Expression Of ORF2 Gene Of PCV2 And Development Of ELISA For PCV2 Antibody

Porcine circovirus type 2(PCV2) was the chief causative agent of postweaning multisystemic wasting syndrome(PMWS), it was also proved to correlate with many kinds of porcine circovirus disease,such as Porcine dermatitis and nephropathy syndrome(PDNS), Porcine respiratory disease complex(PRDC), Reproductive failure et al. The Cap protein, coding by the ORF2 of PCV2, was the major structural protein of virus, with satisfactory immunogenicity and was the chief immunogen to resist the infection of PCV2.In this research, two recombinant baculoviruses expressing Cap protein of PCV2 were constructed by Bac-to-Bac Baculovirus Expression System. First of all, several genes were cloned, including the PCV2 ORF2△41 gene(the ORF2 gene absenced nucleic localization sequence which code the first 41 amine acid of Cap protein), the MEL gene(melitten gene)and the 6H gene(six histidine tag gene), and suitable restriction enzyme cutting site was designed for them. Then the genes were inserted into FastBacTM1 transfer vector one by one, and two recombinant vector plasmid were obtained. One was named FBDORF2, which was inserted double ORF2 gene tandem. The other was named FBSDORF2, which was inserted single ORF2 gene. Finally, two recombinant vector plasmid were transformed to DH10 Bac competent E.coli to generate a recombinant bacmid respectively after homologous recombination. The two recombinant bacmid DNA were transfected into sf9 cell by using cellfection reagent in order to generate a recombinant baculoviruses respectively, which named DORF2 and SORF2. It was demonstrated that two recombinant baculoviruses were constructed correctly after identified by PCR and sequencing.The TCID50 of DORF2 and SORF2 were 10-6.33/mL and 10-6 /mL respective.PCV2 direct immunofluorescence and indirect six histidine tag immunofluorescence were carried out to detect the baculovirus infection in sf9 insect cell. The results showed that apparente positive signal could be viewed in both of two recombinant baculoviruses infected cell. It demonstrated that both of the recombinant baculoviruses can express target gene in sf9 insect cell satisfactorily. Suspension cultured TriEx sf9 insect cell was infected by the two recombinant baculoviruses, and supernate of culture was collected after 72 h. The supernate was purificated by using nickel column. Molecular mass of the product of ORF2 gene expressing by DORF2 and SORF2 was 51 KDa and 28 KDa respectively which were anlyzed by SDS-PAGE, coincident with expected. The Cap protein expresseing by both of the recombinant baculoviruses can react with PCV2 polyclonal antibody serum specifically which were proved by Western-blot.An indirect ELISA was developed to detect antibodies of PCV2 based on the purificated Cap protein. Several appropriate conditions of ELISA were obtained: the best concentration of coated Cap protein was 0.31μg/mL, the optimal dilution of serum was 100 times, working concentration of enzymes marked anti-antibody was diluted to 20000 times, the optimal coating condition was 4℃, for 16 h and best confining liquid was the PBS solution of 10% defatted milk powder. The specificity test was indicated there were not cross reaction with antibodies of normal swine diseases, such as PRV, PPV, CSFV, PRRSV, swine erysipelas, Swine paratyphoid and hemophilus parasuis. Serum samples which came from the countryside of Baoding city in HeBei Province were tested by the indirect ELISA and 61.5% samples were positive, the coincidence was 94.8% with PCV2 antibody detection kit of INGEZIM corporation.