家禽傳染性支氣管炎病毒之棘突醣蛋白與血球凝集特性之關係

Abstract

雞傳染性支氣管炎病毒（IBV）之棘突醣蛋白（S）為一病毒封套表面與病毒之附著、血球凝集反應、中和反應、致病性相關的一個重要蛋白質。其中一株台灣一型傳染性氣管炎病毒2575/98於75代雞胚蛋繼代後失去其野外毒株原有之血球凝集特性，發現減毒株之S1基因上有兩個點突變，因此將野外毒株與減毒株之S1蛋白基因選殖並且轉染至昆蟲表現細胞中；重組桿狀病毒不論是野外毒株或減毒株之重組S1蛋白都只能表現出血球吸附之現象而沒有血球凝集之能力，懷疑因為S2蛋白可能會影響S1蛋白之三級結構。本實驗之主要目的為研究在全段S蛋白上減毒株之兩個點突變是否對於血球凝集之消失扮演一個重要角色，運用桿狀病毒表現系統表現野外株、減毒株之全段S蛋白與具有點突變之S蛋白（分別為第166與280個核苷酸；P56T、A94S），另刪除與血球凝集相關之S1-D區域（DS1D）；結果顯示rBac-2575S與rBac-PM166S分別具有512倍與16倍之血球凝集力價，rBac-2575-75S、rBac-DS1D與rBac-PM280S則沒有，由此可知第166個核苷酸點突變可能會降低血球凝集力價，而280則是血球凝集力價消失之主要原因；細胞吸附之結果上，除了rBac-DS1D處理組沒有血球吸附能力之外，被其他重組病毒感染之細胞仍然能夠表現血球吸附之現象。結果顯示166與280兩個核苷酸點突變對於IBV之血球凝集性是有影響的。

Spike (S) protein of infectious bronchitis virus (IBV) is a major mediator of cell attachment, hemagglutination (HA) activity, neutralization and pathogenicity. One of IBV strain 2575/98 loses its HA activity after serial attenuating passages in SPF chicken embryos and attenuated 2575/98 strain containing two point mutations in S1 protein. However, insect cell cultures transfected with the recombinant baculoviruses carrying either the wild-type or attenuated form of IBV 2575/98 S1 protein exhibited the hemadsorption (HD) activity but no HA activity. It is considered that S2 protein might influence the tertiary structure of S1 protein. The main objective of this research is to investigate whether these two point mutations on full S protein gene are the crucial factors that are responsible for HA activity. The baculovirus expression system is used to express the full IBV S gene with two point mutations (166th and 280th nucleotides; P56T, A94S). The rBac-2575S and rBac-PM166S possessed HA titers of 512 and 16, but rBac-2575-75S, rBac-DS1D and rBac-PM280S do not. The results show that the point mutation on 166th nucleotide cuts down the HA titer, and 280th nucleotide is the main reason to make it lost. Moreover, the cells that are infected by all the recombinant baculoviruses bear hemadsorption activity except rBac-DS1D. In conclusion, the two point mutations account for the HA activity of IBV.