| Xinjiang is one of the main animal husbandry areas in China,and livestock and people working in the animal husbandry are hazarded by ticks.Dermacentor marginatus is an obligate blood-sucking ectoparasite which is a competent vector of many kinds of zoonotic pathogens.In recent years,D.marginatus is gradually expending in distribution over Xinjiang region,and activity of this tick species contributed to the increase of tick-borne diseases posing a serious threat to the health of local residents and livestock.Thus,it is necessary to control the population of ticks and reduce the transmission of tick-borne pathogens among livestock,wild animals and ticks.Mastering the distribution pattern and disclosing the protective antigen sequence information of D.marginatus in Xinjiang is a research focus home and abroad,and it is also a bottleneck of the vector tick comprehensive prevention and control approach waiting to be solved.In the current study,the MaxEnt species distribution model,genetic engineering technology and molecular biology methods were applied to study the distribution pattern and important protective antigens of D.marginatus in Xinjiang hoping to reveal the key prevention and control areas of the tick species and important protective antigens for effective vaccine candidates against the tick species.The main contents of the research are as follow:（1）Study on MaxEnt prediction distribution of D.marginatus.In order to study the potential distribution pattern of D.marginatus in Xinjiang,88 GPS localities and 988 adult ticks were collected by literature reviewing and field tick sampling.In total 756 out of 988 ticks collected from four sites were identified as D.marginatus.MaxEnt model uses maximum entropy algorithm for species distribution prediction.There were88 GPS presence records derived from literature review and field sampling,and 19 bioclimatic variables were assembled in the model to construct the distribution map of D.marginatus in Xinjiang with software running by the best model configuration.The model is verified by the AUC value of ROC curve.The results showed that the model is most accurate when run with the feature combination"LQ"and regularization multiplier 1.5,and the AUC value of the best prediction model after running by 10-fold cross-validation is0.838±0.063.The prediction model shows that the suitable distribution areas of D.marginatus are in the Yili River Valley,the west side of Altai Mountain,Zhunger Basin and the northern foot of Tianshan Mountain.（2）The transcriptome analysis of D.marginatus.The gene sequence information and target gene exploration of D.marginatus were conducted through RNA-seq sequencing.Based on laboratory pure tick species rearing,female adult ticks after molting,long-term starvation and engorged state were studied,and the RNA-seq data were obtained through the Illumina Hi Seq 4000 platform.The results showed that 449 349046 effective reads were sequenced assembling 30 251 unigenes,which greatly enriched the gene sequence information of D.marginatus.KEGG pathway analysis showed that blood feeding of female ticks could induce up-regulation of gene expression in chitin synthesis,defense response,oxidative stress and vitellogenesis.The long-term starvation period can up-regulate the expression of genes related to protein hydrolysis,defense response,detoxification metabolism and other pathways in female ticks.The results of RT-q PCR verification showed that the expression patterns of ferritin 1,ferritin 2,HSP70,GST,galectin and Dm86 genes were consistent with the trend of RNA-seq data obtained in the study.（3）Cloning of GST genes of D.marginatus and analysis of candidate antigens against the tick.In this study,the GST genes in RNA-seq data was used as template,and specific primers were designed for cloning,expression analysis and antigenicity prediction of GST family genes of D.marginatus.The（CDs）,sequence analysis and antigenicity prediction of the coding region of GST family genes highly expressed in female ticks during blood-feeding period were amplified by PCR and analyzed with bioinformatics software.The gene expression profile was analyzed by RT-q PCR to determine the relative expression of GST genes.The results showed that seven GST genes of D.marginatus were amplified and cloned.The nucleic acid sequences were between 651 and 717 bp,and the number of amino acids encoded by the proteins were between 216and 238 aa.Phylogenetic analysis of GST protein sequences showed that 4 of the 7 Dm GSTs belonged to mu class GST,and each for epsilon class,zeta class,and omega class GSTs.Protein sequence alignment showed that there were conserved regions among mu class GST.B cell linear epitopes prediction also revealed similar sequence patterns of epitopes.The conformational epitope analysis showed that the antigenic epitope region of mu class GST was larger than that of other GST subtypes.Finally,according to the analysis of the expression profiles of GST genes in different developmental stages and blood-feeding stages of female ticks,Dm GSTm1 and Dm GSTm2 were considered suitable for candidate antigens for anti-tick vaccine development in theory.（4）Construction of chimeric expression vector of GST and ferritin 2（Fer2）gene and detection of immunogenicity of the recombinant protein.In order to further exploit the potential of GST as an anti-tick vaccine,the chimeric protein expression plasmid was constructed using Dm GSTm1 and Fer2 as templates,and the recombinant protein was expressed and purified.The immunogenicity of the protein and the titer of immune antibody in rabbit serum were detected.The results showed that the truncated Dm GSTm1 and Fer2gene fragments could be linked by overlapping PCR,and the chimeric expression vector p ET-28a-Dm GSTm1-G（7）-Fer2 was successfully constructed.The recombinant chimeric protein can be effectively expressed by E.coli Rossita（DE3）strain in the form of precipitation.The molecular weight of r Dm GSTm1-Fer2 is about 32 k Da.Western blot showed that the recombinant chimeric protein had antigenicity.The results of mass spectrometry showed that the recombinant protein was expressed correctly.The serum immune antibody level of rabbits in r Dm GSTm1-Fer2 experiment was somehow low as a whole,and the antibody titer reached 1:12 800.The overlapping PCR method used in this experiment can be used to connect different tick gene sequences and provide the research basis for the anti-tick experiment of chimeric protein immunity.（5）The anti-tick vaccine trial of recombinant GST.In this study,Dm GSTm1 was used as the research object to construct a prokaryotic expression system,expression of Dm GSTm1 level and GST enzyme activities in different developmental stages,tissues and organs and different blood-feeding states of female adult ticks.The recombinant Dm GSTm1（r Dm GSTm1）was expressed in E.coli and effectively purified by His-trap affinity chromatography.The antigenicity of the recombinant protein was verified by Western blot,and the anti-tick effect of immunity was conducted on experimental rabbits.The results showed that r Dm GSTm1 was mainly expressed in soluble form and the enzyme activity of r Dm GSTm1 was 10.81±1.22U/mg prot.The trends of relative gene expression and enzyme activity were identical.Blood feeding of female ticks could induce the up-regulation of Dm GSTm1 expression and the increase of GST activity（over4 folds compared with the enzyme activity of its unfed status）.The results of Western blot showed that r Dm GST had good antigenicity and could induce rabbits to produce high titers of immune antibodies.The results of tick challenge experiment showed that the average fecundity of engorged female ticks in r Dm GST immunization group decreased by 25.4%（t-test,P<0.05）,the hatching rate of eggs decreased by 20.3%（t-test,P<0.05）,and the total anti-tick effect was 43.69%. |
PDF Full Text Request