Development Of Rapid Assay For Canine Parvovirus By Loop-mediated Isothermal Amplification Method And Cloning And Prokaryotic Expression Of Vp2 Gene

Canine Parvovirus disease is an acute,feverish and contagious infectious disease of canine parvovirus(CPV).CPV can be found in dogs of different ages and species all over the world.The incidence rate and death rate caused by this disease are particularly high in weaned puppies.The clinical symptoms are vomiting,hemorrhagic enteritis and leukopenia.Therefore,it is especially important to establish the early and rapid assays for detecting disease.At present,the main methods to detect canine parvovirus(CPV) are serological test and molecular biological examinations which are supplied powerful technology for detecting CPV. Following the development of molecular biotechnology,some new technologies,such as Loop-mediated isothermal amplification method(LAMP),are developed in the recent years and supply new skills for the early and rapid examination of CPV.The VP2 gene of CPV was downloaded from GenBank and analyzed using MegAlign tools of software DNAStar.The outer,inner and loop primers of LAMP were then designed in their conservative region using software Primer Explorer V3.The fragment of VP2 gene was also amplified and cloned serving as the positive standard and LAMP optimized reaction condition and reaction system in this experiment.The result showed that the LAMP assay had a detection limit of 2～3 DNA copies.The primers used in this method were shown to be specific for CPV and did not amplify Porcine Parvovirus,Gosling Plague Virus,Canine Distemper Virus and canine rotavirus. The process of LAMP was conducted with a step amplification using a water bath and the amplification results were visualized by adding EvaGreen,which made it more simple and convenient.In clinic test,the positive rate is up to 100%.Therefore,the rapid and simple assay is a potential useful technique for CPV detection in field conditions. VP2 protein of canine parvovirus was the major structure proteins and protective antigen which could induce canine neutralizing antibody against CPV infection.In order to research the serology test,VP2 gene from strain YM of CPV was cloned and expressed,the highly purity of VP2 recombinant protein was harvested.In this research,the VP2 recombinants was sub-cloned into the expression vector pET-32a and identified by digestion of EcoRⅠand XhoⅠ,it showed the ORF of VP2 gene and been transformed into E.coli Rosetta(DE3).The recombinant bacteria E.coli Rosetta(CVP2 ) was selected and cultured to induce expression by 1 mM IPTG at 30℃.The culture was treated by supersonic wave and centrifuged.The pellets of the bacteria were washed by 4 M urea and denaturated using 8 M urea. The expressed VP2 protein was purified by Nickel agarose gel FF and has 87 kDa of molecular by SDS-PAGE.The special band was found in the Western blot of the expressed VP2 protein with the polyclonal antibody of CPV.The results showed that E.coli Rosetta(CVP2) expressed the VP2 protein of CPV-YM and have the value in developing CPV colloidal gold kit for the detection of Ab against CPV.