| Objective:Helicobacter pylori is a Gram-negative microaerobic bacterium that can colonize human gastric mucosa.H.pylori infection is an important cause of gastrointestinal diseases such as chronic gastritis,peptic ulcer and gastric cancer.As a highly conserved virulence island of H.pylori,cag pathogenicity island(cag PAI)encodes the important virulence protein CagA of H.pylori and the type IV secretion system(T4SS)responsible for transport and delivery of CagA.The expression of these virulence factors encoded by cag PAI is regulated by transcriptional regulatory proteins and is closely related to the occurrence and development of digestive system diseases.The genome of H.pylori is relatively small,and the only known transcription regulatory proteins are Rpo D,Rpo N,Fli A,Hrc A,Ars R,HP0222,HP0564,Flg R,Hup,HP1021,Hsp R,Fur,Hsr A,Che Y, Nik R.However,so far there have been very few reports on the regulation of H.pylori virulence.In view of this,the team of this research group has carried out research on the regulation and mechanism of these H.pylori regulatory proteins.In this project,five of these regulatory proteins,namely HP1021,Hsr A(HP1043),Ars R(HP0166),Nik R(HP1338),and HP0222,were studied.First clone and express the five regulatory proteins,and screen for proteins that bind to the upstream promoter sequence of CagA and T4SS components.On this basis,further study the effect of this transcriptional regulatory protein on the expression of H.pylori virulence factors CagA and T4SS.It aims to reveal the regulatory factors that regulate the expression of related virulence in H.pylori,and provide clues for the development of new drug targets related to H.pylori infection.Methods:1.Construction,expression and purification of transcriptional regulatory protein recombinant plasmids1.1 Construction of transcriptional regulatory protein recombinant plasmidObtain H.pylori NCTC26695 genomic sequence through NCBI,PCR amplify the regulatory protein sequences of hp1021,hp1043,hp0166,hp1338,hp0222 genes,and connect with double-digested linearized p ASK-IBA7 plus vector fragments to construct a transcriptional regulatory protein recombinant plasmid,Transform E.coli BL21 and conduct amp resistance screening.The accuracy of transformant inserts was verified by colony PCR and DNA sequencing.1.2 Expression and purification of transcriptional regulatory proteinsThe successfully constructed recombinant plasmid was induced to express the target protein with Strep tag by tetracycline,purified by Strep-Tactin purification column and the molecular weight of the regulatory protein was verified by SDS-PAGE.2.Screening of regulatory proteins combining CagA and T4SS important gene promoters2.1 Screening of regulatory proteins combined with the CagA promoter sequenceThe 200 bp promoter sequence fragment upstream of CagA was selected,ligated to the p LB vector plasmid after PCR amplification,and amplified by PCR with a universal primer with Alexa Fluor 700 fluorescent marker to obtain the CagA promoter probe "promoter-cag A".The five expressed regulatory proteins were incubated with the CagA promoter probe at 37℃,and the regulatory proteins that could bind to the probe were screened by gel migration experiment(EMSA).2.2 HP0222 can bind T4SS important gene promoterSelect the important T4SS components upstream 200bp promoter sequence fragment,connect to p LB vector plasmid after PCR amplification,PCR amplification by universal primers with Alexa Fluor 700 fluorescent marker to obtain T4SS promoter Needles "promoter-cag1","promoter-cag U","promoter-cag Z","promoter-cag L","promoter-cag X".The regulatory protein HP0222 and T4SS promoter probe were incubated together at 37°C,and their binding ability was studied by gel migration experiment(EMSA).2.3 BLAST analysis of HP0222 sequenceNCBI sequence database protein BLAST module was used to compare the homology between HP0222(Gen Bank:AAD07296.1)and other proteins(except Helicobacter)to predict the biological function of HP0222.3.Construction of H.pylori NCTC26695Δhp02223.1 Construction of recombinant plasmid"p Bluescript SK II(-)-hp0222 upstream arm-kana ~R-hp0222 downstream arm"The hp0222 gene mutation vector was designed according to the principle of homologous recombination.PCR amplify the upstream and downstream arm fragments of the hp0222 gene,using the kanamycin resistance gene(kana ~R)as the screening resistance,the shuttle plasmid p Bluescript SK II(-)as the vector plasmid,and construct the"p Bluescript SK"by connecting the multiple fragments of the recombinase II(-)-hp0222 upstream arm-kana ~R-hp0222 downstream arm"recombinant plasmid,transformed into E.coli DH5αand screened for kana resistance.The accuracy of transformant inserts was verified by colony PCR and DNA sequencing.3.2 Construction of NCTC26695Δhp0222The successfully constructed recombinant plasmid was transformed into NCTC26695 by electroporation and cultured and screened on Columbia blood plate containing kana.Extract and verify the bacterial genomic DNA after subculture.4.The