Screening Of Specific Biomarkers For Active Tuberculosis,and Cloning,Expression And Purification Of Rv3480c From Mycobacterium Tuberculosis

Objective: To screen specific biomarkers of active tuberculosis through proteome microarray.Targeted protein was further cloned,expressed and purified from Mycobacterium tuberculosis.Methods: Healthy individuals serum,latent tuberculosis infection serum and active tuberculosis serum were reacted with M.tuberculosis bacterial proteins immobilized on the chip.After washing three times in TBST,arrays were probed with cy5-donkey-anti-mouse antibody and Cy3-strepavadin antibody,arrays were dried in a Slide Wash(Capital Bio)and then scanned in a Gene Pix 4200 A slider scanner.We designed primers for full-length using Primer 5.0,PCR amplification was carried out using a Phusion High-Fidelity PCR Kit,and the purity and identity of the PCR products was verified by gel electrophoresis.PCR products were subjected to connect with p ET28 a as the destination vector,then transformed into competent cells of escherichia coli strain,further transformed into expression competent cells of E.coli BL 21.Proteins were induced expression by different concentration of IPTG,further were verified by coomassie brilliant blue staining and Western blot,purification proteins through 2 M Urea.Results:Fifteen proteins were screened through proteome microarray,including Rv3480 c Ig M?Rv1860 Ig G?Rv2352c Ig M?Rv1597 Ig M?Rv2688c Ig M?Rv0049 Ig M?MT1560Ig G?Rv2511 Ig G?Rv0350 Ig M?Rv0350 Ig G?Rv0270 Ig M?Rv1876 Ig M?Rv0494 Ig M?Rv2031c Ig G and Rv2450 c Ig M.We designed a specific proteome microarray according to the reactive extent,(this chip has applied for national invention patent,number201610089179.1).Verified the specific proteome microarray through different samples(for example,health individual,latent tuberculosis infection and active tuberculosis),the specificity and sensitivity of the proteome microarray were 90.3% and 85.4%,respectively.Mycobacterium tuberculosis protein Rv3480 c is one of the specific biomarkers of active tuberculosis.After the PCR amplification of Rv3480 c gene,electrophoresis results showed that the location of the PCR gene fragment was in agreement with the target gene.The recombinant plasmid was successfully constructed.The best condition of induced expression was 16?,0.2 m M IPTG.The protein was expressed in inclusion body.We obtained the purified protein by urea solubilization of inclusion bodies and dialyzing in low concentration urea and PBS,and western blot was further confirmed the target protein.Conclusion : We successfully screened out fifteen specific serum biomarkers for diagnosis active tuberculosis according to the specific proteome microarray.The combination of these proteins had good specificity(90.3%)and sensitivity(85.4%)for distinguishing active tuberculosis,latent infection and normal population.We successfully obtained Rv3480 c recombinant protein by gene cloning,which can lay a foundation for the study of its structure,function,new anti-TB drugs and the serological diagnosis of tuberculosis.We optimized some of the reaction conditions in the process of constructing E.coli recombinant expression vector system and protein expression and purification,which provided new idea for better purification of inclusion bodies.