Research on the high expression of Rv3881c in E.coli and the antigenecity

Objective The inclusion of Rv3881c in a prototype serodiagnostic test will highly increase assay sensitivity and speciality for M. tuberculosis infection when it is combined with other known immunodominant antigens. To obtain purified recombinant Rv3881c antigen, Rv3881c gene was cloned and inserted into prokaryotic expressing vector pET24b, then efficiently expressed in BL21. Method Primers for Rv3881c gene was designed. DNA fragment encoding Rv3881c antigen was obtained by PCR frozen mycobacterium tuberculosis Genomic DNA and cloned into pGEM-T vectors, then sequenced. DNA fragments encoding Rv3881c were inserted into prokaryotic expressing vector pET24b. The pET24b-48 plasmids were transfered into BL21, then cultured and induced by IPTG to obtain fusion proteins. Recombinant fusion protein with a 6×His tag in the C terminus, is purified by Ni-chelating Sepharose Fast Flow reagent. Antigenicity of rRv3881c was simply evaluated by Western blot through the reactivity with sera from 6 M.tuberculosis patients. Result Rv3881c was sequenced and proved to be correct. The amount of fusion proteins contained Rv3881c antigen and His-tag expressed in BL21 occupy more than 20% of total bacterial proteins. Purified protein can react strongly with 6 serum samples from M.tb patients, but not with serum samlpes from healthy control. Conclusion The results indicate that this kind of recombinant antigen show high speciality to sera of M. tuberclosis patients and might be a candidate of diagnostic antigens.