| Avian influenza is caused by Orthomyxoviridae of avian influenza A virus which is a kind of poultry deadly infectious diseases. Once the outbreak of avian influenza, there will be not only poultry industry was brought huge economic losses, but also will pose a threat to the world public health and safety. Now, the commonly used measures to control of avian influenza outbreaks is immuning chickens. However, due to the avian influenza virus contains a variety of isoform and the variability of the virus are very quickly, so the vaccine prevention effect is difficult to achieve succeed. So it’s the major scientific significance and application valuepre to paration of anti-AIVs transgenic poultry by importing immune-related genes or specific shRNA which against avian influenza virus, that could make the poultry itself has the ability to resist avian influenza virus and effectively inhibit the replication of avian influenza virus and spread in poultry individual. For preparing anti-AIVs transgenic chickens successfully, this study will build an effective vector which is capable of inhibiting the spread of avian influenza virus and replication, and improve the transgenic efficiency.First, prepare anti-AIVs double expressing vector CS-RfA-EG-DP-RIG. Clone the cDNA of the immune regulatory genes RIG-I to construct the eukaryotic expression vector CS-RfA-EG-RIG. At the same time, according to the conserved amino acid sequence of the avian influenza virus gene to constructe6kinds of shRNA expression vectors of pENTR4-H1-DP1～DP5. By using the simulation avian influenza virus replication in luciferase reporter gene system and culturing the cells on the interference test to filter out the best shRNA expression vector pENTR4-H1-DP1; On this basis, through the Gateway LR reaction transfer shRNA expression sequence HI-DPI to vector CS-RFA-EG-RIG that successfully build the expression of RIG-I gene and the anti-AIVs shRNA double expression vector CS-RFA-EG-DP1-RIG. Introducing the vector into293T cells, use the quantitative PCR to test the RIG-I RNA overexpression. Co-transfected the double expression vector and analog avian influenza virus replication reporter gene system plasmids into293T cells, Luciferase assay results showed that the vector CS-RfA-EG-DP1-RIG inhibit the function of the avian influenza virus replication simultaneously. This study provides important material foundation for the preparation of transgenic chickens against avian influenza.Second, improve the efficiency of transgenic chicken. Although subgerminal cavity microinjection of lentivirus is the most effective chicken transgenic approaches, but it is a technical bottleneck that there is a low gene transfer efficiency and it’s difficult to obtain transgenic offspring. Therefore, this research was designed to improve the efficiency of the blastoderm cells infected with lentivirus two technical routes. First, mixing the virus solution and trypsin solution into one concentration, then inject the mixture into chicken embryo. And after the trypsin digestion, embryo cells were dispersed and the number of embryo cells contact with virus could increase. The result show, the blastoderm cells can sporadic and regrouped in the0.25%trypsin, and after hatching, there are no affect the normal development of the embryo. Injection of fluorescent protein-expressing recombinant lentivirus into the trypsin-worked embryo, after4days incubation, the fluorescence-positive cell ratio of embryonic cells was0.22%, while the direct injection of lentivirus chicken in fluorescence positive cells accounted for0.29%, that indicate the role of trypsin treatment to improve chick embryos viral transfection efficiency is not significant, the reason lies in its biological titer after lentivirus contact trypsin affected, therefore, to take protective measures to make the virus from trypsin future researchtopics. Second, put the embryos which injected lentivirus at16℃,22℃and28℃conditions for24hours, and then transferred to a temperature of38.5"C incubation, hatching the eggs4days and use the0.25%trypsin digestion the embryo into single cells, observing a ratio of fluorescence positive cells in the chick embryo. The control group of after inject the lentivirus directly into the incubator there is only one cell fluorescence was observed average per1000cells (0.09%), and the ratio of fluorescence positive cells in experimental group of16℃,22℃and28℃, respectively, are2.30%,0.86%and0.55%. The results showed that:the gene transfection efficiency increased20times in16℃for24hours after the embryo injection of lentivirus than direct incubation conventional, it’s expected to get highly chimeric transgenic chickens. take advantage of this improvement measures.This study provides an effective measure to prepare of transgenic chickens against avian influenza virus, and explore possible ways to improve the transfection efficiency in transgenic chicken. |
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