傳染性支氣管炎病毒S1醣蛋白質與血球吸附、血球凝集關係之探討

Abstract

中文摘要 於台灣所分離到的家禽傳染性支氣管炎病毒毒株當中也有數個毒株經過神經胺酸酶的處理後會表現出血球凝集 (Hemagglutination, HA)的特性。其中一台灣一型家禽傳染性支氣管炎病毒2575-5在經過神經胺酸酶處理過後會表現出血球凝集的特性，但該毒株之減毒株在神經胺酸酶處理後則不會表現出血球凝集的特性，為瞭解此血球凝集特性之改變，本實驗將減毒株之S1蛋白基因進行選殖，另將2575-5的S1蛋白基因進行點突變，並將此點突變與選殖之產物放入pFastBacHT A質體中，接著利用同源重組將在pFastBacHT A內的S1醣蛋白轉移至bacmid上並轉染Sf-9細胞產生重組之桿狀病毒，以桿狀病毒蛋白表現系統進行表現蛋白，經過繼代三代後以蔗糖梯度離心純化重組之病毒之後進行血球凝集與血球吸附試驗，經由此實驗可以表現出所欲研究之S1蛋白並且將兩者進行比較來了解該蛋白上之特定胺基酸改變是否與其血液凝集特性改變有關，且可瞭解所表現之蛋白是否與傳染性支氣管炎之S1蛋白有相同之生物特性。

Abstract A Taiwan local infectious bronchitis virus strain 2575 TW-I showed hemagglutination activity after neuraminidase treatment while its attenuated strain did not. In order to investigate this altered hemagglutination activity, we cloned the S1 gene of the attenuated strain. The nucleotide differences between original and attenuated strains were mutated in the S1 gene by site direct mutation. The S1 gene in the pFastBacHT A was transferred to the bacmids in DH10Bac. The Bac-to-Bac baculovirus expressing system was used to express both wild and attenuated strain S1 proteins. After transfection and passages, the P3 stock of recombinant baculovirus was generated and purified by sucrose gradient separation. The hemagglutination (HA) activity and cell-associtaed hemadsorption (Hd) activity of the expression proteins were examined. Both recombinant baculoviruses showed Hd but no HA activities. The Hd activities can be inhibited by anti-IBV antiserum. Through this method, desired S1 protein mutant could be expressed with the baculovirus expressing system and further studied for biological activity and immunological reactivity.