| Classical swine fever is probably the economically most important viral infectiousdisease of pigs caused by classical swine fever virus, all ages of domestic and wildpigs can be infected. CSFV is classified as an important member of the genusPestivirus within the family Flaviridae, a single stranded RNA genome with positivepolarity. CSFV possesses a12.5kb genome encoding a single large polyprotein. Thetranslated polyprotein is processed by viral as well as cellular proteases to the matureviral proteins of four structural and eight non-structural proteins. The E2envelopeglycoprotein of classical swine fever virus is the most immunodominant proteinresponsible for the induction of neutralizing antibodies and the main protein for themarker vaccine,subunit vaccine and detection method of classical swine fever. The E2envelope glycoprotein is the most intensively studied of classical swine fevervirus,including the signal peptide of N-terminus, the membrane-spanning domain ofC-terminus and the amino acids in the middle. Envelope E2protein forms eitherE2-E2homodimer or E2-E1heterodimer in the surface of virus to be functional. Fourantigenic domains, A-D, have been identified on the N-terminal half of the E2glycoprotein.The research suggests that the C-terminal of E2glycoprotein also has theantigenic domain. All of antigenic domains are main domains responsible for theinduction of neutralizing antibodies.This research amplified the534bp antigenic regions (A-D) of E2envelopeglycoprotein by PCR, subcloned this suquence into the prokaryoutic high-expressingvector pGEX-4T-1and constructed a prokaryotic expression vector pGEX-4T-1-△E2for expressing this suquence. For the purification of protein, the His-Tag had beenadded in the C-terminal of E2glycoprotein. Then the recombinant plasmid pGEX-4T-1-△E2was transformed intoEscherichia coli(E. coli) Transetta(DE3) forexpression of E2glycoprotein. The results of SDS-PAGE and western-blot suggestedthat△E2protein of classical swine fever viruscould express in E.coli in form ofinclusion bodies. The amount of expression was higher at30℃0.1mM of the IPTGconcentration, cultured8h. The denatured protein was purified by Ni-NTA His affinitychromatography. The result of SDS-PAGE suggested that when the imidazoleconcentration was250mM, the purification effects of△E2was the best. After therenaturation of△E2protein via the method of gradual dialysis its density wasdetermined as0.3042mg/ml via BCA method.The indirect ELISA for detection of classical swine fever antibodies wasestablished using the purified△E2protein as coat antigen, through the optimizationof the conditions. The optimal reaction conditions of the indirect ELISA for detectionof classical swine fever antibodies was determined. The amount of coated antigen was0.3125μg. The blocking condition was incubating at4℃for24h. The dilution ofserum samples was1:60, incubated at37℃for30min. The working concentration ofHRP-labed goat anti-swine IgG was at a dilution of1:2000, incubated at37℃for1h.TMB was incubated away from light for10min, terminated with0.5mM ofconcentrated sulfuric acid and readed the value of OD450. The standard of the indirectELISA is that the value of OD450≥0.390is the positive sample, the value of OD450≤0.350is the negative sample and the value of OD450between0.390and0.350is thesuspicious sample. When the sera is the suspicious sample, we can redetect andresample to detect. The result of the repetitive experiment of the indirect ELISA fordetection of classical swine fever antibodies showed that this method had wellrepetition. To test the specifity of this method, we examined samples of porcinereproductive and respiratory syndrome virus (PRRSV), porcine circovirus2(PCV2)and japanese encephalitis virus by this method, all the OD450values of these sampleswere less than0.350and the result showed that this method had favourable specifity.Detecting141clinical sera by both the indirect ELISA for detection of classical swinefever antibodies and IDEXX classical swine fever virus antibody test kit, the coincidence rates between the two methods were greater over80%. The researchsuggested that this method had good prospects for clinical application, laying thefoundation of detection of classical swine fever antibodies as well as the preventionand control of classical swine fever. |
