Molecular Survey Of Anaplasma Phagocytophilum Infection And Identification Of Type Ⅳ Secretion Substrates Of A.phagocytophilum

Objective:Anaplasma phagocytophilum,which is transmitted by ticks,can cause serious disease by infecting neutrophils of human and animals.In China,since first case was identified in 2006,the number of infected patients was increased every year.In recent years,there was few report about the Anaplasma phagocytophilum infection in Suzhou in spite of many investigations throughout the country.In this study,we investigated the infection rate of A.phagocytophilum in dogs from pet hospital in Suzhou by polymerase chain reaction(PCR),using specific primers which target A.phagocytophilum 16 s rRNA and msp3.Thus,epidemiological data of A.phagocytophilum infection is generated,which could be used as a reference when local authority takes measures to prevent the infection.The effectors of the type IV secretion system(T4SS)play a key role in the pathogenesis of some infectious diseases.A.phagocytophilum has functional T4 SS.In this study,we analyzed the physico-chemical properties and protein structure of seven potential effectors of A.phagocytophilum T4 SS by bioinformatics.We also produced polyclonal antibodies against these potential effectors and explored the distribution of APH0919 and APH1067 in HeLa cells,by which we partially characterized the T4 SS substrates of A.phagocytophilum.Methods:Part One.Molecular survey of Anaplasma phagocytophilum infection1.Extracting genomic DNA from dog blood.DNA was extracted from blood by blood genomic DNA kit and stored in-20 °C.2.Molecular survey of Anaplasma phagocytophilum infection in dogs in Suzhou.To detect A.phagocytophilum infection in dogs,we designed specific primers to amplify A.phagocytophilum 16 s rRNA and MSP3 genes by PCR from extracted DNA.Samples with positive PCR results were further confirmed with other two sets of PCRs,which specifically amply gltA and MSP4 genes of A.phagocytophilum.To make sure the PCR reactions were specific,PCR products were cloned into pUCm-T vector for sequencing analysis.Part Two.The bioinformatics analysis of T4 SS substrates1.Prediction of protein isoelectric point,hydrophobic,extinction coefficient,half-life,instability index by ProtParam.2.Prediction of signal peptides,transmembrane,antigenic sites,phosphorylation sites and subcellular location by bioinformatics software.3.Prediction of structure by bioinformatics software.Part Three.Prokaryotic expression of potential type IV secretion substrates and production of polyclonal antibodies1.Preparation of prokaryotic expression vectors.According to the nucleotide acid sequences,we designed primers for seven potential effectors.We amplified potential type IV secretion substrate genes by PCR,and then ligated these PCR products into prokaryotic expression vectors after digestion with restriction endonucleases.The APH0832,APH0833,APH0847,APH1067 genes were cloned into pET-28a-c(+),others were into pGEX-4T-1.Finally,the plasmids with correct inserted DNA sequences were transformed into E.coli BL21(DE3)for expression.2.Induced expression of potential effectors.E.coli BL21(DE3),which contains recombinant plasmids,was inoculated in LB liquid culture medium and cultured overnight.Then the cultures were 100-fold diluted in 200 ml fresh LB media,followed by the addition with IPTG after 3.5 h,and harvest by centrifugation after 7.5 h.3.Purification of recombinant proteins.Bacteria,which were suspended in lysis buffer,were lyzed by ultrasonic crusher,and solubility of recombinant proteins were determined by SDS-PAGE.4.Production of polyclonal antibodies.The recombinant protein bands were cut down from the gel after SDS-PAGE.Then the gel bands,which contain recombinant proteins,were mixed with adjuvant,and injected into mice.5.Determination of the titer and specificity of polyclonal antibodies.The titer and specificity of the polyclonal antibodies were determined by Western Blot.Part Four.Eukaryotic expression of potential type IV secretion substrates1.Preparation of eukaryotic expression vector.According to the nucleotide acid sequences,we designed primers to amplify two potential type IV secretion substrates by PCR,and then ligated the PCR products with eukaryotic expression vector after digestion with restriction endonucleases.The ligation product was transformed into E.coli DH5α.The transformant,which contains correct recombinant plasmid,was inoculated into LB liquid culture medium,and cultured for 12 h.Finally,plasmid was sequenced after extracted from E.coli DH5α.2.Eukaryotic expression of recombinant proteins.The recombinant plasmids were transfected into the HeLa cells,and then observed distribution of recombinant proteins under fluorescence microscope.Results:Part One.Molecular survey of Anaplasma phagocytophilum infection in dogs in Suzhou.In this survey,total of 223 dog blood samples were determined by PCR,and 7 of them were positive.The infection rate is 3.14%.Sequence analysis showed the PCR reactions were specificPart Two.The bioinformatics analysis of type IV secretion substratesPhysical and chemical properties,subcellular location,and of the seven potential effectors were analyzed by bioinformatics software.We selected APH0919 and APH1067 for further study.Part Three.Prokaryotic expression of potential type IV secretion substrates and production of polyclonal antibodies1.Preparation of prokaryotic expression vectors.Sequence analysis showed that all the genes of interest were cloned into prokaryotic expression vectors.2.Purification of recombinant proteins.Solubility analysis showed that APH0832,APH0833,APH0847,APH1067,and APH0907 were in inclusion body,others were in cytoplasm.SDS-PAGE showed that APH0723,which was prone to be degraded in purification,could not be purified from bacteria.But,others were purified successfully.3.Production and determination of polyclonal antibodies.Western Blot showed that polyclonal antibodies were produced in mice.But,the antibodies against APH0832,APH0847 and APH1067 need to be purified.Part Four.Eukaryotic expression of potential type IV secretion substrates1.Preparation of eukaryotic expression vector.Sequence analysis showed that aph0919 and aph1067 genes werecloned into eukaryotic expression vector.2.Eukaryotic expression of recombinant proteins.Fluorescence microscopy showed that potential effectors of A.phagocytophilum distribute irregularly in HeLa with punctate pattern.Conclusions:1.Molecular survey was performed to investigate the infection rate of A.phagocytophilum in Suzhou dogs.PCR results showed the infection rate is 3.14%.This study may help medical professional recongnize the possibility of A.phagocytophilum infection.2.The physical and chemical properties,structure of potential effectors were analyzed by bioinformatics software online,which can provide theoretical basis for the characterization of type IV secretion substrates.3.Recombinant proteins were purified successfully after cloning of their genes into prokaryotic expression vector.Then the polyclonal antibodies were produced from mice,which will serve as tools for the identification and characterization of type IV secretion substrates.4.APH0919 and APH1067 were expressed successfully in eukaryotic cells,and their distribution was studied,which will assist the characterization of biological function of these two effectors in future.