| Neuraminidase (NA) is one of the surface glycoproteins from the avian influenza virus, which is a glycoside exonuclease with digestion activity. NA plays a very important role in the invasion and dissemination process of virus. The primary role of NA is to eliminate the sialic acid of the virus particle surface and the infected cell surface, which is conducive to the releasing of the viral particle and preventing the virus particle from aggregation. In other words, NA is capable of degrading host cell receptors identified by influenza virus-sialic acid residues. NA also has immunogenicity, which can induce the body to produce specific antibodies that can provide complete protection against the same virus attacks, meanwhile provide cross protection against the virus attack of the same iso forms with different strains. As the unique features of NA, it is of great significance to fight against AIV, make it one of the hot against the AIV research at home and abroad, also provides a new idea for the preparation of transgenic poultry against avian influenza.Mx gene, namely Myxo-virus resistance gene, is one member of interfero-induced antiviral protein family that can generate antiviral Mx protein if induced by the type I (alpha/beta) and type III (lambda) interfern、double-stranded RNA or virus, that is currently the only gene found with the antiviral role of avian influenza. Present report in poultry shows:Mx protein in duck was found without antiviral activity. The antiviral specificity of chicken Mx protein is determined by an amino acid substitution at position631, Mx has antiviral activities when it is Asn; while it is Ser, Mx has not antiviral activities. This study aimed to clone the passively induced Mx protein gene and NA gene that can produce actively induced immunity, then constructed the single gene expression vector separately and Mx-NA co-expression vector, compared antiviral resistance of the NA gene, Mx gene, Mx-NA co-expression product. At last, the co-expression plasmid was mediated through testis injection to produce transgenic chickens as to open a new way of cultivating anti-avian transgenic chicken. The main research contents are as follows:1. Cloning, sequence analysis of NA and Mx genes, construction of recombinant eukaryotic co-expression vector. NA and Mx gene specific primers were designed and synthesized. cDNAs of NA gene of H5N1avian influenza virus isolated from diseased chicken and China Langshan chicken Mx gene were cloned by using PCR method. NA and Mx genes were cloned into pMD-19T vector.,the digestion and sequencing results confirmed that the pMD19-T-NA and pMD19-T-Mx were constructed correctly.Then NA and Mx genes were cloned into the eukaryotic expression vector PVITRO2to construct the single gene recombinant expression vector PVITRO2-Mx with PVITRO2-NA and co-expression vector PVITRO2-Mx-NA,the digestion confirmed that PVITRO2-Mx, PVITRO2-NA and PVITRO2-Mx-NA were constructed correctly. Sequence analysis revealed that:①The homology between NA gene of H5N1avian influenza virus isolated from chicken and that from human was the highest, the hydrophilic NA protein was mainly located in the cytoplasm and near the plasma membrane which belongs to secretory transmembrane protein.②The homology between Langshan chicken Mx gene and that from broiler was the highest, Mx protein was hydrophilic non-secretory proteins which was mainly distributed in the cytoplasm.2. Studies of PVITRO2-MX-NA recombinant expression in vitro and subcellular localization. First, the recombinant eukaryotic expression vector of pcDNA3.0-Mx was injected to immunize BALB/c mice to initially prepare chicken Mx protein antibody serum, and the serum antibody titer was determined of1:100. PVTTRO2-Mx-NA was then transfected into NIH-3T3cells by electroporation mediated method which were screened at48h after transfection, then total RNA of obtained positive cells was extracted. The mRNA and total proteins were detected to identify the expression in NIH-3T3cells by RT-PCR and indirect immunofluorescence method (IFA) respectively. Through inverted fluorescence microscope, it is found that Mx and NA proteins were both distributed in the cytoplasm,and there was no expression in nucleus, it showed that recombinant expression vector pVITRO2-Mx-NA successfully expressed the recombinant Mx and NA proteins. All these above provided a foundation for the establishment of stable-expressing cell lines and preparation of transgenic chicken.3. Study on antiviral activity of recombinant expression vector of "PVITRO2-Mx-NA". PVITRO2-Mx-NA was transfected into chicken fibroblast cells (CEF) through electroporation mediated method,.48h after transfection total RNA of CEF cellwas extracted, Mx and NA gene expressions could be detected by RT-PCR assay. Then the NDV-CEF mini-cytopathic effect inhibition assay system was used to evaluate the plus or coordinate antiviral activity for recombinant Mx protein and NA protein, the single gene antiviral group was also established and then statistical analysis was used for the data of the experimental groups to detect the antiviral effects differences of each group; at the same time antibodies for Mx and NA proteins we had fabricated were used in antiviral experiment as a positive control. Two kinds of protein against Newcastle disease virus (NDV) infection analysis displayed that:the combined application of Mx and NA protein can effectively improve the resistance of cells on the Newcastle disease virus, and better than that of single gene resistance; this result was also verified by the attack results of two combined antibodies.4. Preparing of resistant transgenic chicken through testis injection method. By liposome transfection method, the linearized recombinant plasmid PVITRO2-Mx-NA was injected into the mature cock’s testis by random dot. Repeated injection was conducted after one week after the first injection, and semen was collectted after injection of25d,50d,60d,70d, the semen of genomic PCR positive individuals was used for artificial insemination in hen to produce F1offspring. Conventional PCR, genomic PCR and Western blot technique were used for the detection of stable integration ability of exogenous Mx and NA genes in the cock body. Results showed that, PCR positive rate of6cocks used in the experiment was detected by33.33%, genomic PCR positive rate of F1generation blood was31.43%, positive rate of total protein Western blot of F1generation was28.6%. It can be confirmed that the exogenous combined NA and Mx gene can be integrated into the chicken genome and passed on to the next generation. |
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