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

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42 d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。

Abstract:

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.