Gene Cloning,prokaryotic Expression And Identification Of Anaplasma Phagocytophilum T4SS Effectors And Novel Diagnostic Antigens For Human Granulocytic Annaplasmosis

Objective:Human granulocytic annaplasmosis(HGA),caused by the infection of Anaplasma phagocytophilum(Ap),is a potentially fatal disease.Now,this disease spreads to all over the world and becomes an important public health issue.To investigate the pathogenesis of HGA,thirty-five Ap hypothetical proteins were screened as type Ⅳ secretion system(T4SS)effector candidates by bioinformatics analysis in part one of this study.Eight potential effectors of these thirty-five candidates and the outer membrane protein,P44,were expressed and purified,and the polyclonal antibodies against these nine proteins were prepared.In addition,we tried to establish a Cre reporter assay for translation(CRAFT)to verify these putative Ap T4SS effectors.Furthermore,we identified two proteins named APH1384 and APH0812 from seventeen specific proteins of Ap species in part two of this study,which can be used as the novel diagnostic antigens to improve the sensitivity of diagnosis for HGA.Methods:Part One.Clone,expression and identification of Ap T4SS putative effectors1.Screening,Gene cloning,expression,purification,and antibody preparation forAp T4SS effector candidates1.1 Ap T4SS effector candidates were screened by bioinformaticsAccording to the common features including species specificity,carboxyl terminal charge of proteins,GC content and Coiled-coil domain shared among Ap T4SS effectors,Ap T4SS effector candidates were screened with bioinformatics analysis from610 hypothetical proteins encoded by Ap genome.1.2 Construction of prokaryotic expression system for Ap T4SS effector candidatesDNA fragments of eight effector candidates and P44 were amplified by PCR with Ap genomic DNA as template and the specific primers incorporated cleavage sites and nucleotide sequences encoding 6×His tag in this study.These DNA fragments were cloned into prokaryotic expression vector pET28a(+)or pGEX-4T-1 followed by digestion of all these target genes and vectors with enzymes,and ligation with T4 DNA ligase.The recombinant plasmids were sequenced and transformed into E.coli BL21(DE3)competent bacteria for expression.1.3 Expression and purification of Ap T4SS effector candidatesE.coli BL21(DE3)culture with recombinant plasmids were induced by IPTG at37 ~oC to express proteins.All the proteins were purified by Ni-chelating affinity chromatography and then,detected by SDS-PAGE.1.4 Preparation of polyclonal antibodyAll the purified recombinant proteins were used to immunize ICR mice for ten weeks to prepare polyclonal antibody.The specificity of these antibodies was determined by Western Blot.2.Preliminary construction of CRAFT systemAp,as intracellular bacterium,has not suitable methods for its genetic modification.Therefore,the identification of T4SS effectors relies on the Cre reporter assay for translation(CRAFT)system in surrogate host.The CRAFT system contains a donor bacterium(E.coli S17-1 lamp pir)and a recipient bacterium(E.coli K-12 MG1655)as the conjugation system,consisting of transport channel,chimeric coupling protein and reporter genes.The T4SS of donor bacterium,providing the transport channel,can mediate the transfer of effectors;The TraG-Vir D4,as the chimeric coupling protein,can capture the effectors of Ap expressed in the donor bacterium,and transfer to the T4SS channel;The report genes contain fusion gene cre-aph and 5’-cat-loxp-tet-loxp-cat-3’,which were cloned into the donor bacterium and recipient bacterium respectively.The recipient bacterium inserted with 5’-cat-loxp-tet-loxp-cat-3’displays tetracycline resistance,but lack resistance for chloramphenicol,due to the insertional inactivation of cat.Cre recombinase can translocate into recipient bacterium to mediate the recombination of the cat on each end of Lox P sites when it fuses with T4SS effectors,which leads to the chloramphenicol resistance phenotype in recipient bacterium.Therefore,the T4SS-dependent effectors can be verified by the conversion of the antibiotic resistance in recipient bacterium from tetracycline to chloramphenicol.2.1 The