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目的原核表达、纯化甲型副伤寒沙门菌外膜BtuB蛋白,并检测其免疫原性和免疫保护性。方法采用PCR从甲型副伤寒沙门菌(CMCC 50973)基因组DNA中扩增btuB基因,插入原核表达载体pET-30a中,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经分子筛和亲和层析纯化后,免疫BALB/c小鼠,ELISA法检测小鼠血清中抗BtuB IgG抗体效价,并用最小绝对致死剂量活菌攻毒,计算小鼠的保护率。结果重组原核表达质粒pET-30a-btuB经双酶切和测序证实构建正确;重组蛋白的相对分子质量约为67 000,表达量约为菌体总蛋白的25%,主要以包涵体形式表达;纯化的重组蛋白纯度大于90%,可与鼠抗His-Tag单抗特异性结合;纯化的重组蛋白免疫BALB/c小鼠可获得高效价的抗BtuB IgG抗体,且可获得60%的保护率。结论成功原核表达并纯化了甲型副伤寒沙门菌外膜BtuB蛋白,重组蛋白免疫原性良好,并能够为小鼠提供一定的保护作用。
Objective To purify the outer membrane BtuB protein of Salmonella paratyphi A (Salmonella typhimurium) in vitro and test its immunogenicity and immunoprotective properties. Methods The btuB gene was amplified from genomic DNA of Salmonella paratyphi A (CMCC 50973) by PCR and inserted into prokaryotic expression vector pET-30a. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed recombinant protein was purified by molecular sieve and affinity chromatography, BALB / c mice were immunized, the titer of anti-BtuB IgG in serum was detected by ELISA, and the protection rate of mice was calculated by live attenuated minimum lethal dose . Results The recombinant plasmid pET-30a-btuB was confirmed by double enzyme digestion and sequencing. The relative molecular mass of the recombinant protein was about 67 000 and the expression level was about 25% of the total bacterial protein. The recombinant protein was mainly expressed in inclusion bodies. The purified recombinant protein was more than 90% pure and could specifically bind to mouse anti-His-Tag monoclonal antibody. BALB / c mice were immunized with the purified recombinant protein to obtain high titer anti-BtuB IgG antibody with a protection rate of 60% . Conclusion The BtuB protein of Salmonella paratyphi A was successfully expressed in prokaryotic cells and purified, and the immunogenicity of the recombinant protein was good, which could provide a protective effect for mice.