Expression And Application Of PPE68 Protein Of Mycobacterium Tuberculosis

Objective:To construct prokaryotic expression plasmid and eukaryotic expression plasmid of PPE68 gene of Mycobacterium tuberculosis. Observe the expressions of PPE68 gene in E. coli BL21 and murine macrophage RAW264.7 cells. Purify the PPE68 proteins of expression in E. coli BL21 by affinity chromatograph. Rabbit antiserum against PPE68 was prepared by hypodermic immunization. This provide an experimental basis for the further research of PPE68 gene and the the construct of PPE68 gene recombinant BCG.Methods:1 PPE68 gene was amplified by PCR from the genome of Mycobacterium tuberculosis H37Rv strain,and cloned into pET32(+) vector, then transformed into E. coli BL21. The recombinant proteins were expressed with IPTG induction. The rPPE68 was identified by SDS-PAGE and Western-blot and purified.2 Mycobacterium tuberculosis PPE68 gene was cloned into pBudCE4.1 to construct recombinant plasmid pBudCE4.1-PPE68. The recombinant plasmid was transient transfection into RAW264.7 cells,and the expression of PPE68 gene was detected by RT-PCR, Western-blot at the transcriptional and translational level.3 Rabbit antiserum against PPE68 was prepared by hypodermic immunization, within purified PPE68 protein mixed with Freund'incomplete adjuvant. The serum was collected at the 7th days after the third time immuned. The specificity of the rabbit antiserum was determined by agglutination test and Western-blot. The antiserum titer was detected by double immunodiffusion.Results:1 After identifying by restriction digestion and DNA sequencing,the plasmid pET32a(+)/PPE68 was constructed successfully. A recombinant protein Trx-PPE68 about 57 000 was expressed in BL21.The protein can binding with the corresponding antibody in the serum of the mouse immuned by Mycobacterium tuberculosis. The purity of target protein was 93%.2 The accurate eukaryotic recombinant plasmid pBudCE4.1-PPE68 was identified by restrict endonuclease. The PPE68 gene expression product in RAW264.7 cells was detected by RT-PCR,Western-blot.3 The specificity of the rabbit antiserum was determined by agglutination test and western-blot. The antiserum had a double immunodiffusion titer about 1:16.Conclusions:1 The prokaryotic expression vector pET32a(+)/PPE68 was constructed successfully, and the rPPE68 proteins was obtained.2 The recombinant plasmid pBudCE4.1-PPE68 was successfully constructed. The protein can be expressed in murine macrophage RAW264.7. The prokaryotic gene PPE68 expressed in eukaryotic system successfully, which provide an experimental basis for vivo expressed of the PPE68 recombinant BCG.3 Rabbit antiserum against PPE68 gene was prepared successfully, which may be applied in PPE protein further function research.