The Effect Of NF-κB、TNF-α、IL-6mRNA Expression In RAW264.7Macrophage By PPE37Protein Of Mycobacterium Tuberculosis

Objective After the completion of the whole genome sequencing ofMycobacterium tuberculosis, researchers found that the PE and PPE family genes accountedfor10%of the coding region of Mycobacterium tuberculosis and it’s expressed differently inpathogenic mycobacteria and non-pathogenic mycobacteria. The PPE37protein located atMycobacterium tuberculosis virulence-related RD area. The purpose of this thesis is toresearch the immunomodulatory mechanisms of PPE37protein in Mycobacteriumtuberculosis infected with human.Methods1. In order to predict the immune regulation mechanisms of PPE37proteinin the process of Mycobacterium tuberculosis infected with human,we use bioinformaticssoftware（uniprot、protparam、GOR4、SignaIP4.1、Bcepred、NetMHC3.2Server） analysisits structural features.2.According the ppe37gene sequences logged on Genebank, to design and synthesis itsprimers.Using genetic engineering techniques to build the pEasyE2-ppe37recombinant clonecarrier,after confirmed correct by PCR、 restriction analysis、gene sequencing cloned it intothe expression vector pET30a.The pET30a-ppe37recombinant expression vector is inducedby IPTG. To analysis the expression form of recombinant protein by SDS-PAGE. To analysisthe specificity of recombinant protein by Western blot. Using alpHaImager2200software toanalysis the purification rate of the recombinant protein, after purified by Ni-NTA agarose.3.To establish the mouse RAW264.7macrophage model stimulated by recombinantPPE37protein, The concentration of recombinant PPE37protein is100ng/mL、500ng/mL、5000ng/mL. After collected the cells respectively (24h,48h,72h), extract total RNA and anti-transcribed into cDNA using reverse transcription primer.Using genetic engineeringtechniques to build cytokines (β-actin、NF-κB, TNF-α, IL-6)standard plasmid and draftstandard curves. To detect the NF-κB, of TNF-α, of IL-6mRNA expression various of theabove group by Real-Time PCR.Results1. The PPE37protein has stable structure, strong hydrophilic and no signalpeptide analyzed by bioinformatics software. The online software Bcepred shows PPE37protein dominant epitopes area in B cells at191-192;288-294;332-336;376-387. The onlinesoftwareNetMHC3.2Server shows PPE37protein dominant epitopes area in Tells at0-9;144-153;156-165;239-248.These results prove PPE37proteins can be recognized by Bcell/T cell in the process of Mycobacterium tuberculosis infection with human,which playsan important immune regulatory mechanism.2.The recombinant expression vector pET30a-ppe37was constructed correctly afterconfirmed by PCR, restriction, gene sequencing.After transformed into E.coli BL21andinduced by IPTG, the recombinant protein was expressed abundantly in the form of inclusionbody proteins.The purification rate of the recombinant protein is96.2%analyzed byalpHaImager2200software after purified by Ni-NTA agarose.