressing SpaA and CbpB of E.rhusiopathiae was successfully constructed,and the recombinant strain laid a foundation for the development of oral live vector vaccines for swine erysipelas.

关 键 词：红斑丹毒丝菌 SpaA蛋白 CbpB蛋白 枯草芽孢杆菌 

分 类 号：S859.797[农业科学—临床兽医学] Q78[农业科学—兽医学]