Heterologous expression of Hemagglutinin and Neuraminidase of Avian Influenza virus in Kluyveromyces lactis

• Main Authors: Li-Tzu Chin, 秦儷慈
• Other Authors: Yu-Chung Chiang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/08692336466229051826

碩士 === 國立屏東科技大學 === 生物科技研究所 === 98 === Avain influenza virus（AIV）, belongs to the family Orthomyxoviridae. This viruses are enveloped and possess 8 segmented genome of single-stranded negative sense RNA. The glycoproteins on the envelope, hemagglutinin (HA) and neuraminidase (NA) are classified antigenically into at least 16 HA groups and 9 NA groups. AIV are divided into highly pathogenic avian influenza (HPAI) virus or low-pathogenic avian influenza (LPAI) virus, some of the H5 and H7 subtypes have been characterized as HPAI, which caused the death of poultry, wildlife and the lethal human infection, threatening the poultry industry and human health worldwide.We used Kluyveromyces lactis expression system to produce Hemagglutinin (HA) amd Neuraminidase (NA) of AIV. The expression of DNA fragment amplification by PCR and clone into the vector pKLAC1 follow expression protein by Kluyveromyces lactis. The expressed proteins were identified by western blotting and purified by Ni-agarose beat. Molecular weight of NA was 51 kDa, but HA larger than predicted size (64 kDa) was about 100 kDa. We presumed the glycosilation occurs in yeast. Pretreatment HA protein by PNGase F was remove N-linked carbohydrates and shift the molecular weight to about 80 kDa. In addition, both HA and NA protein produced from K.lactis have native enzyme activity of AIV. This result may provide further research in detection and diagnosis in avain influenza viruses.