Screening Of Serum Markers For Drug Resistant Tuberculosis And Cloning,Expression And Purification Of Rv0350 Protein

Object: To screen the serological specific biomarker of drug-resistance Mycobacterium tuberculosis and cloning,expression and purification of Rv0350 in vitro Method: We collected serums samples from patients with tuberculosis and normal controls in Affiliated hospital Zunyi medical college from 2011 to 2015,which were hybridized with protein chips of Mycobacterium Tuberculosis.Small protein-chips were made of the specific proteins that were screened by the first hybridization and preliminary experiment.We collected serum samples again,which were hybridized with small protein-chips and the candidate markers for drug resistant tuberculosis were screened out.To design propose gene primers,amplify propose gene fragment and fuse it to pET-28 a construction recombination plasmids,which were transformed to BL21cell(DE3).To induce propose protein by IPTG in vitro,the recombination protein was purified by Ni-Affinity Chromatography.Result: 100 specific proteins were screened out by primary screening,8 specific proteins for drug resistant tuberculosis were detected by the second hybridization,including Rv0363 c,Rv2396,Rv1100,Rv3509 c,Rv3653,Rv0350,Rv0865 and Rv2031 c.The specificity and sensitivity of combined diagnosis were 80.4% and 80%,respectively.We had applied for the national invention patent(application number CN201610089177.2).Rv0350 was cloned,expressed and purified in vitro.Conclusion: We succeeded in screening out 8 candidate proteins which were specific antigen for drug resistance tuberculosis.Among which Rv0350 has higher specificity and sensitivity.We successfully cloned,expressed and purified Rv0350 protein via molecular biological technique,which laid the foundation for developing detection kit for drug resistance tuberculosis.