[Prokaryotic expression, purification, and immunogenicity analysis of Mycobacterium tuberculosis specific excretive proteins].

Objective To obtain the recombinant rv1837c and rv3803c of Mycobacterium tuberculosis using gene engineering technology and explore their prokaryotic expression,purification,and immunogenicity. Methods The Mycobacterium tuberculosis rv1837c and rv3803c genes were amplified by polymerase chain reaction,and then cloned into the vector pTA2,followed by the subclone into the expression vector pET30a(+). The resulting plasmids,named pET30a(+):rv1837c and pET30a(+):rv3803c,encode recombinant protein containing a hexa-histidine tag on its N-terminus. pET30a(+):rv1837c and pET30a(+):rv3803c were introduced into E. coli BL21 (DE3) by transformation respectively,and the recombinant gene was induced with 0.4 mmol/L isopropyl-D-thiogalactopyranoside. The expressed products were identified by Western blot with hexa-histidine tag antibody and serum from tuberculotic patients. The histidine tagged protein was purified by nickel nitrilotriacetic acid His-Bind resin. Rabbits were immunized with purified recombinant Rv1837c and Rv3803c proteins. Then the purified recombinant Rv1837c and Rv3803c proteins were used to detect antibody in rabbit serum,which had been immunized by Western blot. Results After transformation of the E. coli and induction with 0.4 mmol/L of isopropyl-D-thiogalactopyranoside,recombinant target proteins Rv1837c (relative molecular mass: 92 000) and Rv3803c (relative molecular mass: 38 000) were expressed in pET30a(+): rv1837c and pET30a(+): rv3803c system. The expressed protein existed in cytoplasm in an unsoluble form and amounted to 30% and 50% of the total proteins of E. coli. The purity of the purified protein reached 90%. The immunogenicity of the recombinant proteins Rv1837c and Rv3803c was strong,as identified by Western blot. Conclusion The prokaryotic expression recombinant plasmids pET30a(+): rv1837c and pET30a(+): rv3803c was successfully constructed and the recombinant proteins Rv1837c and Rv3803c were obtained,which laid a basis for the optimized diagnosis of active tuberculosis.