The Prokaryotic Expression And Application Of CFP-10 And Rv2626c Proteins Of Mycobacterium Tuberculosis

Tuberculosis is a major health problem throughout the world, resulting in about 3 million deaths annually. This contagious disease, though preventable, has had an increased incidence in recent years, mainly due to its association with human immunodeficiency virus disease and also due to the occurrence of multidrug resistance.Accordingly early diagnosis of tuberculosis (TB) accurately and rapidly is one of the key methods in curbing the epidemic.In order to evaluate the potential of the antigens in serodiagnosis of TB, we have undertaken cloning and expression of a large number of CFP-10 genes and Rv2626c genes in Escherichia coli and purification products.DNA encoding CFP-10 and Rv2626c antigens were separately amplified by PCR using primers which were designed in accordance with the reported CFP-10 and Rv2626c gene sequences by Primer Premier 5.0. The CFP-10 and Rv2626c genes were respectively inserted into the prokaryotic expression vector pET-30a with the N-terminal 6His-tag. The recombinant protein vectors pET-30a-CFP-10 and pET-30a-Rv2626c were respectively transformed into BL21(DE3) cells via chemical transformation. The positive clone was screened by means of PCR. The two target proteins were expressed in E. coli after induction with IPTG. The highest amount was reached at 37℃, after 4 hours. The best IPTG concentration for recombinant CFP-10 protein expression was 2mmol/L and for recombinant Rv2626c protein expression was 1.5mmol/L. The solubility analysis showed that the two recombinant proteins existed as soluble protein in the E. coli. The two recombinant proteins were purified by affinity chromatography.Early detection of tuberculosis was very important to curtail the spread of TB infection. Serodiagnosis was one of the methods of the diagnosis of TB. It was shown that the use of recombinant M.tuberculosis specific antigens may enhance the specificity and sensitivity of serodiagnosis when used in a panel of recombinant antigens. ELISA results shows the sensitivity and specificity of recombinant CFP-10 protein combinated with recombinant Rv2626c protein were 77.1% and 32.3% respectively.Taken together, our results indicated that the CFP-10 protein combinated with Rv2626c protein was a potential promising candidate as diagnostic reagent for the early detection of TB infection.