Cloning, Sequence Analysis And Expression Of Partial Fragment Of VP2 Gene Of Canine Parvovirus Nanjing (CPV-GN) Strain

Canine parvovirus (CPV), firstly isolated in 1978 in the USA, is the causative agent of acute hemorrhagic enteritis and myocarditis in dogs. CPV belongs to the feline parvovirus subgroup of the autonomous parvoviruses. Analysis of CPV isolates by monoclonal antibodies and restriction enzymes have shown that after the first emergence of CPV (CPV-2) it evolved to give rise to new antigenic types, which were designated CPV type 2a and type 2b. These new types have replaced the original CPV type 2 worldwide, and CPV type 2b predominates overwhelmingly relative to CPV type 2a and is endemic in most populations of domestic canids in China.CPV virion is assembled from a mixture of capsid proteins VP1, VP2, and VP3. VP2 is the major component, and has been shown to be able to confer protection on the infected dogs.The research work present in this paper fall mainly into the following three parts: Identification and Genotyping of Nanjing Isolates of canine parvovirusGenotyping and identifying the subtypes of canine parvovirus will serve as guidance to prophylactic work of CPV infection, as well as making contributions to the epidemiological investigation. The present studies , by employing conventional virological techniques such as HA test, virus isolation, and other physco-chemical analyses (e.g., chloroform and trypsin resistance assays , acid challenge test, etc.), were first dedicated to identifying stool specimens from CPV-infected dog suspects. All these specimens have shown features that are characteristic of CPV infections. Subsequent PCR genotyping with specific CPV-type-differential primers has ascribed these viruses to CPV type-2b.Cloning and Sequence Analysis of the VP2 Gene of CPV Nanjing Isolate, CPV-GNThe present studies involve T-A cloning of major capsid- subunit-encoding VP2 gene of canine parvovirus Nanjing isolate, CPV-GN. In this study, pMD 18-T cloning vector was used and the 1548-bp-long VP2 gene successfully obtained. Following T-A cloning, accurate nucleotide sequence of the target gene was determined bysequencing and, the resulting data analyzed and compared with American CPV reference strains CPV-d CPV-15, and etc. Results: Unique base alterations do occur at positibns 18, 354, 405, 1465, and 1543, with T→A, A→G, G→A, G→A, and T→A changes, respectively. More interestingly, G→A point mutation at position 1456 leads to valine₄89 → ileucine substitution, and the T→A point mutation at position 1543 triggers serine₅15 → threonine substitution.Cloning, Expression, and Antigenicity Analysis of the Immunologically Responsive Fragment of VP2 Gene of CPV-GN StrainFollowing comparison and antigenicity analysis of CPV-GN VP2 gene with the counterpart of other CPV reference strains, a central portion of VP2 gene, with desirable antigenic potential, was located (between positions 706 and 1239) and designated VP2' fragment. A primer set specifically designed to amplify the VP2' fragment was synthesized and the target obtained by a thermo-cycler. The PCR product was inserted into pMD 18-T vector by T-A cloning and the resulting recombinant plasmid double-digested with EcoR I and Sal I. Expression vector pET32-VP2', designed to propagate and express the fusion protein in BL21, was constructed by subcloning VP2' into linearized pET32a (+) vector. Transformation of the recombinant construct into the host strain BL21 was performed and, the desired fusion protein with a molecular weight of 34kD detected by SDS-PAGE upon induction with IPTG. Subsequent Western bloting assay revealed that the recombinant protein was capable of being recognized by CPV-2b-raised antiserum, and thus has retained the major epitopes of the native protein VP2 of CPV capsid subunit.