Isolation Of A Field PRRSV Strain And Expression Of Recombinant GP5 Envelop Glycoprotein And The Analysis Of Its Immunogenicity

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)was the pathogeny of Porcine Reproductive and Respiratory Syndrome (PRRS) represented by abortion of sew and respiratory obstacle of piglets. For its infectiousness as well as the immune repression and intercurrent brought to pigs, PRRSV caused huge economic loss to pig's feeding industry. Moreover, the mutants of PRRSV genes increase the difficulty to control the illness. Glycoprotein 5(GP5) is the primary structural protein of PRRSV. It shows the function of combining the virus to the receptor on the host cell as well as inducing the apoptosis of the cell and novel vaccine was designed according to its ability to induce the neutralization antibody. So GP5 provides the virus with important physiological functions and shows great value on the control of PRRS.Standard virological methods were employed to detect and isolate PRRSV from lung tissue samples collected from Shandong Taian pigs with high fever and severe respiratory syndrome in 2007. Based on the cytopathic effect and IFA test, the PRRSV was a filed isolate in Shandong province and designated SD-4. The primers for ORF5 gene of PRRSV North American (NA) strain VR-2332 were synthesized to clone ORF5 gene by RT-PCR. The cloned ORF5 gene was sequenced and analyzed in comparison with PRRSV isolates reported in GenBank, which provided the data for the research on epidemic of PRRS. The E.coli expressed GP5 was also acquired and its antigenic character was investigated; Meanwhile, because some kinds of vaccine failed to protect the piglets from infection, an indirect ELISA system for PRRSV GP5 antibody detection was established to evaluate the level of neutralization antibody in serum. And such study paved way for developing the kit to evaluate the effect of PRRS vaccine.Total RNA was extracted from the lung tissue of pig infected by the PRRSV and cDNA was synthesized by RT-PCR. The DNA of ORF5 was subsequently cloned into the pMD18-T vector and subjected to sequencing. The result of DNA sequence analysis showed that ORF5 consisted of 603 bp nucleotides andencoded the protein containing 200 amino acid residues. PRRSV NA strains can be divided into two subgroups. SD-4 ORF5 gene shares 87.4%-89.9%,92.7%-99.2% and 63.7%-63.8% nucleotide identity with subgroup I, II and European strain, respectively; and the amino acid sequence homology are 85.1%-91.0%,90.5%～99.5% and 56.7%-57.7%. Phylogenetic analysis based on the ORF5 gene showed that SD-4 is more close to JXA1,HUB1,HEB1 and SY0608, which were identified as highly pathogenic PRRSVs. SD-4 isolate belongs to PRRSV NA strain subgroup II and is a possible highly pathogenic PRRSV strain. On the other hand, another pair of primers were designed to amplified tORF5 DNA encoding the truncated GP5 (tGP5)which lacked 26 amino acid residues using the recombinant plasmid pMD18-T-ORF5 as the template. The resulting tORF5 DNA was inserted into the multiple cloning sites of pGEX-6p-1 plasmid. After the recombinant expression vector pGEX-6p-tORF5 had been transformed into the E.coli BL21 and induced with IPTG, truncated GP5(tGP5)was expressed in the form of inclusion body, and showed the molecular weight of about 40kDa as confirmed by SDS-PAGE and Western-blot probed by anti GST .The recombinant protein contributed to about 18% of the total bacterial protein, and after being purified using gel-cutting and electroeluting,its purity reached 95%.Purified GST-tGP5 showed specific reaction with the PRRSV positive serum while the GST tag failed to react with. Purified protein was used as the antigen to detect the anti PRRSV GP5 antibody in the serum by means of ELISA. Checkerboard titration was performed to deduce the optimal experimental condition of ELISA, and when it was coated at 100 ng per well and the serum was 400 folds diluted, the P/N ratio reached the highest level. PRRSV negative serum from 62 samples was tested under the determined ELISA condition and the average S/P ratio was evaluated. The value of 0.4 was proposed as the critical point for distinguishing negative and positive serum. S/P ratio higher than 0.4 suggests that the serum is GP5 antibody positive, while below 0.4 means negative. The result of such detection system showed 94.12% consistence with the result of neutralization detection. Purified GST-tGP5 was also used to immunise mice for anti GP5 serum preparation. After purification, the anti serum showed the titer of more than 1/20000. However, further study is needed to identify if the anti serum can neutralize the PRRSV and prevent its infection to piglets.