Established About Methods To Detection The Secretion Protein Of The Mycobacterium Tuberculosis Its Applied Research

Currently in human and animal infectious diseases, TB remains an important threat to humanand animal health, its diagnostic technology included bacteriological diagnostic, immunologyand molecular biology diagnostic。Tuberculosis bacteriological examination remains the goldstandard in laboratory diagnosis of TB, but its smear-positive rate is low, and time-consuming islong;.Although molecular biology is rapid, sensitive, because of cumbersome operating, and thehighly technical requirements, it is difficult to use widespreadly. Immunology diagnostic valuelied in its simple, quickly and have a certain sensitivity and specificity。Therefore it iscomplementary approaches in diagnostic TB. In recent years, there has been investigated in thefiltrate about MTB, the separation of a wide variety of protein secretion, such as MPT64, MPT70,MPT63, MPT53, Ag85, CPF10, ESAT6. The CFP10, ESAT6 have good immunization, andunique, BCG and non-pathogenic MTB lacking. Thus the distinction between the two types ofprotein can Judgment whether a animal be BCG vaccinated. My paper study on PCR amplifiedthe gene encoding protein CFP10-ESAT-6 from my cobacterium tuberculosis H37RVchromosomal DNA.PCR products were cloned into pET21a and recombinantpET21a-CFP10-ESAT6 were transformed into E.coli BL21. the target proteins of the 25KD wasproduced by induction using IPTG..Western blot analysis of the specificity of recombinantproteins. Secondly we use a recombination CFP10-ESAT6 protein immunized New Zealandrabbits, through ELBA tested titer of antibody, high titer of antibody was pured by DEAE-32anion exchange pillar, part of antibody were used for coating, another part of the antibody usingenzyme-labelled for testing. Lastly we establish a double-mAb sandwich ELISA to detect culturesupernatant in the 17 species of mycobacteria and 341 clinical serum samples of patients andpreliminary assessment the feasibility of the method.Results show that we construct CFP10-ESAT-6 expression vector, and gain fusion protein ofCFP10-ESAT6, They produced good immunoreactivity with serum from tuberculosis patient bywestern blot. CFP10-ESAT-6 immunized rabbits to produce antibody with high titer, whenCFP10-ESAT6 multi-cloning antibody was diluted to 1:16000, the high titer of antibodybasically reached 1.0％at OD450, after purification and enzyme-labelled, when the dilutions ofantibody for coating and enzyme-labelled were 1:5000 and 1:3000 respectively, The minimumdetectable concentration of CFP10-ESAT-6 is 25 ng/ml。Then we used CFP10-ESAT-6 to detect17 mycobacteria culture supernatant, only mycobacterium tuberculosis and Mycobacterium bovis are positive. The specificity and sensitivity of serodiagnosis were 40.1％, 99.2％respectively.Based on successful expression of a fusion protein on the basis of CFP10-ESAT6, and gainedthe high titer of antibody afer immuned rabbit, established a double-mA sandwich ELISA, it isclinically practical to serodiagnosis of Mycobacterium tuberculosis.