Clonging And Function Preliminary Analysis Of Two Component System Phop Gene From Mycobacterium Avium

Objective:Two-component regulatory signal transduction systems are widespread in bacteria, controlling virulence very important in pathogenic bacteria.This system can respond to a variety of in vivo and vitro microenvironment changes, regulating the expression of genes in pathopoiesis process during pathogen infection of the host. PhoP is a reaction regulator of the PhoPR two component system in Mycobacterium avium, it can control a range of regulation of target gene transcription., affecting the mycobacterial virulence and ability to survive in the host macrophage cells. Mycobacterium avium are intracellular pathogens,they can often spread and infect patients with the last stages of AIDS. To reveal the pathogenesis mechanism of Mycobacterium avium, we have cloned the PhoP gene of the two-component system in Mycobacterium avium and have preliminary study the regulation of target gene promoters mechanism of PhoP gene. Lay the foundation for elucidating the mechanism of Mycobacterium avium in macrophage survival and future potential drug targets to Treatment of Mycobacterium avium disease.Methods:1. According to M. avium PhoP gene sequences designed a pair of primers, Laboratory saved Mycobacterium avium DNA as template. PhoP gene sequencing of full-length was amplified by PCR and T vector link before Sequence Analysis.Analysis the PhoP gene and DNA binding domain sequence.2.According to PhoP DNA binding domain sequence designe primers to amplify PhoP DNA binding domain gene sequence of Mycobacterium avium by PCR and expression vector pGEX-4T-3 after the connection was transformed into E. coli BL21 for expression, use the SDS-PAGE for recombinant proteins analyzed. Use Glutathione Agarose for recombinant protein purified, Preparation of GST-PhoP DNA binding domain protein.3.According to M. avium PhoP gene promoter sequence, downstream genes MAV-0127, PhoU, Amt promoter sequence designe primers, By PCR amplification of gene promoter fragment （270bp）. Use （EMSA） promoter sequence to analysis the combination of bGST-PhoP and PhoP, MAV₀127, PhoU, Amt binding domain.Results:1.The homology of PhoP gene and amino acid sequence between Mycobacterium avium and Mycobacterium tuberculosis H37Rv were 86% and 94%. The 212 amino acid residues of Mycobacterium avium PhoP is serine residue, the same as virulent strain of Mycobacterium tuberculosis H37Rv.2.According to EMSA, Mycobacterium avium PhoP protein can bind PhoP, MAV₀127 and Amt gene promoter, can not bind with PhoU. In this study,Mycobacterium avium two componet system PhoP regulation of transcription mechanism do a preliminary study.Conclusion:1. Showing the clinical isolates of Mycobacterium avium and Mycobacterium tuberculosis have close affinities, and indicating that the function of PhoP in them is the same.2.1n EMSA of study have showed PhoP protein not only regulated downstream of MAV₀127 and amt gene for transcription level, but also regulate the transcription of its own gene. EMSA also shows, Mycobacterium avium PhoP protein unable to bind PhoU, showing PhoP can not regulated other two component system PhoU。...