Construction Of Subtracted CDNA Library By Suppression Subtracted Hybridization For Differentially Expressed Genes In Mycobacterium Tuberculosis

ObjectiveTo screen differential expressed genes between Mycobacterium tuberculosis H37Rv and H37Ra, analyze their functions and construct two cDNA libraries using suppression subtractive hybridization (SSH) in order to construct a new type of Mycobacterium tuberculosis mucosa vaccine.The study includes five parts:PartⅠ: Mycobacterium tuberculosis culturesPartⅡ: Mycobacterium tuberculosis RNA extraction and purificationPartⅢ: Suppression subtractive hybridizationPartⅣ: Purified and clone and identified productions of subtractive hybridizationPartⅤ: Hybrid product of sequencing and bioinformatics analysis and conduct sequence analysis Methods1: Use biomerieux BacT ALERT R○MP system to culture Mycobacterium tuberculosis H37Rv and H37Ra.2: According to Trizol and PCR production purification Kit instructions, Extract and purity the RNA productions of Mycobacterium tuberculosis H37Rv and H37Ra.3: Use suppression subtractive hybridization (SSH) to clone the differential genomic genes between Mycobacterium tuberculosis virulence strain H37Rv and attenuated strain H37Ra. After two times of subtractive hybridization and two times of PCR, the products of last PCR amplification were inserted into pGEM-T Easy vector and be transformed into the E.coli DH5αand screened of blue and white clones of the transformants, the subtracted cDNA library of differentially expressed genes identified by RT-PCR.4: Choose these genes including masculine colonies and analyze their sequences. As soon as,we use Blast software to compare their homology and functions.ResultThe cDNA libraries of A and B of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed. 90% of the colonies were white colonies, the single band of the colonies of A library was 75% and B library was 80%. Eleven high or specifically expressed genes had been obtained by SSH in H37Rv. All of them, One is the fragment of a gene coding PPE family protein, one is the fragment of a gene coding virulence factor mce, six gene fragments code 6 kDa early secretary antigenic target (ESAT-6) and isoleucyl-tRNA synthetase and methoxy mycolic acid synthase and ribonucleoside-diphosphate reductase and probable isocitrate dehydrogenase respectively, one codes membrane protein, one for leucy-tRNA synthethetase, one codes short-chain type dehydrogenase/reductase, the last one is hypothetical protein. EAST-6 and PPE family protein are Mycobacterium tuberculosis protective antigens. ConclusionDifferential expressed genes between H37Rv and H37Ra were identified using SSH technique. SSH may used in comparing differential gene expressions between the strong toxin micro-organism and the weak toxin micro-organism and search the pathopoiesis related genes and specific gene fragments. The cDNA libraries of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed by using SSH technique, which lays a solid foundation for screening and cloning new specific differentially expressed genes in them. The method is feasible and reduplicative.