| Xinjiang is one of the major livestock provinces in China,and the healthy development of livestock is the key to regional economic development.Ticks are blood sucking arthropods second only to mosquitoes in the transmission of pathogens.It is reported that ticks can transmit multiple pathogens,including some zoonosis,which has brought great harm to the breeding industry and the health of farmers.Dermacentor spps is the dominant tick genus in Xinjiang.Among them,Dermacentor marginatus is one of the dominant species.The tick can transmit at least six pathogens.At present,the main way for herdsmen to control ticks is to use acaricide to kill ticks in this area.However,acaricide and other chemical reagents containing various harmful substances will pollute the local grassland and water sources in the process of spraying ticks,and make ticks resistant.Therefore,the comprehensive management of ticks needs vaccine to control,such as GST,Bm86 and subolesin,At present,it has a protective effect on the invasion of a variety of ticks.Female ticks will cause oxidative stress after sucking a lot of blood.GST,as a kind of glutathione peroxidase,can alleviate the oxidative stress of ticks.At present,there are 35 GST genes in Ixodes acromii.In order to explore the candidate genes of GST family anti tick vaccine of Dermacentor marginatus,this study carried out systematic evolutionary analysis by amplification and sequencing to identify the genes and gene spectrum of GST family,analyzed the physical and chemical characteristics and structural characteristics of 6 kinds of GSTs by genetic method,measured the relative expression of Dermacentor marginatus in different reproductive age,key blood sucking period and 4 different organs by RT-q PCR,expressed 4 different proteins to prepare multi antibody,and tested its immunogenicity,to provide technical support for the research of anti tick vaccine against Dermacentor marginatus.(1)In this study,GST genes related to oxidative stress were screened by using transcriptome data of Dermacentor marginatus obtained in the early stage of the laboratory and NCBI comparison results of Dermacentor silvarum tick genome data.Six target genes were amplified by designing primers and sent for sequencing,and the phylogenetic tree was established by blast comparison.Six target genes such as Dm GSTE2,Dm GSTE3,Dm GSTK1,Dm GSTM5,Dm GSTM6 and Dm GSTZ2 were obtained.The login numbers after uploading were MW280833,MW280834,MW280835,MW280836,MW280837 and MW280838 respectively;The biological information of six genes was predicted by online software,among which the theoretical isoelectric points of Dm GSTE3 and Dm GSTK1 were 7.55 and 9.67 respectively,indicating that the proteins corresponding to these two genes are basic proteins and the others are acidic proteins;The instability coefficient of Dm GSTE3 is 31.44 < 40,indicating that the Dm GSTE3 is stable in solution;The prediction results of signal peptide and transmembrane region showed that only Dm GSTM6 had signal peptide and transmembrane region.The results of allergy prediction showed that the six proteins were non allergens.(2)Through pure tick culture in the laboratory,we collected four organs of the female tick at different developmental stages of Dermacentor marginatus and in the two states of semi-engorged female tick and engorged female tick.The RNA of the sample was extracted according to the general Trizol method,and the RNA was reverse transcribed to obtain c DNA according to the operation of the kit;The specificity of the designed primers was verified by the absence of bimodal peaks in the RT-q PCR melting curve and the single band without spurious bands in the RT-q PCR product running gel electrophoresis.Use EF-1 α Gene as an internal reference gene,six designed RT q PCR primers were used to determine the expression of four organs of Dermacentor marginatus at different developmental ages,key blood sucking stage,semi-engorged female stage and engorged female stage.The results showed that the expression of Dm GSTK1 was the highest in the period of blood engorged female ticks(305 516099);The expression level of Dm GSTZ2 female tick(184497)was the highest at 96 h;The expression of Dm GSTE3 gene was the highest in the ovary of semi-engorged female ticks(1098414).It can be inferred that Dm GSTEK1,Dm GSTZ2 and Dm GSTE3 are more suitable as candidate genes of anti tick vaccine.(3)In this study,when the strength of the real antigen is not clear,four different subtypes of six genes obtained in the previous experiment are selected for expression,The optimal expression conditions for the four proteins were Dm GSTM5(temperature 16 ℃,induction time 17 h,inducer concentration 0.6 mmol / L),Dm GSTZ2(temperature 16 ℃,induction time 19 h,inducer concentration0.8 mmol / L),Dm GSTE3(temperature 16 ℃,induction time 16 h,inducer concentration 0.8 mmol / L),Dm GSTK1(temperature 16 ℃,induction time 17 h,inducer concentration 0.8 mmol / L)was used to detect the activities of four proteases.It was found that the highest enzyme activity was Dm GSTK1,followed by Dm GSTZ2,indicating that the spatial folding of the expressed protein was correct.Mice were immunized with four proteins.The results of ELISA showed that the titer of Dm GSTK1 antibody was the highest,followed by Dm GSTZ2 antibody.Western blot showed that the four proteins had immunogenicity and reactivity.According to the immune experiment,among the four subtypes,kappa subtype GST has relatively strong antigenicity.(4)Based on the results of bioinformatics analysis,antigenicity prediction,expression assay,ELISA and enzyme activity test,Dm GSTK1 can be used as one of the candidate genes of anti tick vaccine. |
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