Expression And Immunological Properties Of Mycobacterium Tuberculosis Antigenic Proteins And Their Application In The Detection Of Bovine Tuberculosis

Tuberculosis (TB) is one kind of zoonosis characterized by chronic respiratory infections mainly caused by Mycobacterium tuberculisis (MTB) complex. The key members of the complex including MTB mainly caused human tuberculosis and Mycobacterium bovis (M. bovis) mainly caused bovine tuberculosis. A resurgence of TB has occurred all over the world due to a range of factors such as population growth, migration, the human immunodeficiency virus (HIV) coinfection and broad spread of aggressive and resistant new strains since the80s of last century. Globally, about2billion people are infected with MTB which including more than20million active pulmonary TB patients,8to10million develop active disease and2million die from TB every year. Bovine tuberculosis has not only affected the healthy development of the cattle industry seriously, but also may cause some human TB occurrence. TB are increasingly becoming a serious worldwide public health problem and attracting the international community’s attention as the disease contributes considerably to illness and death burden around the world.The global control of TB requires effective vaccines and reagents for specific diagnosis. Mycobacterium bovis bacillus Calmette-Guerin (BCG) as the only vaccine currently available for humans has proven efficacy against childhood TB and disseminated TB. However, the efficacy of BCG against pulmonary TB in adults has varied between0and80%, and furthermore, vaccination with BCG cannot be distinguished from infection with MTB. Thus, there is a need to identify MTB specific antigens to develop new protective vaccines and specific diagnostic reagents against TB. Comparative genome analysis provided lots of information with antigens specific to MTB, immunological evaluation of which was predicted to identify antigens of MTB important for developing specific diagnostic reagents and vaccines to control TB. In this study, we expressed and purified7different kinds of antigenic proteins from MTB through the prokaryotic expression system, and immunologic characteristics of5among them were analyzed by animal experiment. Meanwhile, their potential capacity in the detection of bovine tuberculosis was evaluated through used as stimulants in an IFN-y test as well as coated antigens in an indirect ELISA assay.1. Expression and identification of MTB antigenic proteinsThe coding genes of6target proteins, including MPT64, MPT63, MPT83, CFP32, MPT51and MPT53were cloned by PCR amplification and sequencing analysis, and then the recombinant prokaryotic expression plasmids based on pET30a were constructed and transformed into E. coli BL21(DE3). After induced by IPTG, these6fusion proteins as well as ESAT-6-CFP10were purified using affinity chromatography assay. Furthermore, the concentration and purity of the furified fusion proteins were determined, and their immunoreactivity was analyzed in western blotting assay.SDS-PAGE analysis demonstrated that all7fusion proteins were successfully expressed mainly in soluble form after induced by IPTG, which accounts for30%of total supernatant proteins (34.8%,32.7%,27.4%,27.8%,30.6%, and31.5%for each protein respectively), meanwhile, gardation analysis showed a high purity more than90%(92.7%,95.6%,96.6%,91.8%and92.3%for each protein) except for MPT53protein (69.8%). Results of western blotting assay which only one specific band appeared indicated that the purified proteins could well react with polyclonal antibodies and proved their efficacious immunoreactivity.2. Cellular immunological characteristics of these five expressed fusion proteinsC57BL/6mice were administered subcutaneously with MPT64, MPT63, MPT83, CFP32and ESAT-CFP10fusion protein, respectively, and the splenocytes were collected and analyzed from these mice after14days of second injection in two-week intervels. FACS results of these splenocytes labeled with PE-CD4/CD69-FITC and APC-CD8a/CD69-FITC showed that all the five proteins can significantly up-regulated CD69molecule expression on both CD4+and CD8+T cell subgroups, MPT63and ESAT-6-CFP10proteins had higher capacity to stimulate CD4+T cell to express CD69than other proteins. An ELISPOT assay was used to detect the specific IFN-y-secreting cells and IL-4-secreting cells which were activated by the fusion proeins used as stimuli, respectively. The results indicated that ESAT-6-CFP10and MPT63proteins have the strongest capacity of inducing IFN-y produce, while ESAT-6-CFP10and CFP32proteins induced the highest IL-4. In contrast, the protein MPT64had the lowest capcity to induce both IFN-y and IL-4. Overall, the results showed that both ESAT-6-CFP10and MPT63proteins can induce higher level of IFN-y than IL-4and biased toward Thl response while the MPT83and MPT64proteins induced a higher level of IL-4than IFN-y and showed the tendency to Th2response. However, the specific immune response induced by protein CFP32showed the balanced Thl and Th2responses, which registered as the frequency of IFN-y-secreting cells was close to that of IL-4-secreting cells. A sandwich ELISA assay was also used to detect the concentration of IFN-y and IL-4in cell culture supernatant after stimulated with the same five fusion proteins, respectively, and the results were consistant with those of the ELISPOT assay.3. Potential application of five fusion proteins in the detection of bovine tuberculosisIn a stimulational experiment, fusion proteins of MPT64, MPT63, MPT83, CFP32and ESAT-CFP10were used as stimuli as well as Bovine PPD and Avian PPD to stimulate peripheral blood from the vena caudalis of milk cow, and then detected the level of IFN-y in the culture supernatant by BOVIGAM Gamma Interferon Test Kit. Results of average value and linear regression analysis indicated that the fusion protein ESAT-6-CFP10had the highest interrelational tendency to Bovine PPD used as stimulant while MPT63protein showed the secondary high interrelation. When0.280was defined as the cut-off value of positive reaction stimulated by those fusion proteins, the level of positive compliance rate with that of IFN-y test decreased sequentially from ESAT-6-CFP10, MPT63, MPT83and MPT64to CFP32.The serodiagnosis potential applications of the five proteins in the detection of bovine tuberculosis were evaluated. An indirect ELISA assay was established using those fusion proteins as coating antigen for the detection of their specific antibodies in bovine sera. The positive rates were8.40%(20/238) in MPT64,9.66%(23/238) in MPT63,17.65%(42/238) in MPT83,11.34%(27/238) in CFP32and2.52%(6/238) in ESAT-6-CFP10, respectively. The higher positive rate of MPT83and CFP32protein indicated the higher sensitivity among these 5proteins. Furthermore, all5proteins’indirect ELISA results had a negative coincidence rate more than90%compared to the results of IFN-y test (96.84%in MPT64,94.68%in MPT63,90.43%in MPT83,90.43%in CFP32and100%in ESAT-6-CFP10, respectively), which indicated that these five proteins had a high specificity as a supplementary detected assay in serodiagnosis of bovine tuberculosis.