Prokaryotic Expression And Immunological Properties Of Seven Mycobacterium Tuberculosis Detection Proteins

According to a WHO report, one-third of the population has been infected withMycobacterium tuberculosis worldwide, approximately10%of which were active TBdisease, and most were latent TB infection. When people have weakened immunesystems, latent TB infections may become active TB disease. The identification andtreatment of people with latent TB is an important part of controlling tuberculosisdisease. In this study, the resuscitation-promoting factors (RpfA, RpfB, RpfC, RpfD,RpfE) and the two latency-associated antigens (Rv3407and Rv0569) were expressedin prokaryotic expression system. We stimulated the dormant Mycobacteriumsmegmatis with the five renatured Rpf proteins, which was to detected the biologicalactivities. After immunizing the mice with the seven proteins in different ways, wedetected the level of antibody and IFN-γ by ELISA to study the immunologicalproperties of the seven proteins and selectly study the immune effects of the mixedproteins, Which laid the foundation for developing new methods to detect latent TBinfection and provide a theoretical basis for the study on the dormance and recoverymechanism of Myccbacterium tuberculosis.The seven genes were amplified by polymerase chain reaction (PCR) fromgenome DNA of Mycobacterium tuberculosis H37Rv strain. They were inserted intocloning vector pMD-18T for sequencing. After verifying the sequence of the sevengenes were correct, they were digested by restriction endonuclease and cloned into theprokaryotic expression vector pET-28a and pET-22b. The seven recombinant plasmidswere transformed into E. coli DE3for prokaryotic expression. The seven expressedproteins were confirmed by SDS-PAGE and western blot analysis with mouse-specificmonoclonal antibody to6×His. The five Rpf proteins were inclusion bodies and theprotein Rv3407and Rv0569were soluble proteins. Then the seven proteins werepurified by AKTA HPLC System and HisTrap FF Crude as description in the protocol.We detected the biological activity of the five Rpfs through observing the growthcurves of dormant Mycobacterium smegmatis stimulated with the proteins. The resultsshowed the effects of the protein RpfA, RpfB, RpfD and RpfE could effectivelystimulate the dormant Mycobacterium smegmatis to grow.The BALB/c mice were immunized three times at two weeks intevral with theseven proteins. The results showed the titers of the specific antibody in the BALB/cmice immunized with protein RpfA, RpfB, RpfC, RpfD, RpfE, Rv3407and Rv0569were1:1600,1:3200,1:800,1:3200,1:1600,1:1600,1:1600, respectively, and theimmunogenicity of protein RpfC was the worst. The IFN-γ was obtained from thecultured supernatant of spleen lymphocytes in the immuned mice stimulated with theseven proteins, then was detected by IFN-γ ELISA Kit. The IFN-γ level in the miceimmunized with RpfA, RpfB, RpfC, RpfD, RpfE, Rv3407and Rv0569were546.80±15.97pg/ml、814.34±22.14pg/ml、97.65±12.78、936.45±28.74pg/ml、745.23±34.25pg/ml、1061.49±20.90pg/ml and1135.36±26.01pg/ml, which weresignificantly higher than that of negative control(P<0.05), except RpfC (P>0.05). TheIFN-γ level of RpfD, Rv3407and Rv0569groups were higher than other groups(P<0.05).The BALB/c mice were immunized three times at two weeks intevral with theprotein RpfD, Rv3407, Rv0569, mixed protein RpfD+Rv3407, mixed proteinRpfD+Rv0569and mixed protein Rv3407+Rv0569. The titers of specific antibodyand the IFN-γ level were detected by ELISA. The results showed the titers of specificantibody in the mice immunized with the protein RpfD, Rv3407, Rv0569,RpfD+Rv3407, RpfD+Rv0569, Rv3407+Rv0569were1:3200,1:1600,1:1600,1:3200/1:800,1:3200/1:800,1:1600/1:1600. The IFN-γ concentrations of the miceimmunized with RpfD, Rv3407, Rv0569, RpfD+Rv3407, RpfD+Rv0569andRv3407+Rv0569were864.79±39.18pg/ml,1101.51±35.96pg/ml,1183.80±22.38pg/ml,958.67±32.02pg/ml,928.52±24.29pg/ml,1275.12±19.26pg/ml, whichsignificantly higher than that of negative control (P<0.01). The IFN-γ concentrationsof the mice immunized with mixed protein RpfD+Rv3407and RpfD+Rv0569werebetween that of the mice immunized with RpfD, Rv3407and Rv0569alone (P<0.05). The IFN-γ concentrations of the mice immunized with mixed proteinRv3407+Rv0569were higher than that of the mice immunized with protein Rv3407and Rv0569alone (P<0.01).The seven expression vector were correctly constructed and successfullyexpressed the seven proteins. The protein RpfA, RpfB, RpfD and RpfE couldeffectively promote the dormant Mycobacterium smegmatis to grow. The sevenproteins and the mixed proteins could induce humoral immunity and cellularimmunity, and the immunogenicity of protein RpfC was the worst. The IFN-γ levelinduced by mixed protein (Rv3407+Rv0569) was higer than that induced by theprotein Rv3407and Rv0569alone. Above all, they may be the candidate antigens todetect latent TB infection and be worthy of further study.