| Porcine reproductive and respiratory syndrome (PRRS) was originally documented in United States of America in 1980, rapidly spreading to other pig-breeding countries and causing huge economical loss. In China, this disease was firstly confirmed in the south in 1995 and successively became epidemic in most of the pig farms within several years. PRRS was characterized by late term abortion, early farrowing, stillbirth and the birth of weak piglets, and is associated with the porcine respiratory disease complex in combination with secondary infections.Genomic analysis indicated that PRRSV had two major genotypes, the the North American serotype represented by strain VR-2332 and European serotype represented by strain LV. As PRRSV naturally infects macrophages and traditional strategy of vaccine development does not work very well to the disease. Therefore, development of safe and effective vaccine with genetic engineering technology would be a promising approach for prevention from the disease. GP5 and M proteins are the two main structural proteins of PRRSV, they play important role in viral pathogenecity, virus replication, and in inducing protective immune response. GP5 forms heterogous dimer with M protein in the endocytoplasmic reticulum and packaged into viral particles. Breaking the disulfate bond may destroy the infectivity of the virus. Generally, both GP5 and M proteins are in relatedness with the pathogenecity and immunity.Canine Adenovirus (CAV) infects a wide range of animals in canis and felis. The genome is 31kb in full length and is infectious. The E3 region is not neccessary for viral replication and its deletion permit the insertion of foreign gene and therfore the CAV is an excellent vector for carrying and expressing protective antigens.In this study, we utilize the common vector of CAV-2 virus with partial E3 deletion to express the structural proteins GP5 and M, intending to develop a potential vaccine candidate for PRRSV infection. In this study, we utilize the common vector of CAV-2 virus with partial E3 deletion to express the structural proteins GP5 and M, intending to develop a potential vaccine candidate for PRRSV infection.1. Isolation, identification and gene cloning and analysis of PRRSV strain JL-0605The clinical signs and status in a pig farm were investigated. Based on the clinicalsymptoms, those pigs were premarily diagnosed as PRRS. The virus, JL-0605, was isolated by blind passages on Marc-145 cells using the lung specimens, and confirmed by orrhology and morphology test. GP5 and M gene amplification and sequencing demonstrated that this virus is PRRSV. GP5 and M genes were also amplified by RT-PCR. The TCID50 of this virus was 105.0.2. Cloning and analysis of GP5 and M genesIn this study, according to the published sequence of PRRSV genome, VR-2332 GeneBank Number: PRU87392), two pairs of primers were designed, these primers were designed by molecular biology. The fragments of JL-0605 strain's GP5 and M gene were obtained by RT-PCR, respectively. The products of PCR were purified and cloned into pMD18-T vector, and then transferred, the recombinant plasmid sent for sequencing. Then the sequences of GP5 and M genes of JL-0605 strain were compared with the sequences and deducted amino acid sequence of other domestic and foreign PRRSV strains has published in GeneBank by the biology software such as DNAStar. The results showed that the homology of deduced amino acid sequences of GP5 and M gene was highly homologous to the VR2332 strain.3. Recovery and characterization of GP5 and M protein recombinant canine adenovirus type-2The expression cassettes of GP5 and M and the fragments containing the E3 region were constructed into plasmids and then subcloned into the whole genome of CAV-2, formed recombinant infectious genome. After transfection in MDCK cells, the recombinant virus was obtained. The GP5,M-recombinant virus was further confirmed to be pure and harbor the GP5 and M genes. The recombinant virus was proved to be stable with a TCID50 of 105.25. The lysate of the infected cells was identified to be reactive with the specific antisera, demonstrating that the indirect immunity fluorescence test antigens have been expressed.4. Immune response induced by GP5 and M protein recombinant CAV-2 in miceTotally 75 mice of 6-8 week-old were randomly divided into 5 groups, 15 in each group. Group 1,2 and 3 were subcutanously with the recombinant virus CAV2-△E 3-GP5-M, CAV2-△E 3-GP5 and CAV-2, respectively, 0.5mL/mouse, and boostered 14d after the prime vaccination. The 4 and 5 group injected with PBS and PRRSV vaccine strain as control. The sera were collected 2, 4, 6 and 8 weeks after the booster and tested for neutralizing antibody using ELISA kit. Cellular immunity was assayed by lymphocyte proliferation test using the lymphocytes from spleen. Specific antibodies against the PRRSV GP5 and M proteins were detectable 2 weeks after the booster vaccination in mice, the antibody reached peak at 6th week and slightly declined at 8th week.This demonstrated that the recombinant virus induced humoral immune response in mice. In vitro neutralizing test showed that the neutralizing antibody existed in the sera collected 2 weeks after booster with a level of 1:4-1:16. PRRSV specific lymphocyte proliferation test showed that on 2 weeks after the booster, the lymphocyte proliferation was enhanced in vitro, and reached peak on 4 weeks after the booster, compared with the controls of CAV-2 and PBS, indicating the mice produced specific cellualr response.5. Immune response induced by GP5 and M protein recombinant CAV-2 in pigsAfter intramuscular inoculation of 3-month-old pigs with 3×105.25 TCID50 of GP5, M-recombinant CAV-2, the serum samples were collected 4, 6, 8, 10, and 12 weeks post vaccination and titrated by neutralization test. The results showed that at 6 weeks after inoculation, the specific antibody started to appear, 8 weeks after the inoculation, the antibody reached a higher level, then the antibody decreased and maitained at a detectable level, demonstrating that this recombinant virus may effectively induce and sustain PRRSV specific antibody.Different from the previous design of monovalent PRRSV subunit vaccine, GP5 and M proteins encoded by the ORF5 and ORF6, respectively, were designed to be co-expressed in this study in consideration of the formation of dimer between GP5 and M. Also, canine adenovirus was used in this study due to its wide range of hosts. The results showed that the recombinant canine adenovirus expressing the GP5 and M proteins was immunogenic. In previous experiment, GP5 or M protein alone can only induce a lower level of neutralzing antibodies, while in this trial, the neutralzing antibody titer was higher due to the co-expression of GP5 and M proteins, probably the reason that the fusion expression of the two proteins altered the stereo-structure that enhance the production of neutralzing antibody. Furthermore, the recombinant virus was able to produce cellular immune response as demonstrated by the lymphocyte proliferation test. As to which of the GP5 and M protein is involved in the cellular immune response needs further demontration.Summarizing the above results,it could be concluded that the GP5, M- recombinant CAV-2 were able to elute immune responses specific for PRRSV and restrain the reproduction of PRRSV. Virus vector vaccines have important effects on the anti-viral immune responses and weakening the lesions caused by the virus. These data would be helpful for future vaccine design against PRRSV. The above study layed foundation for PRRS specific immunity and provided a way for screening effective antigen and preventive products.
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