開發單株抗體以準確偵測H5亞型禽流感病毒血球凝集素

Abstract

2014年起在歐洲、亞洲、非洲與美洲等地的野生鳥類和家禽中，檢出了H5N8高致病性禽流感病毒（highly pathogenic avian influenza virus, HPAIV）。2015年台灣亦首次檢測到H5N8 HPAIV，自此每年都有檢出該病毒。因此，開發快速檢測試劑與中和抗體對於減少農業和經濟損失至關重要。血球凝集素（hemagglutinin, HA）是流感病毒表面的醣蛋白，是檢測感染樣品中流感病毒亞型的主要標地。本研究利用大腸桿菌表現系統，去表現並純化出H5N8 HA1和HA2的重組蛋白，以應用於單株抗體的製備，並順利篩選出7H6C和YC8兩株能分別辨識H5N8 rHA1和rHA2的單株抗體。其中7H6C可以結合H5N1和H5N8的rHA，但不能結合H1N1、H3N2和H7N9的rHA，顯示它具有辨識H5亞型的特異性。而YC8則可以結合H1N1、H5N1和H5N8的rHA，但不能結合H3N2和H7N9的rHA，顯示它具有辨識H1和H5亞型的特異性。經分析YC8之抗原結合序列後，發現其所辨認之HA2序列於H1和H5亞型中具有高度保守性的區域。此外，7H6C和YC8還具有抑制H5N8 rHA之血球凝集能力，因此具有開發成診斷試劑和中和抗體的潛力。

In 2014, the highly pathogenic avian influenza A (H5N8) virus (HPAIV) was detected in wild birds and poultry in Asia, Africa, Europe, and America. H5N8 HPAIV was also detected for the first time in Taiwan in 2015. Since then, the threatness caused by H5N8 HPAIV still exists. As a result, the preparedness for epidemic prevention and decreasing the agricultural and economic lost is extremely important. Hemagglutinin (HA), a glycoprotein found on the surface of influenza viruses, is considered as the major target for detection of the influenza virus subtype in the infected samples. In this study, the recombinant H5N8 HA1 and HA2 proteins were expressed in E. coli, and were utilized to generate two monoclonal antibodies, named 7H6C and YC8. 7H6C was generated by rHA1 immunization and can bind the rHA proteins of H5N1, and H5N8, but can not bind the rHA proteins of H1N1, H3N2 and H7N9, indicating that it has H5-subtype specificity. In contrast, YC8 was generated by rHA2 immunization and can bind the rHA proteins of H1N1, H5N1, and H5N8, but can not bind the rHA proteins of H3N2 and H7N9, indicating that it has H1-subtype and H5-subtype specificity. The antigen binding sequence of YC8 is highly conserved among the H1 and H5 subtypes. Both of 7H6C and YC8 have great capability to inhibit hemagglutination, therefore they can be applied in developing the diagnostict kit and neutralizing antibodies in the future.