| Tuberculosis which is the result of the infection by Mycobacterium tuberculosis is one of the infectious disease that causes extremely high lethality rate, the key step in preventing and controlling tuberculosis is early diagnosis and accurate pharmacy which present the urgent requirement for the early diagnosis of tuberculosis. In recent years, serodiagnosis methods are widely utilized in the early diagnosis of tuberculosis in laboratories and have brought about a series of perfect results, meanwhile, there are also sets of intractable problems in these research programs, among them, one of the core bottleneck is the failure in the discovering of a specific antigen with high efficiency, therefore, the finding of a antigen with both high sensibility and efficiency has become a hot research field in the serodiagnosis of tuberculosis.The two antigens utilized in this set of experiments were located in the divergent region between M. tuberculosis and BCG. they were firstly expressed by genetic engineering methods and then undergone the analysis of the effect. These series of methodologies could simplify the operation procedures as well as find new proteins that could not be purifed in the filtrate of M tuberculosis.In the present study. I am aimed to insert the sequence of TB15.3 and TB 16.3 into expressional plasmids, then induce them to expression in E. coli BL21(DE3) cells. The expressional products were then purified in order to acquire a large number of recombinant antigens, in the meantime, the immunoreactions of these two antigens were analyzed and defined. These set of experiments had thus paved the way for the further utilization of (?)combinant peptides in the serodiagnosis of tuberculosis in the future.By the utilization of PrimerPremier 5.0 software and according to the DNA sequences of the No proteins. I had successfully designed two pairs of primers, then by PCR amplification by using H37Rv gene as the template, the two segments were generated and then inserted into the expressional plasmids pET-30a. the two recombinant plasmids were therefore successfully constructed. The two recombinant plasmids were then transformed into the competent cells of E..coli BL21(DH3) strains, after colonal PCR screening, the positive translbrmants were selected and by IPTG inducement and SDS-PAGE analysis, the engineering strains were proved to successfully express the recombinant antigens. The gradient tests had proved that the optimum temperature for the expression was 28℃, the expressional time plateau was 4h, and the optimum concentration of IPTG was 1mM. Solubility analysis had proved that both of the recombinant peptides are expressed as soluble protein in host cells and thus could be directly purified by affinity chromatography.The combination of a multi-antigen system could apparently promote the sensibility and efficiency of the results of serodiagnosis of tuberculosis. In the present study, the two recombinant antigens TB15.3 and TB 16.3 were combined and utilized in the ELISA system, the result had suggested that the sensibility of TB15.3 was44.6%, and the specificity toward healthy sera was99%; whereas the sensibility of TB16.3 was 71.2%, and the specificity toward healthy sera was85.8%;The sensibility of the combined antigen of the two proteins was65.3%. and the specificity toward healthy sera was96.9%. These series of results had showed the potential value of the combination of antigens utilized in serodiagnosis system of tuberculosis. |
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