Isolation And Identification Of Mycobacterium Avium And Its PhoR Gene Expression And Protein Function Studies

Objective:Nontuberculous mycobacterial (NTM) infections continue to emerge inwith the growing epidemic of AIDS/HIV patients. They have very similarclinical symptoms comparing with TB, easily lead to misdiagnosis in the earlydiagnosis, while there are so obvious difference with their TB treatmentprograms. To develop fast and accurate identification technology is importantfor early diagnosis and promptly treatment of NTM disease. Mycobacteriumavium disease is the major complication in AIDS/HIV patients. In order to betterunderstand the mechanism of the MAC survival retention in AIDS/HIV patients,15cases of experiments from AIDS/HIV patients with sputum culture positivespecimens were identified by using16S rRNA sequencing to obtainMycobacterium avium, and preliminarily to study the PhoR gene function ofMycobacterium avium.Methods:1. Isolation and identification of Mycobacterium avium In this study,15cases of experiment from AIDS/HIV patients with sputumculture positive specimens were identified by using16S rRNA sequencing.2. Prokaryotic expression of Mycobacterium avium PhoR geneSearching in the NCBI database to obtain104strains of Mycobacteriumavium PhoR gene sequence, in which both sides join BamH I and EcoR Irestriction sites were designed and synthesized primers for PCR amplification;the PCR product and pGEX-4T-3expression vector with gel recycled weredouble digested, T4ligase transformed and recombinant plasmid pGEX-4T-3-PhoR were constructed. To verify the correct sequencing of recombinantplasmid by PCR and restriction enzyme digestion. bioinformatical analysiswould be done after sequencing results been dealt with splicing and comparing,then pGEX-4T-3-PhoR recombinant plasmid was transformed into E. coli BL21(DE3), induced by IPTG, SDS-PAGE electrophoresis analysis conducted aftercell disruption.3. Prokaryotic expression of PhoR gene affect recombinant bacteria to surviveunder three tolerance conditionsE. coli BL21(DE3) and E. coli BL21(DE3)containing with pGEX-4T-3plasmid, respectively tolerance threshold experiment under three conditionsabout SDS, NaNO2and H2O2to determine their maximum tolerated threshold.The IPTG induced culture of recombinant bacteria BL/pGEX-4T-3-PhoR andBL/pGEX-4T-3for2h, cultured in LB was adjusted to OD600after0.8, E. colirespectively in0.4%SDS,7mmol/L NaNO2and1mmol/L H2O2survivaltolerated under the influence experimental conditions. Results:1. The isolation and identification of Mycobacterium aviumThe results showed that there were9cases of Mycobacterium tuberculosis,4cases of Mycobacterium avium and2cases of abscesses mycobacteria in these15cases. Sequencing results showed four identical sequences of496bp length.NCBI BLAST comparison found that the104strains of Mycobacterium aviumwith16S rRNA sequence essentially the same, there is only one base differencein the first81sequence judged as Mycobacterium avium.2. Prokaryotic expression of Mycobacterium avium PhoR geneTemperature gradient PCR amplified by using PhoR gene, its actual sizewas approximately1500bp, and basically was consistent with the expected size.After sequencing correctly for recombinant plasmid pGEX-4T-3-PhoR,prokaryotic expression would been done later. SDS-PAGE results showedcontrol group, appears clear protein band with the expected GST-tagged proteinband (26.8kD) closely. The size of the fusion protein expression in theexperimental group was about85kD, slightly larger than the expected proteinsize77.4kD.3. PhoR prokaryotic expression of recombinant bacteria surviving under thethree conditions affect tolerance(1)At0.4%SDS tolerance condition, the bacterial growth of theexperimental group BL/pGEX-4T-3-PhoR as control group BL/pGEX-4T-3, areunable to survive.(2)At7mmol/L NaNO2tolerance condition, the experimental groupBL/pGEX-4T-3-PhoR bacterium could continue to grow, and the control groupcannot survive. (3)At1mmol/L H2O2tolerance condition, the experimental groupBL/pGEX-4T-3-PhoR still grows, and the control group basically cannot survive.Conclusions:(1)The15cases of AIDS/HIV patients with culture-positive sputumsamples successfully isolated and identified， and obtain a strain ofMycobacterium avium104.(2)Successfully constructed the pGEX-4T-3-PhoR recombinant plasmid.(3)PhoR gene expression in E. coli under the condition of SDS maximumtolerance threshold tolerated no effect on bacterial survival.(4)PhoR gene expression contributes to the survival of E. coli in NaNO2and H2O2maximum threshold tolerance conditions.