Prokaryotic Expression Of Mycobacterium Tuberculosis RD2Antigenic Proteins And Their Immunologic Characteristics And Protential Application In The Detection Of Bovine Tuberculosis

TB (Tuberculosis, TB) caused morbidity and mortality in both humans and animals, and resulted in major economic loss worldwide. Early accurate diagnosis and timely treatment of patients with active tuberculosis, are the most important measures to reduce TB morbidity and mortality. More than95%of TB infection is latent tuberculosis infection (LTBI). In some cases, like HIV infection, suffering from autoimmune diseases or other conditions which result in poor immunity of human beings, LTBI would futher develop into active TB and which is a major source of infection. TB infection detected in timely is critical for the TB control, especially in low-incidence areas. Currently, tuberculin skin test (TST) is the most widely used diagnostic method. Mainly purified protein derivative (PPD) or Old tuberculin (OT) was injected subcutaneous. The specificity of the diagnostic method is poor, especially the high false positive rate. Therefore, there is an urgent need for researchers to develop a novel method with both specificity and easily application. In recent years, the research progress of mycobacterium tuberculosis (MTB) specific antigen diagnostic value was very noticeable, for a large number of specific antigens were provided by comparative genomics. More than a dozen specific areas (RD) were found. In this study, five of RD2encoded antigen proteins were selected for the prokaryotic expression and purification. The immunological characteristics of all the five antigens were preliminary analysed, four of them were evaluated in the diagnost is of bovine tuberculosis.1. Expression, purification and identification of5different antigenic proteins from RD2of mycobacterium tuberculosisTo obtain5target proteins:the coding genes of rv1981.rv1983、rv1985、rv1986and rv1988were cloned and idenfified, then transformed into the prokaryotic expression vector pEASY-E1、 pET-30a(+) and pET-32a(+) after sequencing analysis. The recombinant plasmids were identified and transformed into E. coli DE3, and the positive clones were screened and induced by IPTG The expression fusion proteins were purified by affinity chromatography technique,and identified by SDS-PAGE and Western blotting assay. The result of SDS-PAGE demonstrated that all fusion proteins were successfully expresse.Western blotting assay showed that the fusion purified proteins could well react with specific polyclonal antibodies and indicated their good immunoreactivity.2. Analysis of immunogenicity of the fusion proteins and their potential application in the diagnosis of bovine tuberculosisC57BL/6mice were immunized subcutaneouly with Rv1981、Rv1983、Rv1985and Rv1986fusion protein respectively, and the splenocytes were collected from these mice after14days of second immunization in two-week intervel. An ELISA assay was used to detect the specific IFN-y-secreting cells、IL-4-secreting cells and IL-2-secreting cells which were activated by the fusion proteins used as stimuli respectively. The results indicated that Rv1985protein had the strongest capacity of inducing IFN-y produce while Rv1981protein showed the highest IL-4-secreting cell induced compared to the protein of Rv1986had capacity of inducing both in IFN-γ and IL-4.The serodiagnosis potential for bovine tuberculosis of Rv1981、Rv1983、Rv1985and Rv1986proteins were further evaluated through the detection of specific antibodies in clinical cattle plasma supernatant by an indirect ELISA assay. The positive rates were22%(15/68) for Rv1981、35.3%(24/68) for Rv1983、36.7%(25/68) for Rv1985and32.4%(22/68) for Rv1986, repectively which indicated that these proteins had a high specificity and low sensitivity relatively in serodiagnosis of bovine tuberculosis. In another experiment, these4fusion proteins were used as stimuli to stimulate peripheral blood from milk cow vena caudalis and detected the produce of IFN-y by BOVIGAM Gamma Interferon Test Kit, a positive coincidence rate was20%、30%、50%and35%respectively compared to the IFN-y test results.All the results suggested that these fusion proteins have the potential use as antigens for the diagnosis of bovine tuberculosis.