| Objective：To induce and express the genetically engineered Escherichia coli withAg85A-pET30a and Ag85B-pET24b, to purify and obtain recombinant Ag85A (rAg85A)and recombinant Ag85B (rAg85B) proteins, and to evaluate the immunogenicity ofMycobacterium tuberculosis rAg85A and/or rAg85B protein combined with M. vaccaevaccine.Method：The genetically engineered Escherichia coli with Ag85A-pET30a andAg85B-pET24b were induced by IPTG (Isopropyl β-D-Thiogalactoside). The rAg85Aand rAg85B proteins were purified by Ni2+resin, and were identified by SDS-PAGE.Sixty female BALB/c mice were randomly divided into the following6groups:(1)Negative control group: Phosphate buffer solution (PBS),(2) Positive controlgroup: M. bovis Bacille Calmette-Guerin (BCG),(3) Positive control group: M. vaccaevaccine (MV),(4) Experimental group: rAg85A+MV,(5) Experimental group:rAg85B+MV, and (6) Experimental group: rAg85A+rAg85B+MV. The mice wereimmunized3times at2weeks intervals. Changes in body weight of mice were recordedweekly. The mice were killed at14days after the last immunization. The lungs, livers andspleens of the mice were weighed. The spot number of spleen T lymphocytes secretedinterferon γ (IFN-γ) were detected by enzyme-linked immunospot (ELISPOT) assay. Thelevels of antibodies against Ag85A/Ag85B IgG, IgG1, and IgG2ain sera from the mice weredetermined by enzyme-linked immunosobent assay (ELISA) at1day before theimmunization,10days after the first and the second immunizations, and14days after thethird immunization. The percentage of Th1secreted IFN-γ and Th2secreted interlukin4(IL-4) by mononuclear cells in whole blood were detected by flow cytometry. The levelsof IFN-γ and IL-4in cultural supernatants of spleen lymphocytes were detected by ELISA.Results：The genetically engineered Escherichia coli with Ag85A-pET30a andAg85B-pET24b were induced and expressed successfully. The rAg85A and rAg85Bproteins were expressed in the form of inclusion bodies. The purified rAg85A and rAg85Bproteins were obtained。The body weights of the mice in6groups were significantly higher at14days after the last immunization than that at the beginning of the immunization (P<0.001), andsignificantly higher in three experiment groups than in three control groups. The weightsand weight indexes of the spleens and livers of the mice had no differences between eachgroups (P>0.05), but the index of lung in rAg85B+MV group was significantly higher thanthat in the other five groups (P<0.05).The result of ELISPOT showed that the number of T lymphocyte spots in the threeexperimental groups and two positive control groups were significantly higher than that inthe negative control group (P <0.05).The levels of specific antibodies IgG, IgG1and IgG2ain6groups increasedsignificantly after3times immunization (P<0.001). The levels of specific antibodies inthree experimental groups increased gradually as the time extension of immunization. Thelevel of IgG, IgG1and IgG2ain the three experimental groups were significantly higherthan those in the three control groups(P<0.001).The result from the flow cytometry showed that the percentage of Th1inrAg85A+rAg85B+MV group was significantly higher than that in the other five groups (P<0.001), and the ratio of Th1/Th2in three experimental groups were also higher than thatin the other three control groups (P <0.05).The levels of IFN-γ in the cultural supernatants of spleen lymphocytes in threeexperimental groups and two positive control groups were higher than that in the negativecontrol group (P <0.05), and that in rAg85A+rAg85B+MV group was highest (P<0.01).The levels of IL-4in the cultural supernatants of spleen lymphocytes were not detected.Conclusion：The genetically engineered Escherichia coli with Ag85A-pET30a andAg85B-pET24b expressed rAg85A and rAg85B protein in the form of inclusion bodies.MV had better effect as an adjuvant. The rAg85A and rAg85B protein could enhance theimmunogenicity of MV, combined with MV could induce high levels of Th1type of cellimmune response. |
PDF Full Text Request