肺炎链球菌丝氨酸-苏氨酸激酶基因的原核表达及免疫血清的制备

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。

Prokaryotic expression of STKP and preparation of anti-STKP antiserum

Objective To express and purify of STKP and prepare anti-STKP antiserum. Methods STKP gene of C- terminal domains was amplified from S.pn by PCR, and cloned into pMD19-T vector and confirmed by sequencing.The enzyme-digested target fragment was sub-cloned into PET-32a （＋） expression vector and transected into E.coli. BL21 （DE3）, in which STKP expression was induced.After the soluble protein was purified by Ni^2＋ affinity chromatography, the recombinant protein was identified by Western blot,and used to immunize rabbit. The antiserum was obtained and identified by indirect ELISA and Western blot. Results The result of DNA sequence analysis showed that the cloned stkp gene sequence was identical with Gene Bank data.SDS-PAGE and Western blotting showed that the expressed STKP fusion protein was about 55×10^3 Mr,existed as soluble form in E.coli, and could be purified by Ni^2＋ affinity chromatography.The titer of the antiserum to the purified protein was 1：100 000 by indirect ELISA and Western blotting confirmed that the antiserum reacted specifically to the STKP. Conclusion A recombinant STKP protein and the specific antiserum have been obtained, which provides a basis for establishment of polyvalent protein vaccine.