Cloning And Prokaryotic Expression Of Rv3407 Gene Of Mycobacterium Tuberculosis

Tuberculosis(TB)is a chronic respiratory infectious disease caused by pathogen Mycobacterium tuberculosis(MTB).In recent years,factors such as the unsuccessful protection of BCG,increasing frequency of drug-resistance MTB,incidence of HIV-associated tuberculosis and increasement of population movement,have aggravated further the opportunity of people worldwide face to the threat of TB.Currently. MTB has infected approximately two billion individuals in total.TB remains one of the great killers,causing between 2 and 3 million deaths,and an estimated more than 8 million new infections a year.There are two main strategies for reducing the transmission rate:(1)rapid diagnosis and promptly treatment of active TB;(2)preventing TB occurrence with an effective vaccine.There fore,it is very important to develop new vaccine,or enhance BCG effection and diagnosis reagent for finding TB patient timely and specially.For the past ten years,people's has conducted the extensive research to bacillus tuberculosis nutrient fluid proteinand has obtained the inspiring result.The tubercidin bacillus growth period secretion's protein to tuberculosis's protection immunity is very important.The domestic and foreign research material indicated that the tubercidin bacillus Rv3407 protein is highly conservative has the conservative sequence hypothesis protein(Conserved hypothetical protein),is the bacillus tuberculosis vegetal period secretion protein,(99 aa) is composed of 300 bp,the code molecular is 10kDa,has the cell and humoral immunity activeness,domestic has not seen its structure and the function aspect research,research to it,may provide certain experiment basis for the tuberculosis vaccine development.This research aims to study foreign scholars on the basis of the introduction of modern molecular biology techniques to Mycobacterium Determination of Rv3407 gene sequence and expression in Escherichia coli as the main research objective is to study the molecular people Mycobacterium biological characteristics, In order to develop an effective set of DNA-based vaccines.In this study,Mycobacterium tuberculosis Rv3407 gene was amplified from M.tuberculosis H₃₇Rv genomic DNA using PCR and use pGEM-T vector system successfully cloned human Mycobacterium Rv3407 protein genes,the identification and digested PCR,confirmed the the cloned gene consistent with the size of the target gene.Sequencing and DNASTAR through analysis found that the cloned Rv3407 protein gene sequence with Genbank publishes of Human Mycobacterium H₃₇Rv strain Rv3407 protein gene sequence homology of 100 percent,exactly the same amino acid sequence also showed that the gene in Mycobacterium is highly conserved.Further cloned in the pGEM-T-Rv3407 protein gene was subcloned into pGEX-4T-1 expression system to construct a highly efficient prokaryotic expression plasmid,in which the expression plasmid match E.coliBL21(DE 3)by IPTG induction 4 h,the expression level reached a peak,so as to further research to it,human genetic engineering Mycobacterium subunit vaccine and DNA vaccine for a certain foundation.