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目的构建植原体免疫主导膜蛋白A(IdpA)的原核表达载体,表达并纯化目的蛋白,制备抗血清。方法以重组克隆质粒pMD18-T-IdpA为模板PCR扩增IdpA基因片段,经酶切连接将IdpA基因克隆到原核表达载体pET-28a(+),重组质粒转化感受态E.coli BL21(DE3),PCR和双酶切进行鉴定。IPTG诱导重组菌表达IdpA蛋白,并进行纯化和鉴定,以纯化获得的IdpA蛋白为抗原免疫BALB/c小鼠制备抗血清,并采用ELISA和Western blot法检测抗血清的效价和特异性。结果成功构建了原核表达载体pET-28a(+)-IdpA,并在大肠杆菌中能稳定表达IdpA蛋白,经纯化获得了纯度大于90%的高纯度目的蛋白,用纯化的IdpA蛋白免疫BALB/c小鼠,获得了效价高于1∶320 000的强特异性抗IdpA蛋白抗血清。结论成功进行了IdpA的原核表达并制备了抗血清。
Objective To construct the prokaryotic expression vector of immunophenotypic membrane protein A (IdpA) of E.coli and express and purify the target protein to prepare antiserum. Methods The IdpA gene fragment was amplified by PCR using the recombinant plasmid pMD18-T-IdpA as template. The IdpA gene was cloned into the prokaryotic expression vector pET-28a (+) and the recombinant plasmid was transformed into competent E. coli BL21 (DE3) , PCR and double enzyme digestion. IPTG was used to induce the expression of IdpA protein in the recombinant strain. The recombinant was purified and identified. The purified IdpA protein was used to immunize BALB / c mice to prepare antiserum. The titer and specificity of antisera were tested by ELISA and Western blot. Results The prokaryotic expression vector pET-28a (+) - IdpA was successfully constructed and the IdpA protein was stably expressed in E. coli. The purity of the target protein was over 90%. The purified IdpA protein was used to immunize BALB / c Mice, a strong specific anti-IdpA protein antiserum with a titer higher than 1: 320 000 was obtained. Conclusion The prokaryotic expression of IdpA was successfully prepared and the antiserum was prepared.