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 Cterminal 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 Ni2+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 ×103Mr,existed as soluble form in E.coli, and could be purified by Ni2 +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.