Preparation Of Targeting Dairy Goat With Swine Fever Virus Structural Protein E0 Gene

Classical swine fever is a deadly infectious disease which can cause huge economic losses of worldwide. So far, our country did not fully grasp the method of complete eradication of classical swine fever. In our country, the method of controlling classical swine fever virus is mainly using the traditional vaccines. But due to the epidemic form of classical swine fever occurred great changes, the traditional vaccines could not play a very good protection against classical swine fever,therefore, the research of the new gene engineering vaccine is the trend of the times. The structure protein of classical swine fever virus which has the value of immunization is E0 protein. Structural protein E0 of CSFV can induce neutralizing antibodies,so that the body produce protective immunity of lethal CSFV. The nucleotide sequence of encoding CSFV E0 protein is a high degree of conservation, so E0 protein of classical swine fever virus(CSFV E0) can be used as a target protein to prevent and control classical swine fever. In this paper, we use TALENs gene targeting technology to take structural proteins E0 gene of CSFV site-specific inserted in goat beta lactoglobulin gene first exon. It was verified that it could induce the expression of CSFV E0 in goat mammary epithelial cell, which was prepared for the high expression of CSFV structural protein E0 gene of goat mammary gland bioreactor and with CSFV E0 gene cells as nuclear donors for nuclear transfer making transgenic animals has laid the foundation.1. The expression of CSFV E0 gene targeting vector in mammary epithelial cells.TALENs expression vector for the first exon of the goat beta lactoglobulin gene and containing structural protein E0 gene of CSFV targeting vector which laboratory have been constructed good for were co transfected into goat mammary epithelial cells by using liposome mediated transfection method, then by drug(G418) screening to obtain monoclonal which needed to the identified of 5 ’and 3 ’ end. Those clones which were identified as positive clones were induced by hormone, and culture medium and cells were collected after induction. By RT-PCR and blot Western technology, it is proved that the transgenic positive cells can express and secrete CSFV E0 protein.2. The preparation of transgenic goats with CSFV E0 geneTALENs expression vector for the first exon of the goat β-lactoglobulin gene and containing CSFV E0 gene targeting vector which laboratory has been constructed good for were co transfected into goat fetal fibroblast cells by using electroporation method, then cell clones were screened with a drug resistance by drug(G418), by the 5 ’end and the 3 ’ end PCR to identify CSFV E0 gene is inserted into goat beta lactoglobulin gene in the expected site, then choose good shape cell lines. Using somatic cell nuclear transfer technology of preparation containing CSFV E0 gene cloned embryos were constructed 138 cloned embryos, transplantation 6 receptor goats, and each only 20-25 pieces of cloned embryos were transpianted and got a transgenic goat, by the 5 ’end and the 3 ’ end PCR identify that transgenic goat was targeting goats with CSFV E0 gene.In summary, using TALEN editing techniques maked CSFV E0 gene integrate into the first exon of the goat β-lactoglobulin gene, and combined with the somatic cells for transplantation and embryo transfer technology to prepare mammary gland bioreactor which contained CSFV E0 gene. Goat mammary gland secreted expression of CSFV E0 protein, which laid the foundation for the preparation of CSFV E0 gene engineering vaccine.