论文部分内容阅读
目的制备抗肺炎链球菌(S.pn)丝氨酸-苏氨酸激酶(STKP)基因的特定区域的免疫血清,为疫苗研究奠定基础。方法 PCR扩增S.pn STKP蛋白C末端314个氨基酸区域的基因,将其克隆入pMD19-T载体,将鉴定正确的STKP基因从pMD19-T载体亚克隆至PET-32a(+)原核表达载体进行诱导表达;获得的可溶性蛋白经Ni2+柱纯化及Western blot鉴定后,免疫家兔制备抗STKP血清,间接ELISA鉴定抗体效价,Western blot鉴定免疫血清特异性。结果成功将STKP基因特定序列克隆入PET-32a(+)原核表达载体并诱导表达,形式以可溶性表达为主,经Ni2+柱层析法纯化STKP,纯度达92%,免疫家兔制备出STKP免疫血清,间接ELISA检测抗体效价为1∶100 000,Western blot证实免疫血清能与目的蛋白特异结合。结论获得STKP重组蛋白及免疫血清,重组表达产物具有免疫原性,为S.pn多价联合蛋白疫苗研制奠定了基础。
Objective To prepare immune sera against specific regions of Streptococcus pneumoniae (S. pn) serine - threonine kinase (STKP) gene and lay the foundation of vaccine research. Methods The 314 amino acid region of S.pn STKP protein was amplified by PCR and cloned into pMD19-T vector. The correct STKP gene was subcloned from pMD19-T vector into PET-32a (+) prokaryotic expression vector The soluble protein was purified by Ni2 + column and identified by Western blot. Rabbit was immunized to prepare anti-STKP serum. The antibody titer was identified by indirect ELISA. The specificity of the immune serum was identified by Western blot. Results The specific sequence of STKP gene was successfully cloned into prokaryotic expression vector PET-32a (+) and expressed in soluble form. STKP was purified by Ni2 + column chromatography with a purity of 92%. Immunized rabbits were immunized with STKP Serum, indirect ELISA detection of antibody titer of 1: 100 000, Western blot confirmed that the immune serum can be specifically combined with the target protein. Conclusion The recombinant protein and immune serum of STKP were obtained. The recombinant protein was immunogenic, which lays the foundation for the development of S.pn multivalent conjugate protein vaccine.