effect of HP0222 on the expression of cag PAI4.1 The effect of HP0222 on the expression of CagAUse NCTC26695 and NCTC26695Δhp0222 in the logarithmic growth phase.Bacterial RNA was extracted and the expression of cag A gene at the transcription level was detected by qPCR.The bacterial protein was extracted by ultrasonic cracking,and the protein expression of CagA was detected by Western blot.4.2 The effect of HP0222 on the expression of T4SSExtract the NCTC26695 and NCTC26695Δhp0222 bacterial RNA in the logarithmic growth phase,select the T4SS genes cag1(hp0520),cag C(hp0546),cag F(hp0543),cag M(hp0537),cag P(hp0536),cag Q(hp0535),cag S(hp0534),Cag U(hp0531),cag V(hp0530),cag L(hp0539),the expression of all T4SS operons at the transcription level was detected by qPCR.4.3 The effect of HP0222 on the expression of CagA after H.pylori infection in AGS cellsCollect NCTC26695Δhp0222 and NCTC26695 in the logarithmic growth phase,respectively,and adapt to AGS cells after 4h in serum-free cell culture medium.Extract total protein 9h after infection,and detect the CagA and phosphorylated CagA levels by Western blot.5.Statistical methodsSPSS 22.0 was used for paired sample T test for data analysis.Results:1.Construction,expression and purification of transcriptional regulatory protein recombinant plasmids1.1 Construction of transcriptional regulatory protein recombinant plasmidThe results of DNA gel electrophoresis showed that the size of the amplified fragment was the same as that of the target fragment,and the results of DNA sequencing confirmed that the target fragment had no mutation sites,and a recombinant plasmid with transcriptional regulatory protein was successfully constructed.1.2 Expression and purification of transcriptional regulatory proteinsThe results of SDS-PAGE Coomassie brilliant blue staining showed that the molecular weight of the purified protein was consistent with the size of the target protein.Five transcriptional regulatory proteins with Strep tags were successfully expressed.2.Screening of regulatory proteins combining CagA and T4SS important genepromoters2.1 Screening of regulatory proteins combined with the CagA promoter sequenceThe EMSA results showed that the regulatory protein HP1021 with known function and the regulatory protein HP0222 with unknown function both have theability to bind the upstream promoter sequence of CagA.In this project,HP0222,whose function is unknown,was selected as the research object to further study its binding ability with T4SS important gene promoter.2.2 HP0222 can bind T4SS important gene promoterEMSA results show that HP0222 has the ability to bind the promoter of T4SS important genes,suggesting that HP0222 has the role of regulating the expression of CagA and T4SS.2.3 BLAST analysis of HP0222 sequenceBLAST analysis showed that there is no homology between HP0222 and proteins other than Helicobacter.3.Construction of H.pylori NCTC26695Δhp02223.1 Construction of recombinant plasmid"p Bluescript SK II(-)-hp0222 upstreamarm-kana ~R-hp0222 downstream arm"The results of DNA gel electrophoresis verification showed that the amplified fragment had the same size as the target fragment,and the DNA sequencing results confirmed that the target fragment had no mutation site,and the transcriptionrecombinant plasmid"p Bluescript SK II(-)-hp0222 upstream arm-kana ~R-hp0222downstream arm".3.2 Construction of NCTC26695Δhp0222The results of DNA gel electrophoresis showed that the target gene hp0222 was successfully replaced by kana ~R,and the gene mutant NCTC26695Δhp0222 wassuccessfully constructed.4.The effect of HP0222 on the expression of cag PAI4.1 The effect of HP0222 on the expression of CagAThe qPCR results showed that the expression of the cag A gene at the transcription level was increased by 1.489±0.186 times compared to NCTC26695 and NCTC26695Δhp0222.Western blot results showed that compared with NCTC26695,the expression of CagA in protein level of NCTC26695Δhp0222 increased by 1.498±0.099 times.4.2 The effect of HP0222 on the expression of T4SSThe qPCR results showed that compared with NCTC26695,NCTC26695Δhp0222,the T4SS component genes cag M(hp0537),cag S(hp0534),cag V(hp0530),and cag L(hp0539)were up-regulated by 1.766±0.103,1.417±0.173,1.271±0.166,1.836±0.157 times.4.3 The effect of HP0222 on the expression of CagA after H.pylori infection in AGS cellsWestern blot results showed that compared with NCTC26695,the expression of CagA protein in NCTC26695Δhp0222 infected AGS cells increased by 3.526±0.834times,and the level of phosphorylated CagA transported into cells increased by 3.534±1.045 times.Conclusion:1.HP0222 has the ability to bind CagA promoter and T4SS important gene promoter "promoter-cag1","promoter-cag U","promoter-cag Z","promoter-cag L","promoter-cag X".2.HP0222 inhibits the expression of Cag M,Cag S,Cag V,Cag L and CagA3.HP0222 inhibits the expression of CagA and the level of phosphorylated CagA after H.pylori infection in AGS cells... |
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