Expression Of Mycobacterium Tuberculosis FurB And Preparation Of Its Monoclonal Antibodies

Tuberculosis (TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). TB remains an important global public health problem because there are 8 million new cases and 3 million deaths annually. In recent years, factors such as increasing frequency of drug-resistance MTB, the unsuccessful protection of BCG, incidence of HIV-associated tuberculosis and increasement of population movement, have aggravated further the opportunity of people worldwide face to the threat of TB.The growth and pathogenicity of MTB are relative with many metal elements and iron is one of them, which are necessary to keep the normal metabolism of MTB. The metal ions are mainly as coenzyme or activators to take part in the redox reactions in the growing of MTB. Restricton of iron can delay the growth of MTB and iron can be an index for expression levels of some virulence genes. There are four kinds of iron-depending regulators inMTB genome and they belong to two families respectively, Fur and DtxR. The genes of furA and furB encode the proteins of Fur family while IdeR and SirR encode the proteins of DtxR family. furA is located at the upstream of katG and participates in the modification of the expression of katG, which is closely related with the resistence of isoniazid, but the function of furB remains unknown. The analysis of gene sequence showed that there is high homogeneity between the gene of furB in MTB and fur in E.coli. So it was deduced that FurB has similar function with Fur in E.coli. FurB may be regulats expression of many genes including the virulent gene in MTB. Therefore, to study the gene furB and its expressed products FurB in MTB is very important in clarifying the mechanism of drug-resistence and virulent factors of MTB.In this study, the gene furB was amplified by PCR from the genome of M. tuberculosis H37Rv strain. Then furB was cloned into pGEM-T-easy vector and the sequence of furB was confirmed. Then the prokaryotic expression vector pQE-80L-furB was constructed and transinfected into E.coli DH5 α . Induced by IPTG, FurB was successfully expressed in fusion form. There are two forms of FurB protein, one is soluble form and the other is inclusion form but the latter is much more than the former. The result of SDS-PAGE was analysised and found that FurB was about 20% of the total proteins. FurB was purified by the Ni-NTA affinity chromatography and detected by Western blot with the His6-specific MAb. The result suggests that the fusion was what we wanted. After that, the purified protein was gently renaturelized in PBS with urea.