Detection Of Deer Tuberculosis By PCR Technique And The Cloning And Expression Of HSP65 Gene

Deer tuberculosis is a sort of bacterial infection between human and animal, it happens in all parts of the world. In order to confirm the incidence of deer-tuberculosis in china, tuberculosis was detected with PCR in 79 samples of deer whole blood form one area. 43.0%of samples were positive, including Mycobacterium tuberculosis, indicating an overspread of tuberculosis existed in deer.To effectually defend and treat Deer-tuberculosis, the gene encoding for protein HSP65 was amplified form Deer-tuberculosis strain chromamal DNA by PCR technique. The PCR product and the PMD18-T were connected by ligase, it was transformed into host strain E.coli DH5a. The Hsp65 was completely identical to GenBank of Hsp65 by sequencing. The PMD18-T-Hsp65 and the prokaryotic expression vector pGEX-6p-1 were digested by the restriction enzyme of EcoR I and SaL I. The recombinant expression plasimids were constructed by T4 ligase and transformed into host strain E.coli DH5α. The Hsp65 was completely identical to GenBank of Hsp65 by sequencing and had the correct open reading frame. The recombinant expression plasimids pGEX-HSP65 was successfully constructed.The right recombinant plastids were transformed into E.Coli BL21 strain. Bacterial lysatrial prepared from lmmol/IPDG induced cultures were loaded directly SDS-PAGE. The result shows that the MW of 86kDa is equivalent to the summation of Hsp60kDa and GST26kDa, it amounted to 30.7% of the total protein of E.coli BL21 by thin-layer scan. The fusion protein GST-Hsp65 was mensurated by Westem-blot and there is special band in 86kDa. The sera from Deer-tuberculosis could specially recognize the expressed protein. The implie the expressed protein showed the high activity of antigen. It will be helpful in futher study diagnosis and therapy of tuberculosis.