| Tuberculosis (TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). It has been one of the most important contributory and mortiferous factors in the world. It is estimated by WHO that one-third of the world population are infected with MTB. In China, the number of TB patients was the second in the world, approximately 400 million people infected. 10% of these people will develop to activity TB, and the most of infected MTB exist in condition of dormant infection. This dormant MTB has strong drug resistance, it can exist in vivo for a long time, and it's difficult to isolate and cultivate. The current vaccine against MTB, M. bovis Bacillus Calmette-Guérin(BCG)has many problems such as ineffective to subclinical infection,short period of protect and low immune response. Current preventive measures and diagnosis methods have no effect on MTB dormant. So it is great significant for studying new diagnosis reagents, therapy methods and vaccines to prevent TB, especially for dormant infection.Recent studies showed that the resuscitation promoting factor(Rpf) which secreted by MTB played a important role in promoting the resuscitation and growth of dormant mycobacterium, Rv0867c(RpfA),Rv1009(RpfB),Rv1884c(RpfC),Rv2389c(RpfD)and Rv2450c(RpfE). Rpf was first discovered in Micrococcus luteus(M.luteus). Homology analysis suggests that Rpf-like protein widely distribute among high G+C Gram-positive bacteria. It has been found the domain of Rpf has the same biological characteristics as Rpf-like protein. It also has been demonstrated that Rpf-like protein can not only stimulate the resuscitation of stationary-phase mycobacterial, including M. bovis BCG and MTB, but also a target antigen of immune system of host. In this study, we cloned, expressed and purified M.luteus Rpf protein, M.luteus Rpf domain protein and RpfB domain protein, generated MAb of M.luteus Rpf domain and RpfB domain. Furthermore, we studied the biological function and immunological characteristics of them, in order to evaluate the possibility of using them to rapidly isolate and cultivate MTB, detect related antigen, and become candidate vaccines.AIM: To express and purify M.luteus Rpf protein, Rpf domain and RpfB domain.To generate MAb of M.luteus Rpf domain and RpfB domain .To study the biological and immunological properties of M.luteus Rpf, Rpf domain and RpfB domain.METHODS AND RESULTS:1. Cloning, expression and purification of M.luteus Rpf protein, Rpf domain and RpfB domainThe genes of M.luteus Rpf, Rpf domain and RpfB domain were amplified respectively from genome of M.luteus and MTB H37Rv strain by using polymerase chain reaction(PCR). Inserted the PCR product into pUC-19 vector accordingly and sequenced. The DNA sequence of M.luteus Rpf, Rpf domain and RpfB domain were identical with that published by GenBank. After sequencing was confirmed, Subcloned the three right sequenced genes into pPRO-EXHT prokaryotic expression vector . The recombinant clones were screened restriction enzyme analysis and transformed into E.coli DH5αstrain and induced with IPTG. The analysis of SDS-PAGE showed that there were specific protein expressions at the corresponding positions as we anticipated. The three proteins were further identified by Western-blot using anti-6×His MAb.Purified theproteins by Ni-NTA purification system under denaturing conditions.2. Biological Activity Of M.luteus Rpf protein, Rpf domain and RpfB domain2.1 Construction and identification of MAbs to M.luteus Rpf domain and RpfB domainWe obtained 3 hybridoma cell strains which could stablely secrete antibodies adainst M.luteus, named F3D10,G10D5 and G6C8 from BALB/c mice immunized with. The subclass of F3D10 and G10D5 is IgG1,while the subclass of G6C8 is IgM. The relative affinity of the MAbs are different and the sequence is A9C8>D3A5>B8G11.We also obtained 3 hybridoma cell strains which could stablely secrete antibodies adainst RpfB domain ,named D3A5,B8G11 and A9C8. The subclass of D3A5 and B8G11 is IgG1,while the subclass of A9C8 is IgM. The relative affinity sequence of the MAbs is A9C8>D3A5>B8G11.In order to identify the specificity of the MAb, we constructed eukaryotic expression vector pcDNA3.1(-)-Rpf domain and pcDNA3.1 (-)-RpfB domain, and expressed in COS-7 cells. The specificity of the MAb was confirmed with indirect immunofluorescence.2.2 Cross test of anti-M.luteus Rpf domain and anti-RpfB domain.Cross test of the two constructed MAbs were detected