Expression And Application Of The Main Antigene Epitopes Of VP₂ Protein Of Porcine Parvovirus

The VP2 protein is PPV's main neutralizing antigen. In this study the gene encoding the main antigenic epitopes (VP2I gene) was cloned and expressed and the expressing product was studied for antigen instead of full virion. The main contents are as follows:1. PPV was amplified in PK-15 cell culture. According to the published VP2 gene sequence in GenBank, a fragment with clustered epitopes was chosen as target gene by analysis with NDAstar. A pair of primers was designed with Primer Primier. With this pair of primers a target gene of 868bp was amplified from PPV's nucleotide and was named VP2 I gene. This gene was inserted into pIREShyg and after restriction analysis a eukaryotic expression vector pIRES- VP2I was constructed successfully.2. pIRES- VP2I was transfect to CHO cell line by improved calcium phosphate co-precipitation. Positive clones were obtained with hygromycin.The expression of VP2I gene was detected by IFA and re-assured by RT-PCR in a transcriptional level in CHO-k1 cell line. By PCR method the VP2I gene was confirmed to integrated into cell genome and recombinant CHO/ VP2I cell line was established.3. The recombinant plasmid pET- VP2I was transformed to BL21(DE3) competent cell. According to the optimized conditions the VP2I gene was expressed in a high level. The expressed product was purified with His Bind chromatography. The purified product was used as antigen to establish an indirect ELISA to detect antibodies against PPV. About 70 clinical samples of sera were detected and compared with indirect HA test and this ELISA method was proved to be more sensitive.