Expression Of Glycoprotein E2 Of Classical Swine Fever Virus In Insect Cells And Establishment Of An Indirect ELISA For Detecting Antibodies

Classical swine fever virus(CSFV) is one of the important pathogenic agent caused an economically important highly contagious diseases of swine worldwide. This virus only infects domestic pigs and wild boars.CSFV can cross the placental barrier,and this transplacental transmission usually results in mortality of fetuses and birth of congenitally infected pigs with a late-onset disease and death.CSFV cause acute disease and high mortality and leads to severe economical losses in pig industry. There are many diagnostic methods for CSFV,of which serological detecting methods are used widely,especially for ELISA assays because of having high specificity and sensitivity.Structural and envelope glycoprotein E2 of CSFV is the most antigenic protein being responsible for eliciting neutralizing antibodies and conferring protective immunity.The main antigenic region(E2-1)(containing the high conserved sequences of N terminatio)of classical swine fever virus E2 protein was expressed in insect cells using the Baculovirus Expressing System and is used as the antigen to develop an indirect ELISA.The details as follow:1.Expression of CSFV E2-1 gene in prokaryotic expression system:The DNA fragment was amplified by RT-PCR and was cloned into the contribution vector pFastBac^TMHTA to get the recombinant plasmid pFastBac^TMHTA-E2-1. pFastBac^TMHTA-E2-1 was then introduced into E.coli DH10Bac,which included a shuttle vector,Bacmid.By site-specific transposition,E2-1 gene site was integrated into bacmid,and a recombinant shuttle vector was constructed,named Bacmid-E2-1. The recombinant bacmid was transfected into Sf9 insect cells with Transfast Transfection Reagent Kit.The cell CPE could be observed in 72 hours after transfection.And the pure recombinant baculovirus was obtained by plaque. Expression of E2-1 protein(about 22kDa) was identified by IFA,SDS-PAGE and Western-blotting.2.Establishment of an indirect ELISA for detecting antibodys:The recombinant E2-1 protein was used as antigen for estabishing an indirect enzyme-linked immunosorbent assays(ELISA).92 pig sera have been to detected by the indirect ELISA and IDEXX ELISA antibody test kit.The coincidence rate between the two was determined as 83.7%.CSFV recombinant E2-1 protein was obtained throught genomic technologies. The indirect ELISA had good sensitivity and specificity and it was used to detect 135 clinical pig sera.It provided a quick and easy serological detection method which can monitor the changes of antibody level of the immunized swinery and the investigate epidemiology of CSF.