Analysis Of Diagnosis And Treatment Of Canine Parvovirus Disease.isolation,identification Of CPV-JZ2020 And Prokaryotic Expression Of The VP2 Gene

The canine parvovirus（CPV）is one of the viruses leading to highly infectious and fatal diseases in dogs.VP2 protein,as the most important structural protein in CPV capsid protein,has the function of determining the immunogenicity and coding.In this study,25 cases of canine parvovirus from December 2019 to February 2021in the Teaching Animal Hospital of Yangtze University were collected and analyzed.A canine parvovirus was isolated from the intestinal canal and contents of one of the dead dogs.It was identified as canine parvovirus by biological technology and named CPV-JZ2020.The VP2 gene was sequenced and prokaryotic expression of VP2 gene was carried out.The main research results are as follows:（1）The treatment results showed that the total cure rate of 25 cases of canine parvovirus was 68%,among which the cure rate of comprehensive treatment was 80%,that of cause treatment was 71.43%,and that of symptomatic support treatment was33.33%.（2）The Jingzhou strain of canine parvovirus was isolated from the intestine and contents of dead dogs by using F81 cells,which was named CPV-JZ2020,and could be stably passaged on F81 cells.F81 cells were inoculated with virus,and the cells were observed to have obvious shrinkage after 48 hours.The result of TCID50 showed that the TCID50 of the fifth generation virus on F81 cells was 10^5.0/m L.The VP2 gene with the size of 1755bp was amplified by PCR.Compared with the VP2 gene of CPV strains at home and abroad,the nucleotide homology was 98.39%-99.83%,and the amino acid homology was 97.56%-100.00%.The genetic analysis showed that CPV-JZ2020 strain was CPV-2c type,the application Gen Bank accession number is MW495851.（3）The VP2 gene was cloned into pMD18-T vector,and the recombinant cloning vector p MD18-T-JZ2020-VP2 was successfully constructed by PCR identification,double digestion identification and sequencing identification.The successfully constructed cloning vector was cloned into prokaryotic expression vector,and the recombinant expression vector identified as positive was named p ET-32a-JZ2020-VP2,which was transferred into E.coli BL21（DE3）.SDS-PAGE results showed that protein expression only appeared in the sediment of inclusion body of bacteria solution after ultrasonic fragmentation,it was found that when the IPTG concentration was 0.8 m M,the induction time was 4 h,and the induction temperature was 37℃,the protein expression was the highest.The recombinant protein was successfully expressed by Western blot.To sum up,the best treatment for canine parvovirus disease is to use the comprehensive treatment methods of causal treatment,symptomatic treatment,supplementing body fluid,maintaining body nutrition and regulating body acid-base balance.CPV-JZ2020 is CPV-2c type,which is a more common genotype in China at present,but its genetic relationship with traditional vaccine strains is low,which may be one of the reasons for the current clinical immune failure.In addition,the successful expression of recombinant protein lays a foundation for the development of new vaccines.