| Neuraminidase (NA) is one of the two surface glycoproteins from the avian influenza virus, is a typeⅡglycoprotein and a glycoside exonuclease with digestion activity. NA has a dual function, on the one hand, for the flu virus itself, it can be cracking sialic acid and adjacent sugar residues between theα-glycosidic bond, which is conducive to release progeny viruses, and can prevent progeny virus releasing from the host cell surface aggregating, promote the virus through the mucus covering in the airway epithelial cells to proliferate; the other hand, for the host cell, since neuraminidase (also known as sialic acid, SA.) on the host cell membrane is major receptor of the influenza virus, influenza virus must bind with the SA receptors before the virus can enter the cells. While the NA can degrade the SA receptors, inhibit the virus to enter cells, and protect cells from the AIV infecting. As the unique features of NA, it is of great significance to the fight against AIV, make it one of the hot against the AIV research at home and abroad, also for the preparation of transgenic poultry against avian influenza provides a new idea. This study through cloning NA gene, expressing in vivo and vitro, subcellular localization and detection of antivirus activity, aims to explore the new idea that NA gene is used to develop disease-resistant transgeni poultry. Major contents and results in this study are as follows:1. Cloning, sequencing and prokaryotic expression of NA gene. Design and Synthesis NA gene-specific primers, pGEM-T-NA as a template, using PCR-directed mutagenesis technology to amend NA gene and cloned into pMD19-T Vector, the digestion and sequencing results show that pMD19-T-NA construct correctly, and the NA gene ORF is correct. Then NA gene is cloned into the vector pGEX-6P-1, construct prokaryotic expression vector pGEX-NA; expressed by IPTG induction, SDS-PAGE electrophoresis separation, Western blot analysis showed that the recombinant NA protein obtained expression and the anti-NA serum can be identified by expression products.2. Immune responses in mice induced by plasmid DNA expressing neuraminidase. The NA gene is cloned into the pcDNA3.0 vector to construct expression vector pcDNA3.0-NA, use different immunization methods, inject BALB / c mice respectively with different doses of pcDNA3.0-NA; collected blood at different times after immunization, separate and collecte serum; ELISA detecting and analysing immune effects. The results show that after the plasmid DNA pcDNA3.0-NA immunized the mice, which could induce body to produce specific antibodies.3. Expression of recombinant neuraminidase in vitro and subcellular localization. Construct fusion gene expression vector pcDNA3.0/EGFP-NA carrying the EGFP reporter gene, and with pcDNA3.0-NA transfected into NIH-3T3 cells by PEI.At 48 h after transfection, the former directly was observed the position of the fluorescence in the cell under fluorescent inverted microscope; the latter which is detected by indirect immunofluorescence firstly, and then observed the location of fluorescence in the cell under a microscope. Results are consistent with two methods, fluorescence concentrated in the cytoplasm near the membrane, no fluorescence within the nucleus. Through the distribution of fluorescence in cells, the expressed recombinant NA proteins are located on the cell membrane.4. Study on disease-resistant activity of neuraminidase. Construct eukaryotic expression vector pcDNA3.0-NA, transfected into NIH-3T3 cells with the PEI-mediated method, selected by G418 to obtain positive monoclonal cell lines, NA gene expression can be detected by RT-PCR and Western blot. And then collected and purified cell lysate, using the NDV-CEF micro-cell inhibition test to determine the anti-viral activity of NA protein. The recombinant NA proteins against Newcastle disease virus infection analysis showed that recombinant NA protein has a certain biological activity of anti-Newcastle disease virus.
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