Construction of subtracted cDNA library by suppression subtracted hybridization for differentially expressed genes in Mycobacterium tuberculosis

Objective:To isolate Mycobacterium tuberculosis H37Rv and H37Ra genes especially,and to construct two cDNA libraries by using suppression subtractive hybridization (SSH).Methods:We used 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 DH5α and screened of blue and white clones of the transformants.The subtracted cDNA library of differentially expressed genes were identified by RT-PCR.Results:High or especially expressed genes as tester had been obtained by SSH in correctitude reaction (H37Rv as tester) and reverse reaction (H37Ra as tester),the cDNA libraries of A and B of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed.90% of the colonies were white clones,the single band of the colonies was 75% and 80%.Conclusion:The cDNA libraries of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed using SSH technique,which lay a solid foundation for screening and cloning new specific differentially expressed genes in them.