by ELISA, using Rpf, Rpf domain, RpfB domain, and other Rpf proteins of MTB as antigen to invest culture plate respectively and the two constructed MAbs to be the first antibody. The results showed both MAbs have reaction with all above proteins.2.3 The promotive action of resuscitation and growth of M.luteus Rpf, Rpf domain and RpfB domain on M.luteus and MTBH37Ra and M.luteus dormant strains were at a suitable dilution using culture medium, and then divided into 3 groups randomly. Different concentration proteins were added to each group respectively. Taken 200μl from each group at different time to detected the OD600. The results showed that Rpf protein and Rpf domain could significantly stimulate the growth of M.luteus at concentration of 100pmol/L, while they stimulated the growth of MTB H37Rv notably at concentration of 10pmol/L in Rpf protein and 100pmol/L in Rpf domain. This stimulation was significantly inhibited after adding 1:600 anti- M.luteus Rpf domain MAb. When the concentration of RpfB domain was 1000pmol/L, stimulation to M.luteus was significant, and When its concentration was 500pmol/L, stimulation to MTB H37Rv was also significant. The stimulation was inhibited after adding 1:1000 anti- RpfB domain MAb.3. Immunological activity of M.luteus Rpf protein, Rpf domain and RpfB domainTo test immunological properties of the proteins, BALB/c mice were immunized three times at 2-week interval subcutaneously on their backs with Rpf , Rpf domain and RpfB domain proteins respectively, which were transferred to NC membranes beforehand. The titers of specific antibody in BALB/c mice were detected by ELISA. The results suggested average titers of anti-Rpf was 1:12800, that of Rpf domain was 1:4800, and that of RpfB domain was 1:6400.To evaluate cell-mediated immune response, stimulating index(SI) of the spleen lymphocytes in immunized mice were measured by MTT method, the SI of Rpf , Rpf domain and RpfB domain were significantly higher(which were 2.86±0.12 for Rpf, 2.10±0.09 for Rpf domain, 2.40±0.11 for RpfB domain than that of normal saline controls ( P<0.05 ) , but lower than that of BCG groups(3.50±0.23)(P<0.05).The levels of IFN-γ, IL-10 and IL-12 secretion stimulated by specific antigen were detected by indirect ELISA. IFN-γ, IL-10 and IL-12 concentrations in cultured supernatant of spleen lymphocytes from mice immunized with Rpf protein were 1528±36 ng/L, 485±13 ng/L and 302±14 ng/L respectively. The levels of three cytokines mentioned above stimulated by Rpf domain protein were 1126±36 ng/L, 368±13 g/L and 289±14 ng/L respectively, while thats of RpfB domain protein were 1432±30 ng/L, 503±11 ng/L, and 311±11 ng/L respectively. In group immunized by BCG, the average levels of IFN-γ,IL-10 and IL-12 were 2022±38 ng/L, 578±13 ng/L and 400±10 ng/L respectively; while in normal saline groups, the average levels of IFN-γ,IL-10 and IL-12 were 256±6 ng/L, 76±3 ng/L and 56±4 ng/L. From above results, we found the levels of three cytokines immunized by three proteins were significantly higher than that of normal saline groups(p<0.01), but lower than that of BCG groups. (p<0.05).In order to test our three proteins'efficacies against MTB H37Rv, BALB/c mice were intravenously infected with 105 CFU MTB H37Rv. four weeks after the final immunization, the numbers of MTB CFU in spleens were determined. Compared with the normal saline groups, Bacteria loads in the three protein groups were dramatic reduction(differences were 1.89 log10, 1.61 log10 and 1.78 log10 respectively,P<0.05). But the protective efficacies of mice immunized with proteins were lower than that of BCG vaccination group(2.83 Log10) (p<0.05).CONCLUSION:M.luteus Rpf, Rpf domain and RpfB domain proteins could stimulate the resuscitation and growth of M.luteus dormant and MTB H37Ra dormant. MAb of the two domain could significantly inhibited the effectiveness of growth promotion and specifically identified many kinds of Rpf and Rpf domain. They also could induce high level cell-mediated immune reponse and confer effective protection against TB. So they may be an appropriate candidate for culture medium additives which promote growth of dormant MTB in order to elevate detection rate of subclinical patients. We can establish detection methods on MTB related antigen according to our results. At the same time, they may be new candidate for vaccine to TB control and prophylaxis. |
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