家禽流行性感冒病毒血球凝集素蛋白之表現及其應用

Expression and application of avian influenza virus hemagglutinin protein

陳建豪(Chien-Hao Chen)

指導教授 ： 王金和

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00466

摘要

1986年至2003年間台灣陸續有分離到H6N1、H7N1等弱毒型家禽流行性感冒病毒 (AIV)，在2004年間計有22間養禽場之38萬隻雞因發現弱毒H5N2 AIV潛藏而遭致撲殺。此表示目前台灣已有H5和H6亞型禽流感感染的情形，為瞭解目前於台灣廣泛流行之H5N2和H6N1 AIV是否會經由雞胚胎繼代而改變其HA力價，並且觀察病毒基因與毒力是否有改變，本實驗將家畜衛生試驗所分讓野外病毒3233 (H5N2) 與2838D (H6N1) 毒株分別以SPF雞胚胎尿囊腔連續繼代至45代，選殖出初代與45代之HA1基因進行其序列分析比較，結果僅於3233 (H5N2)毒株第45代之EID50較初代上升約100倍。在HA1基因序列比對分析繼代前後H5有2個胺基酸變異。在HA蛋白表現方面，本實驗也從野外病毒株H5N2與H6N1針對HA基因序列上誘導產生中和抗體區域進行選殖，並分別構築於原核表現系統載體pGEX-5X-1及真核表現載體pcDNA3.1載體上進行蛋白質的表現，以發展次單位疫苗，將原核表現載體表現出的蛋白進行純化後以皮下肌肉注射方式免疫接種於4週齡SPF雞隻，2週後進行補強注射，在免疫及補強注射後2週採其血液以血球凝集抑制試驗檢測H6亞型HI抗體力價呈陽性 (HI抗體力價為24~26)，而H5亞型HI抗體力價則呈陰性。另外，為暸解微脂體複合物是否可協同疫苗而增強免疫效力，本試驗利用市售新城病疫苗以微脂體與幾丁質進行包覆製成複合型疫苗，免疫接種實驗雞隻檢測其新城雞病HI抗體力價，實驗結果顯示疫苗以微脂體和幾丁質包覆後所產生的免疫效果比單獨以微脂體包覆的疫苗來的好。

關鍵字

家禽流行性感冒病毒 ； 血球凝集素蛋白 ； 疫苗

並列摘要

From 1986 to 2003, the avian influenza viruses (AIV) isolated in Taiwan were all low pathogenic. Some low pathogenic H5N2 AIVs were isolated in Taiwan in 2004, resulting in stamping 380 thousands chickens out from 22 poultry flocks. To test if the virus titers increased during passaging, H5N2 and H6N1 isolates were passaged in specific-pathogen-free embryonated eggs. After 45 passages, the virus titer of H5N2 increased 100 times, and two amino acid residues in HA1 of H5N2 virus were changed. The partial fragments of HA1 relating to neutralizing epitopes of the H5N2 and the H6N1 isolates were cloned in pGEX-5X-1 vector (prokaryotic expression) and pcDNA3.1 vector (eukaryotic expression) for expression. The purified recombinant proteins were intramuscualarly injected in 4-week-old SPF chickens for two times. Two weeks after the second immunization, the serum from H6-immunized chickens showed hemagglutination inhibition (HI) titers of 24 to 26, but no antibody was detected in H5-immunized chickens. To test the increase efficiency of liposome compounds on vaccine, Newcastle disease vaccines coated with liposome and with liposome/chitosan were used to immunize chickens. The results showed that Newcastle disease vaccine coated with liposome/chitosan increased HI titers in vaccinated chickens but not vaccine coated with lipsome alone.

並列關鍵字

Avian influenza virus ； Hemagglutinin protein ； Vaccine

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