Study On Proteins Encoded By The Regions Of Deletion Genes Of Vaccine Strain Of Bcg

Tuberculosis (TB) is a chronic disease caused by Mycobacterium Tuberculosis (MTB), Since the mid 1980s,tuberculosis has shown an increasingly upward trend,therefore new diagnostic antigens are needed so as quickly diagnose the patients infected with tuberculosis and treat them timely.H37Rv Mycobacterium tuberculosis and BCG genome are compared by using DNA microarray technology,and many different fragments in the two genomes are found out.These fragments are named RD1-16 .In addition,it turns out that 12 RDs are missing in all the BCG. To Study these proteins coded by the RDs,it is likely that new and valuable specificity of diagnostic reagents and antigens which can used as a new vaccine will be found.In this paper,Rv3874,Rv3875 of RD1 region,Rv0222 of RD4 region,and Rv3615c area of non-RD are studied. Rv3874 and Rv3875 can induce cellular immune responses and humoral immune responses, They exist only in pathogenic mycobacteria,while BCG and other non-pathogenic mycobacteria lack these two genes.Thus,they have become the focus in the researches in TB diagnosis and prevention.It is reported in foreign study that Rv0222 has higher level of specificity and sensitivity than the other antigens. It is also reported that Rv3615c can lead to a strong level of IFN-γresponse in the cell-mediated immunity,but as to the humoral immunity limited is known. Due to the deletion of regions and strains,the appearance of the same antigen is different.Therefore,it is very necessary to choose potential reagents of specific diagnosis from the encoded proteins by BCG lacking genes,and to verify whether antigens of RD area are more immunogenic compared with non-RD region .In this way,tuberculosis serum protein antigen detection technology will truly become an important clinical rapid auxiliary test means.In conclusion,genes including Rv0222, Rv3615c, Rv3874, Rv3875 are amplified respectively and the optimal condition of the polymerase chain reaction (PCR) is determined.Then the vector pET32a (+) is connected with,recombinant plasmidis are expressed in BL21(DE3)after identificationes and recombinant proteins of M. tuberculosis are purified by affinity chromatograph.What is more,the optimal parameters of induction and purification are obtained. The most suitable expression conditions for recombinant protein pET32a-0222,pET32a-3615c,pET32a-3874,pET32a-3875 are as follows: 1.0mmol / L of IPTG,37℃, induced 5h; 1.0mmol / L of IPTG,20℃, induced 6h; 1.0mmol / L of IPTG, 37℃,induced 3h; 1.0mmol/L of IPTG,37℃, induced 4h respectively. Through the Bradford test,the final concentrations of pET32a-0222, pET32a-3615c, pET32a-3874 proteins are determined at 0.518 mg/mL,0.468mg/mL and 1.697mg/mL.All of those proteins pave the way for further design of specific diagnostic antigen,and eventually establish a foundation of the screen antigens for the diagnosis of tuberculosis.