Expression of VP1 of Norovirus and VP1 fusion proteins by baculovirus expressin system

• Main Authors: Fu-Jen Tsai, 蔡馥任
• Other Authors: Tzong-Yuan Wu
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/x7488r

碩士 === 中原大學 === 生物科技研究所 === 105 === Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most toxic proteins causing the food borne disease and listed among the bio-weapon candidates. The molecule is formed by a heavy chain (HC) and a light chain (LC). The heavy chain C-terminal (HCC) is a receptor binding domain, when it binds to the receptors on axon terminal, the toxin will be endocytosed into nerve cells. In recent studies, the HCC domain has been targeted as a recombinant antigen to generate the neutralizing antibodies. In this study, we fuse the HCC domain with viral protein 1 (VP1) of norovirus (NoV) by fusion linker protein, the VP1 can bind to histo-blood group antigens (HBGAs) on intestinal epithelial cells and infect them, and we expect VP1 could carry HCc to pass through the intestinal epithelial, and be detected by the immune system, as a concept of botulinum neurotoxin oral vaccine. The flexible linker (GGGGS) 2 and rigid linker (EAAAK) 3 were chosen for comparing the effect of the virus like particles assembly, and added a histidine tag at the 3’ end of the HCC for the purification. After completing the plasmid, transform them into MAX Efficiency® DH10Bac™ competent cells to produce bacmid, and then transfect bacmid into sf21 insect cells to produce recombinant baculovirus. The virus titer were analysis by end point dilution assay (EPDA). Amplify the viruses by infecting sf21 with M.O.I = 0.5. Analysis the new viruses titer and infect the sf21 with M.O.I = 1 to produce the recombinant protein. We confirmed the VP1 protein by western blot assay, and used the same condition to produce and analysis the fusion protein VP1-linker-BoNT/A HCC-His. The single that we expect at ~120 kDa had confirmed, but there also showed other single at 58~60 kDa, we suppose those are the protein with the broken linker or it only translate the part of the amino acid sequence. In the future, we will purify the fusion protein, and observe whether the virus like particles are assembled by the electron microscopy.