The Isolation And Identification Of Yak Rotavirus And Cloning Expression Of Its VP6 Gene For Application

Rotavirus(RV)infection leads to economical loss for the world cattle industry,which is also an important reason for the yak diarrhea at present.The VP6 protein of bovine rotavirus(BRV)is the major structural protein and group of antigenic protein that plays an important role in maintaining virus particles stabilization and virus replication.VP6 gene sequence is the most conservativein 11 gene segments of RV,which is the preferred target for molecular and serological detection.Also,theresearches have shown that the subunit vaccines based on RV VP6 recombinant protein can provide effective protection against RV infection in recent years.The purpose of this research is to clone and express yak RV VP6 gene,for further study of serological detection method and subunit vaccine.The research has achieved the following results:1.Isolation and Identification of rotavirus from Tibetan YakTwo yak virus strains were isolated from 5 diarrhea stool samples of RV positive collected from Tibet using MA-104 cells,named Yak RVt-1 and Yak RVt-2.These two strains of the virus have been cultured to 10 generations,which hadn’t appeared cytopathic effects.The RT-PCR results demonstrate the presence of RV in each cell cultures;At the same time the isolated strain was also confirmed with yak RV by indirect immunofluorescence assay(IFA).The results of this research laid a material basis to the further study molecular characteristics and vaccine of yak RV.2.Cloning,expression and application of yak RV VP6 proteinAccording to the BRV VP6 sequences in GenBank,primers was designed to extend the yak RV VP6 by software Primer prime 5.0 with the template of Yak RVt-1cellculture,which got the full gene sequence.The full length of Yak RVt-1 VP6 gene sequence is 1338 bp,which sharing 94% ~ 96% homologywith BRV VP6 gene in GenBank.Compared with VP6 gene sequence of BRV NCDV,there were 118 nucleotides mutationand and the mutation rate is 12%,led to the protein 142 sites of amino acid changes.In order to get the recombinant RV VP6 protein in further,the synthetic Yak RVt-1 VP6 gene was ligated with pET-28 a vector and transformed into TOP10.The research had achieved the recombinant yak RV VP6 expression in engineering bacteria with the expression plasmid pET28a-VP6 was transformed into Rosetta(DE3).The recombinant VP6 protein was expressed in the form of inclusion bodies.Western Blot test showed that the recombinant VP6 protein had good immunogenicity to yak RV positive sera,which provided the material basis for the establishment of the detection method and the development of subunit vaccine based on the protein in further.After preparing of rabbit anti-VP6 hyperimmune serum using the recombinant VP6 protein,an IFA method for detecting yak RV was developed,withmouse anti-bovine IgG/FITC as secondary antibody.The method can specific detect yak RV,and there is no specific reaction with bovine coronavirus,bovine viral diarrhea disease virus,and bovine enterovirus,indicating that the IFA method established in this experiment can be used for rapid identification of yak RV.