Construction Of Nucleic Vaccine Vector For Swine's Transmissible Gastroenteritis Virus M And S Gene

Transmissible gastroenteritis virus (TGEV) is the etiological agent of transmissible gastroenteritis(TGE), which is a highly contagious enteric disease in piglets causing high mortality rate of up to 100% and resulting in severe economical losses to the affected farms. There are currently no effective TGEV vaccines which emphasizes the importance of developing new prevention and control methods for this pathogen. How to enhance expression of main antigen is a crucial thing. Between S protein including all sequence and major of the antigenic sites had same expressive lever, particularly the later was better for recombinant gene. N terminate on the M glycoprotein is mainly neutralize antibody. Therefore, In this research, the nucleic vaccine expression vector was constructed, which was consisted of regulating elements regulation region of bovineβ-casein and expression sequence M glycoprotein and S polypeptide cDNA. The results of research is as following:1. By the strategy of directional cloning, cDNA sequence M glycoprotein was fused with the commonly primary expression vector pBCC, then was cloned directionally into expression vector pEGFP-C1, the mammary gland-specific expression vector(pEBM) for TGEV M glycoprotein was constructed, which included resistant gene and EGFP reporter gene, it could also ensure EGFP and aimed gene express respectively.2. By the strategy of directional clone after double digestion by endonucleases, the commonly primary expression vector for nucleic vaccine expression vector was construced ,fristly the fusion of M glycoprotein and S polypeptide ,secondly the fusion of M glycoprotein and S polypeptide was conjoined with the commonly primary expression vector pBCP by endonucleases, then was cloned directionally into expression vector pEGFP-C1, the mammary gland-specific expression vector(pEBSM1) for TGEV was constructed.3. Internal ribosome entry site (IRES) was cloned by PCR, using directional cloning, co-expressive nucleic vaccine vector was constructed which M glycoprotein and S polypeptide only including antigenic sites was conjoined by IRES(pSM2), merging with the 5' and 3' regulation of bovineβ-casein, then was cloned directionally into expression vector pEGFP-C1, the mammary gland-specific expression vector(pEBSM2)for TGEV was constructed, which could also ensure EGFP and aimed gene express respectively.