construction of traG-virD4The gene fragment of traG of RP4 plasmid was amplified by PCR using genomic DNA of E.coli S17-1 lamp pir as template.The specific primers were incorporated cleavage sites including Mfe I and Pci I.The gene fragment of virD4 of Ap was amplified by PCR using genomic DNA of A.phagocytophilum as template.The specific primers were incorporated cleavage sites including Pci I and BamH I.The traG-virD4was constructed by Overlap Extension PCR.The traG-virD4 will be further cloned into pMAL-c5x and transformed to donor bacterium.2.2 Chemical synthesis of creDNA fragment of cre synthesed by chemical method will be cloned into pMAL-c5x vector,followed by the insertion of genes encoding T4SS effector candidates at its 3’-end,creating the fusion gene of cre-aph.This recombinant plasmid will be transformed into donor bacterium.2.3 Construction of 5’-cat-loxp-tet and PCR of loxp-cat-3’According to the gene sequence of 5’-cat,four pairs of specific primers were designed.The gene fragment of 5’-cat-loxp was amplified by PCR using plasmid pACYC184 as template.After PCR amplification,the LoxP site(34 bp)and DNA sequence matching with 5’end of the tet were incorporated in the downstream of the5’-cat.According to the gene sequence of cat-3’,four pairs of specific primers were designed.The gene fragment of loxp-cat-3’was amplified by PCR using plasmid pACYC184 as template.After PCR amplification,the Lox P site and DNA sequence matching with 3’end of the tet were incorporated in the upstream of the cat-3’.Fragment of tet was amplified by PCR using plasmid pACYC184 as template.The5’-cat-loxp-tet was constructed by Overlap Extension PCR.The loxp-cat-3’will be further ligated with 5’-cat-loxp-tet.Part Two.Identification of novel diagnostic antigens for HGABioinformatics analysis screened out thirty-five Ap T4SS putatitve effectors,and antigenicity of these seventeen proteins including APH1153,Ats-1,APH1127,APH0812,APH0847,APH1067,APH0653,APH1384,APH1345,APH1386,APH0832,APH0615,APH 1235,APH1157,APH0261,APH1068 and APH0833 were analyzed.1.Identification of Ap positive seraP44,the outer membrane protein of Ap was used to be the diagnostic antigen to screen the P44 positive sera from 600 clinic samples by Western Blot.The indirect immunofluorescent assay(IFA)was used to confirm the result.2.Identification of the recombinant proteins with strong antigenicityProteins with strong antigenicity among 17 Ap-specific recombinant proteins were screened with Ap positive sera.The serological reaction between proteins with strong antigenicity and P44 were compared by 100 clinic samples.3.Synthesis and antigenicity verification of peptide antigensThe epitopes of P44,APH1384 and APH0812 were predicted by Bioinformatics software named Kolaskar&Tongaonkar Antigenicity of Antibody Epitope Prediction(http://tools.immune epitope.org/tools/bcell/iedb_input).All these epitopes were synthesed by Beijing SBS Genetech,and then verified with Ap positive sera by Dot Blot.Furthermore,the serological reactions between the epitopes of P44 and APH1384were compared by Dot Blot.4.Clone,expression and purification of APH0812 fragments and identification of its epitopeSince the synthesized peptide of APH0812 didn’t react with Ap positive sera,we decide to clone,express and purify APH0812 fragments designated as APH0812-1,APH0812-2,APH0812-3,APH0812-4 and APH0812-4-5 to identify the epitopes.4.1 Clone,expression and purification of APH0812 fragmentsAll these DNA fragments encoding APH0812 were amplified by PCR with Ap genomic DNA as template and 5 specific primers incorporated cleavage sites and nucleotide sequence encoding 6×His tag.These DNA fragments were cloned into prokaryotic expression vector pMAL-c5x.The recombinant plasmids were sequenced and transformed into E.coli BL21(DE3)competent bacteria.Then,the bacterial cultures were induced by IPTG at 37 ~oC to express proteins.All the proteins were purified by Ni-chelating affinity chromatography and detected by SDS-PAGE.4.2 Preliminary identification of the epitope for APH0812Each fragment of APH0812 was probed with Ap positive sera by Western Blot to identify the distribution of the epitope for APH0812.The epitopes including