| Tuberculosis (TB) is a major infectious disease problem with one-third of the world population infected, 8 million people developing the active disease and 2 million dying of TB each year. Our country is one of 22 high-load countries, and the number of TB patients rank second in the world. According to epidemic data of Tuberculosis of our country in 2000, 4 billion people were infected, 5000 thousand get TB per year, and it kills 150 thousand per year. It has influenced people very seriously.Genes of expressed in vivo of pathogen are thought that have a most important role in pathogenic course, which are called in vivo induced gene (rvi gene).It is very possible that these genes will give the new targets in developing vaccine, diagnosis reagent and drug. In vivo-induced antigen technology (IVIAT), brought forward in the past two years, is a new method study the induced genes in vivo with the sera from patients. Compared to the former methods, it is more accurate and convenient.Up to now, there have not been any literatures reported the study on in vivo induced genes of M. Tuberculosis. In this study, IVIAT was employed to find a few ivi genes of M. Tuberculosis and to analyze their functions, and further more, to understand its molecular pathogenic mechanism, and to find a few clues for the development of new vaccine, diagnosis reagent and drug.Methods:1 .Construction of a genomic library of M. Tuberculosis H37Rv: pKK223-8 expressionvector was cleaved by BamH I ,then the linear vectors were obtained and de-phosphatized by CIAP. The H37Rv DNA were cleaved by Sau3A I ,1-4kb DNA fragment was recovered. Then they were ligated into the prepared pKK223-8 expression vectors, the recombinants were transformed into E. coli DH5a.At the end, the library was appraised.2.Screen of the genomic library of M Tuberculosis: sera were pooled from confirmed TB patients who were sputum-positive, and these sera were absorbed with the lysate of H37Rv M Tuberculosis grown in vitro and the lysate of E. coli DH5a. The library was screened with in situ immunochemistry.3.Sequence analysis of the genes of in vivo-induced antigen: Extract plasmid from positive clones, design the and synthesize primers, sequence the foreign DNA fragments, do BLAST on the GenBank.Results:1 .The genomic expression library of M. Tuberculosis have been constructed, its capacity was 1.02×104 clones, which was enough for this study and paved a firm way for the screen the library.2. The sera were absorbed with the lysate of E. coli DH5a and M. Tuberculosis H37Rv grown in vitro. The expression library was screened with these absorbed sera, almost 7000 clones have been screened and get 28 positive clone.3. Sequenced the foreign DNA fragments of these positive clones and did BLAST on the GenBank, found 15 possible genes of in vivo induced antigen. These genes are divided into 5 classes: lipid metabolism, metabolic intermediate, cell walk hypothesis protein, structure protein. Through identification, they were regarded as the in vivo induced genes of M. Tuberculosis H37Rv.Conclusions:1. In China, with IVIAT, we firstly got 15 possible genes of in vivo induced antigen in M. Tuberculosis H37Rv, and paved a way for the further analysis of their functions and provided the new idea for study on controlling Tuberculosis:2. Combined the routine methods applied to immunology and molecular biology, IVIAT is a rapid and convenient new way which can be used to study the in vivo induced genes of pathogens.
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