Production and Immunization of H6 subtype Influenza HA Virus-Like Particles by Baculovirus Expression System

• Main Authors: Yu-Wei Feng, 馮昱維
• Other Authors: Min-Ying Wang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/51600765142645325143

碩士 === 國立中興大學 === 生物科技學研究所 === 100 === Vaccination for eliciting neutralization antibody against HA is the major prophylactic strategy to reduce diseases caused by an influenza virus infection. Virus-like particles (VLPs) are looking as a newly promised vaccine candidates due to their lack of viral genetic materials and morphological resemblance to authentic viruses. In this study, we want to develop a subunit vaccine against H6 subtype influenza viruses, which are frequently identified in wild birds or domestic poultry. For this purpose, HA gene from a local influenza strain, A/chicken/Taiwan/2838V/00 (H6N1), was constructed and expressed in Hi-5 insect cells. The results demonstrated that the expression of HA on cell membrane conferred the infected Hi-5 cells with the function to agglutinate erythrocytes. Furthermore, the hemagglutination was also detectable from the medium, which suggested some HA molecules were released into extracellular environment. The infection has been optimized to have a highest HA expression level using the multiplicity of infection (MOI) of 0.001. 20-60% sucrose density gradient ultracentrifugation analysis was demonstrated that these extracellular HA molecules were released in a particle form with the buoyant densities between 1.16-1.21 g/cm3. The existence of HA-VLPs with a size of 80-120nm and the prominent HA spikes were confirmed by electron microscopy. On the other hand, analysis of the HA protein in the intracellular, the hemagglutination was also detectable from the lysate, which suggested HA protein with function were produced in the intracellular. 20-60% sucrose density gradient ultracentrifugation analysis was demonstrated that these intracellular HA molecules were produced in a particle form with the buoyant densities between 1.13-1.17 g/cm3. The existence of HA-VLPs with a size of 80-100nm were confirmed by electron microscopy. The HA-VLPs have function from pH 6-10 down to 37℃. Animal immunization demonstrated that HA-VLPs elicit high titers of hemagglutination inhibition (HI) antibody to protect chickens. This study demonstrated that expression of influenza HA by baculovirus expression system is functional and can form HA-VLPs, and that can protect chicken.