APH0812(length of amino acids:365-389),APH0812(length of amino acids:395-416),APH0812(length of amino acids:450-485)of APH0812-4-5 fragment(length of amino acids:378-490)were predicted by the online software,Antibody Epitope Prediction.All these epitopes were synthesized in and then probed with Ap positive sera by Dot Blot.Results:Part One.Clone,expression and identification of Ap T4SS effectors1.Screening,gene cloning,expression,purification,and antibody preparation forAp T4SS effector candidates1.1 Ap T4SS effector candidates were screened by bioinformaticsThirty-five Ap T4SS effector candidates were screened by Bioinformatics,8proteins among them including APH1153,APH0819,APH1384,APH1386,APH1235,APH1068,APH1157 and APH1345 and P44 were selected for the further research.1.2 Gene cloning,prokaryotic expression and purification of Ap T4SS effectorcandidatesThe results of colony PCR identification and DNA sequencing revealed the gene of Ap T4SS effector candidates and P44 were cloned into prokaryotic expression vector pET28a(+)or pGEX-4T-1.The SDS-PAGE showed that all the recombinant proteins were expressed successfully.1.3 Preparation of polyclonal antibodyWestern Blot suggested that the polyclonal antibodies of all the purified specific recombinant proteins were prepared successfully.2.Preliminary construction of CRAFT system2.1 The construction of traG-virD4Colony PCR and DNA sequencing revealed that the fusion gene,traG-virD was constructed.Next,the fusion gene will be further cloned into pMAL-c5x.2.2 Chemical synthesis of creDNA sequencing revealed that the sequence of cre is correct.Next,it is ligated with gene of Ap T4SS effector candidates,aph.The fusion gene,cre-aph will be further cloned into pMAL-c5x.2.3 Construction of 5’-cat-loxp-tet and PCR of loxp-cat-3’Colony PCR and DNA sequencing revealed that the fusion gene,5’-cat-loxp-tet was constructed.The results of PCR suggested that the gene of loxp-cat-3’has been amplified.Next,loxp-cat-3’will be ligated with 5’-cat-loxp-tet to construct5’-cat-loxp-tet-loxp-cat-3’.Part Two.Identification of novel diagnostic antigens for HGA1.Identification of Ap positive seraWestern Blot revealed that P44-reactive positive sera were screened from 600clinical samples and IFA confirmed the result.2.Identification of the recombinant proteins with strong antigenicityWestern Blot revealed that five proteins with strong antigenicity including APH1127,APH0812,APH0847,APH1067 and APH1384 were screened by Ap positive sera.The serological reaction between these five proteins and P44 were compared for100 clinical samples and the results showed that APH1384 and APH0812 had obvious sera discrepancy from P44.3.Synthesis and antigenicity verification of peptide antigenDot Blot revealed that all the peptides except APH0812 have positive reaction with sera,and peptides of P44 and APH1384 has the serological discrepancy reaction.4.Clone,expression and purification of APH0812 fragments and identification ofits epitope4.1 Clone,expression and purification of APH0812 fragmentsColony PCR revealed that 5 gene fragments of APH0812 were cloned into prokaryotic expression vector pMAL-c5x,respectively.SDS-PAGE showed that all the recombinant proteins were expressed successfully.4.2 Preliminary identification of the epitope for APH0812Western Blot showed the fragments of APH0812-4 and APH0812-4-5 have reaction with Ap positive sera.However,Dot Blot revealed that all the three newly synthetic epitopes of APH0812-4-5 didn’t react with Ap positive sera,which suggested that the epitope of APH0812 maybe exist in other regions of APH0812-4-5.Conclusion:1.In this study,8 Ap T4SS putative effectors and P44 were cloned,expressed and purified and polyclonal antibodies against these proteins were prepared,which could contribute to the characterization the biological function of Ap T4SS effectors.2.The fusion gene of tra G-virD4 and 5’-cat-loxp-tet were constructed and the gene of cre was synthesized,which could contribute to further construction of CRAFT system to verification of Ap T4SS effectors.3.Two recombinant proteins with strong antigenicity,APH1384 and APH0812were identified,which may be worked as novel diagnostic antigens for HGA.