| Xinjiang is located in the west of China,unique natural and geographical environment,rich in all kinds of resources,livestock development prospects.Under the convenient conditions of the implementation of “the Belt and Road Initiatives” strategy,the rapid development of animal husbandry economy makes animal products unique to Xinjiang,such as animal fur,cream and dairy products,enter the sales market all over the country and even the world,providing convenience for people everywhere.At the same time,climate,vegetation and abundant animal resources also provide suitable conditions for tick breeding and facilitate the transmission of tick-borne diseases.Dermacentor marginatus is a predominant local vector tick carrying a variety of pathogens.The threat of D.marginatus and its transmitted pathogens to animal and human health is increasing day by day.At present,no protective antigen is available against D.marginatus.Therefore,in this study,D.marginatus was taken as the research object to conduct molecular biological identification and detect the common pathogens carried by it.At the same time,Dm86 gene was cloned and expressed to study its immunogenicity,which provided preliminary support for the mining of anti-tick vaccine candidate genes.(?)In this experiment,a total of 90 ticks were collected from Zhaosu County,Xinjiang.After rough classification by naked eyes,species were identified by COI gene.The target fragments of Bc48 gene and 16 S r RNA gene were amplified by PCR to detect the B.caballi and R.raoultii pathogens.The results showed that 90 ticks collected were identified as D.marginal.By sequencing,the similarity between COI gene and D.marginatus was 99.9%,which could be used to identify dermatitis marginal rapidly and accurately.In the 90 dermatitis borderline ticks,the target fragment of Bc48 gene of B.caballi and 16 S r RNA gene of R.raoultii were amplified by PCR at 451 bp and 1 332 bp respectively.The phylogenetic tree showed that the B.caballi was clustered into one branch with the local strain in Xinjiang,and that of gene sequence of R.raoultii was clustered into one branch with the local strain in Harbin.In the ticks collected this time,the detection rates of B.caballi and R.raoultii were 46.6% and12.2%,respectively,and the two pathogens were effectively monitored in Zhaosu country.(?)Screening of protective antigen based on D.marginatus,using PCR amplification D.marginatus truncated Dm86 gene fragments,build Dm86-p MD19-T recombinant plasmid,sequencing identification and use of bioinformatics analysis software sequence analysis purpose,all data to predict protein bioinformatics.According to the target sequence,fluorescence quantitative PCR primers were designed to understand the expression of Dm86 gene.The results showed that 1 773 bp target fragment was obtained by PCR amplification.The Dm86 clone sequence encoded 591 amino acids by biological characteristics prediction analysis.The molecular weight is 64 857.81,the theoretical isoelectric point is6.78,and it is acidic.hydrophilic and had multiple B cell epitopes.Fluorescent quantitative PCR results showed that Dm86 gene was expressed in different stages of D.marginal,and the expression level in satiety stage was higher than that in starvation stage.(?)According to the results of biological information for credit analysis,screening higher hydrophilicity,strong antigenicity and antigen epitope continuous 345 bp DNA fragment,build recombinant plasmids and name Dm86-p GEX-4T-1.After the expression of IPTG induced protein,polyclonal antibodies were prepared to verify its immunogenicity by Dot blotting and Western blotting.Finally,the immune effect of Dm86 recombinant antigen was evaluated by immunization test.The results showed that the recombinant protein was 39 k Da in size and expressed in the form of inclusion body.The antibody titer reached 1 : 25 600,and the recombinant protein could be recognized with certain reactivity and immunogenicity.Finally,rabbits were inoculated with adult to evaluate the immune effect of Dm86 antigen,which laid a foundation for the study of anti-D.marginatus vaccine candidate antigen,which could lay a foundation for the study of candidate antigens of anti-D.marginatus vaccine.In this study,90 ticks collected from Zhaosu County,Xinjiang Province were identified as D.marginatus by molecular biology methods.The detection rates of B.caballi and R.raoultii were 46.6%and 12.2%,respectively.At the same time,the Dm86 gene was cloned and sequenced,and the gene expression was detected by q RT-PCR,and the expression level was different at different developmental stages.Finally,the truncated expression of Dm86 gene was carried out,and it was found that the antigen of Dm86 had a certain effect on the full blood rate and incubation time through animal experiments. |
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