Study On ELISA For Diagnosis Of Porcine Corcovirus Type 2 And Bivalent Genetic Engineering Vaccine Of Porcine Circovirus Type 2 And Pseudorabies Virus

Porcine circovirus type 2 is one of the new animal viruses in recent years. It is associated with various disease syndromes in pigs, especially post-weaning multisystemic wasting syndrome. This disease is first emerged in western Canada in 1991. Clinical signs include progressive weight loss, respiratory disorders, fever, pallor and jaundice. PMWS is widespread in many countries by pathological and serological survey and has a serious economic impact on the swine industry worldwide. To find out the prevalence of PCV2 in our country and develop safe and effective vaccine to prevent and control PCV2 infection in swine, the ELISA assay for diagonosis of PCV2 and the bivalent genetic engineering vaccines against porcine circovirus type 2 and pseudorabies virus were studied. So the following researches were explored.1. Cloning and expressing of PCV2 0RF2 gene in Escherichia coliformThe complete coding region of PCV2 ORF2 gene was amplified by PCR from the plasmid including the entire genomic sequence of porcine circovirus type 2. PCR product of ORF2 gene was cloned into pMD18-T vector to generate the recombinant plasmid pTORF2. There was not any mutation on 0RF2 gene by sequencing. The nuclear localization signal of ORF2 protein in the N-terminal 41 amino acids has been reported. To overcome the defect that intact ORF2 gene can be expressed in a low level or not be expressed in E.coli, the partial nuclear localization signal of ORF2 gene was removed by digestion with restriction enzyme. The truncated fragment of ORF2 was expressed in Escherichia coliform. The method was as follows. The plasmid pTORF2 was digested with Bal I and Sal I and then the small frament including ORF2 gene was extracted and laterly inserted into the region between Sma I and Sal I of pGEX-KG to construct plasmid pGEX-ORF2. This plasmid was transformed to E.coli BL21 and GST-ORF2 fusion protein was expressed after induction with IPTG. The fusion protein was identical to the predicted size and specific to standard positive serum of PCV2, which indicated the fusion protein possessed immunological activity.2. Development and application of ELISA assay for detecting PCV2 antibodiesGST-ORF2 fusion protein exists in supernatant and precipitation of the bacteria with ultrasonication, but that in supernatant is dominating. The protein in precipitation and supernatent was purified in two methods respectively. The inclusion body of fusion protein was dissolved with N-lauroylsarcosine and renaturated with TE (pH8.0) buffer to resume its activity. The soluble protein was purified with glutathione sepharose from the supernatant. The result showed the high quality was the hallmark of this method, butquantity of protein obtained was very low.The optical coating concentration of soluble protein and inclusion body was determined by checkerboard titration respectively. Non-specific reaction between soluble protein and standard positive or negative serum is lower than that between inclusion body and the same serum, therefore soluble protein is suitable as the diagnostic antigen even more. But considering the expenses and decrease of non-specific reaction between inclusion body and the standard serum by increasing the dilution multiple, inclusion body was used as the blocking antigen for ELISA. PCV2 ELISA assay was developed with the optical coating concentration of inclusion body. This assay showed strong specificity, good repeatability and high sensibility with twelve months at 4°C. The result of comparative examination between ELISA and indirect immunofluorescene assay for detecting PCV2 antibodies indicated there was a high positive correlation rate (90.3%), negative correlation rate (94.4%) and total correlation rate (91.8%). On this base, sera from different farms were examined with this ELISA, which revealed the positive prevalence was very high and the positive prevalence had a direct ratio with the year-old of pigs.3. Construction of recombinant pseudorabies viruses expressing ORF1 and ORF2 gene of porcine circovirus type 2ORF2 gene amplified by PCR was inserted into the universal tranfer vector pgG-Uni to construct transfer plasmid pgGORF2. And then this plasmid was co-infected into IBRS-2 cells with genome of PRV TK7gG7LacZ+. When cytopathic effect was occurred in cells, recombinant virus TK7gG7ORF2+ was obtained from the cytopathic cells by plaque purification. Also transfer plasmid pIE0RF10RF2 was constructed by inserting ORF1 and ORF2 deleted partial nuclear localization signal into pIECMV. This plasmid and genome of PRV TK7gE/LacZ+ were co-infected into IBRS-2 cells, and then the recombinant virus TK7gE7gI7ORFl-ORF2+ was obtained by plaque purification after cytopathic effect appeared in cells. The two recombinant viruses were identified with Southern blotting and expression of ORF2 protein or ORF1-ORF2 fusion protein was detected with indirect immunofluorescene assay or Western blotting respectively. Results of TCID50 showed the insertion of ORF2 or ORF1-ORF2 gene had no influence on the propagation of recombinant viruses in IBRS-2 cells.4. Immunogenicity of the bivalent genetic engineering vaccines against PCV2 and PRV in mice and weanling pigletsRecombinant pseudorabies viruses were prepared for the bivalent vaccines according to the procedures of vaccine production. Immunogenicity of the bivalentvaccines was determined in mice and weanling piglets. The results in mice showed that PRV neutralization antibodies induced by TK7gG7ORF2+ corresponded with that by TK7gG7LacZ+, but PRV neutralization antibodies induced by TK7gE7gI7ORFl-ORF2+ were lower than that by TK7gE7LacZ+. However, mice immunized with these two bivalent vaccines could resist lethal pseudorabies virus and possessed the same preventive efficacy to that vaccinated with attenuated vaccines of PRV. Antibodies against PCV2 were very low in mice immunized with bivalent vaccines by ELISA. On this base, immunogenicity of TK/gE7gI7ORFl-ORF2+ was determined in weanling piglets. The result revealed that PRV neutralization antibodies induced by TK7gE7gI7ORFl-ORF2+ were lower than that by TK7gE7 gl\ which accorded with the result in mice, but ELISA antibodies against PRV induced by TK/gE7gI7ORFl-ORF2+ corresponded with that by TK7gE7gI". Antibodies against PCV2 were not detectable in piglets vaccinated with TK7gE/gI7ORFl-ORF2+, but PCV2-specific lymphoproliferation was higher than that in piglets immunized with TK7gE7 gl" and PBS.