ABSTRACT
The capsid structural protein encoded by the gene ORF2 of Porcine Circovirus Type 2 is critical for eliciting the broad protective immunity. We constructed and characterized the recombinant adenovirus encoding the capsid and porcine IFN gamma, designated as rAd-ORF2-IFN-γ. The construct was further confirmed by the enzyme digestion, PCR, sequencing and transfection. The humoural immunity to the capsid protein, induced by the recombinant in the mice, was tested by ELISA. Notably, this recombinant induced a much better ORF2-specific antibody response than that of rAd-ORF2 alone or the adenovirus itself. Clearly, the porcine IFN gamma could strongly enhance the immunogenicity of ORF2. The results showed that the recombinant adenovirus might be an attractive candidate vaccine for preventing the disease associated with PCV2 infection.
Subject(s)
Adenoviridae/immunology , Circovirus/immunology , Interferon-gamma/immunology , Viral Vaccines/immunology , Adenoviridae/genetics , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/genetics , Animals , Antibodies, Viral/blood , Circovirus/genetics , Enzyme-Linked Immunosorbent Assay , Female , Interferon-gamma/genetics , Mice , Mice, Inbred BALB C , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/geneticsABSTRACT
Influenza A virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in the virus life cycle. In the present study, we extracted the viral genome RNAs from allantoic fluid of 9-day-old embryonated chicken eggs infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M1 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into an expression vector pET-28a (+) (designated pET-28a-M1) and confirmed by DNA sequencing analysis. We then transformed the plasmid pET-28a-M1 into Escherichia coli BL21 strain for heterologous expression. The expression of M1 was induced by 1mM isopropyl-beta-D-thiogalactopyranoside. SDS-PAGE analysis of the induced bacterial cells revealed that the recombinant M1 protein was expressed in high yield level. Next, we purified the expressed recombinant M1 using Ni2+ affinity chromatography and immunized Wistar rat with the purified M1 protein for producing polyclonal antibodies specific for M1. Western blotting analysis showed that the produced antibodies were capable of reacting with M1 protein expressed in Escherichia coli as well as that synthesized in SIV-infected cells. We further cloned the amplified M1 cDNA into a eukaryotic expression plasmid p3xFLAG-CMV-7.1 to construct the recombinant plasmid p3xFLAG-CMV-M1 for expressing M1 in eukaryotic cells. Western blotting analysis revealed that the M1 protein was expressed in p3xFLAG-CMV-M1-transfected Vero cells and recognized by the produced anti-M1 antibodies. Using the produced anti-M1 antibodies, we analyzed the kinetics of M1 protein in the virus-infected cells during influenza virus infection and estimated the possibility of M1 as an indicator of influenza virus replication. The recombinant M1 protein, anti-M1 antibodies and recombinant expression plasmids would provide useful tools for studies of biological function of M1 protein and the basis of SIV replication.