ABSTRACT
Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.
Subject(s)
Animals , Female , Mice , Antibodies, Viral/blood , Antigens, Viral/genetics , Body Weight , Cross Protection/immunology , Disease Models, Animal , Epitopes, T-Lymphocyte/genetics , Influenza A Virus, H3N2 Subtype/genetics , Influenza Vaccines/immunology , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology , Peptides/genetics , Random Allocation , Survival Analysis , Vaccines, Synthetic/immunology , Virus ReplicationABSTRACT
Establishment of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) with co-expression E2 Epitope of Classical Swine Fever virus (CSFV) is a crucial step to develop a genetic engineered vaccine against PRRSV and CSFV. Reverse genetic manipulation could be adopted as a com monly used technique. In this study, we focus on using nonessential regions of NSP2 (aa480-532 and aa508-532) as viral vector to express E2 Epitope of CSFV. A neutralizing epitope of classical swine fever virus (CSFV) E2 protein was inserted into the two nonessential region of nsp2 by the method of mutant PCR, basing on the infectious clone of HuN4-F112 vaccine strain. The co-expressed full-length cDNA clones (psk-HuN4-F112-delta508-532 + E2 and psk-HuN4-F112-delta480-532 + E2) were assembled by cloning and splice of the gene fragments. The completely assembled full-length cDNA clones were confirmed by sequence and Swa I enzyme digestion. Capped RNAs were transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into BHK-21 cells by liposome to acquire the rescued virus. The rescued recombinant viruses were passaged on MARC-145 cells. The successfully rescued viruses were tested by RT-PCR, digestion, and genome sequence. The results showed that these rescued viruses could be distinguished from the parental virus (HuN4-F112) with the mutant genetic marker (Mlu I enzyme site of virual genome at 14 667nt was created by synonymous mutation) and the inserted nsp2 gene region. The results of IFA showed that the inserted E2 epitope could be expressed by the recombinant viruses and the E2 epitope gene was stable during the viral serial passage. The results of plaque assay and viral growth curve showed that the recovery viruses possessed similar characterses of viral growth to those of the parental virus. In summary, the full-length infectious cDNA clones containing the marker gene were constructed and the marker recombinant viruses were rescued. The results suggested that these stable infectious clones could be used as an important tool for development of novel vaccine against PRRSV.
Subject(s)
Base Sequence , Cysteine Endopeptidases , Genetics , Epitopes , Genetics , Molecular Sequence Data , Porcine respiratory and reproductive syndrome virus , Genetics , Viral Envelope Proteins , Genetics , Allergy and Immunology , Viral Vaccines , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) of vascular endothelial cells.</p><p><b>METHODS</b>Tryptase-siRNA (small-interfering RNA) vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA (RNAi-P815 group) or pure vector (P815 group) was collected and used to culture bEnd.3 cells. The messenger RNAs (mRNAs) of IL-6 and TNF-alpha in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.</p><p><b>RESULTS</b>IL-6 and TNF-alpha mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, IL-6 and TNF-alpha protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group.</p><p><b>CONCLUSION</b>Reduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-alpha in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.</p>
Subject(s)
Animals , Mice , Cell Line , Culture Media, Conditioned , Down-Regulation , Genetics , Endothelial Cells , Bodily Secretions , Gene Expression Regulation, Enzymologic , Interleukin-6 , Genetics , Bodily Secretions , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , Transgenes , Tryptases , Genetics , Metabolism , Tumor Necrosis Factor-alpha , Genetics , Bodily SecretionsABSTRACT
<p><b>AIM</b>To study the effects of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) on proliferation and migration of bovine coronary artery endothelial cells (BCAEC) in vitro.</p><p><b>METHODS</b>BCAECs were isolated and cultured in vitro, and divided into control group, VEGF group and HGF group. BCACEs proliferation were measured using MTT, and their migration was observed using reverse microscope.</p><p><b>RESULTS</b>The OD value of control, VEGF and HGF group were 0.22 +/- 0.01, 0.40 +/- 0.14, 0.44 +/- 0.15 respectively. The proliferation ratio of BCAECs in VEGF and HGF group was 81.8% +/- 16.9%, 100.0% +/- 21.1% respectively. There was no migration in control group, but significant migration in VEGF and HGF group.</p><p><b>CONCLUSION</b>Both VEGF and HGF can promote proliferation and migration of BCAECs, the effect of HGF is stronger than VEGF.</p>
Subject(s)
Animals , Cattle , Cell Division , Cell Movement , Cell Proliferation , Cells, Cultured , Coronary Vessels , Cell Biology , Culture Media, Conditioned , Endothelial Cells , Endothelium, Vascular , Cell Biology , Hepatocyte Growth Factor , Pharmacology , Vascular Endothelial Growth Factor A , PharmacologyABSTRACT
The proposed registration algorithm based on wavelet transform is a multi-resolution block matching one, which exploits the hierarchical self-similarity of Digital Subtraction Angiography images. This method befittingly trades off between the estimation precision and computational complexity, because the registration precision is from coarse to fine, which reduces the searching complexity of motion vector. This technique has been proved that the convergence speed is exponential. The proposed method takes full advantage of the precise location both temporally and frequently which characterizes the wavelet transform. Both the matching speed and consistency are boosted without the loss of matching performance.