Construction And Immunogenicity Of Recombinant Adenovirus Expressing M Protein Of Porcine Reproductive And Respiratory Syndrome Virus

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens of pigs in the world. The virus infection could produce stillbirth, piglet mortality, respiratory disorders and immunosupression on all categories of pigs. But the current commercial vaccines are only partially effective in providing pigs protection against infection by PRRSV. The M protein is a major structural protein of PRRSV. It might play a role in virus assembly and building, and could induce humoral immune response and T-cell proliferative response in pigs.In this study, a pair of specific primers against the M gene of PRRSV was designed according to the gene sequence of PRRSV Sl(No.AF090173) strain. The total mRNA of PRRSV S1 strain was extracted and used as the template, the M gene of 530bp was amplified by the reverse transcription polymerase chain reaction (RT-PCR), and the PCR product of M gene was inserted into pShuttle-CMV plasmid at KpnI, XhoI sites. The positive clones carrying a M gene were screened on the LB plate with kanamycin after culturing 16-20h at 37℃. The recombinant pShuttle-CMV-M plasmids were identified by PCR. and KpnI, XhoI digestion and sequencing, respectively. The results showed that the M gene was successfully cloned into pShuttle-CMV in correct open reading frame (ORF). The comparison of M gene sequences with PRRSV S1 strain has shown a degree of accordance >99%. Subsequently, the plasmids pShuttle-CMV-M were linearized with PmeI, and linearized pShuttle-CMV-M plasmids were transformed into Ecoli BJ5183 containing backbone vector pAdEasy-1 by elecporitation. At the presence of recombinase of Ecoli BJ5183, homologous recombination between linearied pShuttle-CMV-ORF6 plasmid and pAdEasy-1 vector occurred. Consequently, the M gene fragment was transferred into adenovirus backbone pAdEasy-1 and selected with kanamycin on LB. The recombinant plasmids(pAd-M) were identified and linearized with Pad, transfected into 293 cell with anion lipid transfection reagent. After incubation of the transfected cell for 15 days at 37℃, the CPE was observed obviously. Thenthe recombiant adenovirus was purified with phage forming unit(PFU). The virus titre was 10^7.6TCID50/ml. The expression of M gene was amplified by RT-PCR and indirect immunofluorescent assay(IFN) with antibody to PRRSV, respectively. After the recombinant adenovirus (rAd-M) was passaged in 293 cells by 25 times, it still containing the M protein gene amplified by RT-PCR, the virus titre was 10^7.6 TCID50/ mL, 10^8.1 TCID50/ mL , 10^8.5 TCID50/mL , 10^7.4TCID50/mL. 10^8.0 TCID50/mL at No. 5, No. 10, No. 15, No. 20 and No. 25 passage respectively.The rAd-M and rAd-GP5, expressing PRRSV M and GP5 respectively, were inoculated into 18-20g mice. 120 female mice were randomly divided in 4 treatments, 30 animals per treatment .and inoculated with rAd-A ,rAd-M , rAd-GP5 , rAd-M and rAd-GP5 by hypodermic injection on the back. After inoculation, the presence of antibodies against the viral proteins in the sera of mice was analyzed by ELISA and virus neutralizing(VN) assay, the T-cells proliferative response was detected by MTT assay. The results of animal immune assay showed that the mice inoculated with rAd-M developed antibodies against the viral proteins at 15 days post -inoculation (dpi) as detected by ELISA. The T-cells proliferative response was also detected in mice inoculated recombinant adenovirus at 15 dpi. It indicated that the recombinant adenovirus is able to express antigens of PRRSV and elicit a humoral and cellular immune response in mice. It should be necessary to study on the immunogenicity of the recombinant virus in pigs in further.