| The essence of animal mammary gland bioreactor technology is to obtain the target protein through the animal's milk. The protein, produced and modified by animal mammary gland, was not only high biological safety, but production was greatly improved. Thus, the goal protein can be used for massive production.Porcine circovirus virusis one of the smallest animal virus, mixed with fungi or infection virus, it could cause the pig's nephrotic syndrome, dermatitis, breeding disorder and other diseases. Porcine circovirus virus belong to the wreath virus family, divided into two kinds of serum type, it including PCV-1 and PCV-2, PCV-2 is a type of pathogenic serum. The length of PCV-2 gene is about 1.76 Kb, which includs 11 open reading frames. But the second reading frame ORF2 in its molecular mechanism has played a very important role, it is an important code of PCV-2 Cap protein. Because of its main immunogenicity of porcine circovirus virus, so the study of the second reading frame of PCV-2 is relatively more, and the existing experimental proof their can induce the body's protective antibody, longevity of porcine circovirus virus plays an important role in prevention and treatment.Swine fever virus is clarified as class A infectious disease by the World Animal Health Organization, the death of this disease is as high as 80%-90%. the impact on pig is very huge, and it caused huge economic loss. Swine fever virus gene sequence length of about 12.3 Kbp, single strand RNA virus is chain, belong to the yellow virus when virus genera. Swine fever disease is mainly caused by the swine fever virus, actually made of protein enzyme with the host polymer formed by the four kinds of structure of the protein, the E2 protein gene encoding can reacts with the antigenic determinant. And CSFV E2 gene encoding capsule membrane proteins are the major protective antigen, swine fever virus can cause the body to produce resistance to virus attacks. Therefore E2 capsule membrane protein has been used for swine fever virus prevention and control in the process of the main research object, has become a new vaccines and antigen detection in recent years the main target genes.To obtain the goat fetal fibroblasts which can steady express porcine circovirus virus type 2(PCV-2) ORF2 gene and CSFV E2, the PCV-2ORF2 gene, CSFV E2 gene and the screening NEO expression cassette were inserted into p BC1 plasmid, the recombinant eukaryotic expression vector was named p BC1-ORF2-NEOand p BC1-E2-NEO. Then, the plasmid was transfected into the dairy goat fetal fibroblasts(g FFs) cells by liposome mediated and screened to get the positive cells g FFs with stable resistance by G418. The analysis of RT-PCR indicated that 689 bp ORF2gene,1131 bp E2geneand the 1953 bp NEO expression cassette were both amplified in g FFs, which was consistent with the expected result. Western-Blot analysis showed that the 37 k D and 42 k D expression product was obtained and can also react with protein antibody of rabbit anti PCV-2 and CSFV.Results showed that the ORF2 gene and E2 was successfully inserted into the chromosome of goat fetal fibroblast cells, and the expressed product conducted good reaction ogenicity. Expression product also had good reaction ogenicity, which can provide the theoretical and material basis for building the dairy goat mammary gland bioreactor for Cap protein and E2 protein. |
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