Cloning and Sequence Analysis of Mycobacterium paratuberculosis hsp65 gene

The genomic DNA was extracted from Mycobacterium paratuberculosis(MP) strain C-2.The secreted protein hsp65 gene was amplified with a pair of specific primers using polymerase chain reaction(PCR).PCR product was anapproximate 1 626 bp DNA segment.The clone vector pGEM-T-hsp65 was constructed successfully by the PCR product that was cloned into pGEM-T vector using T-A clone technique, and the recombinant clone was identified by using complementation test,plasmid size test,restrictional enzyme assay,plasmid PCR identification and recombinant plasmid sequence analysis.The analysis indicated that the hsp65 gene was very conservative in MP.These results could serve as a basis for further study on the usefulness of hsp65 gene and immunogenicity characteristic of hsp65 gene expression product.