摘要：
[目的]对从河南某地区发病鸡群中分离出的1株IBDV进行驯化使适应永生细胞DF-1,并对其VP2基因进行克隆和生物信息学分析,对IBDV分离株及其细胞适应株的VP2高变区及七肽区进行比较.[方法]测定IBDV分离株X1对10日龄SPF鸡胚的半数致死量(ELD50);通过SPF鸡胚-CEF-DF-1途径对X1进行驯化,使其适应永生细胞DF-1;根据IBDV VP2基因序列设计1对特异性引物,应用RT-PCR技术克隆IBDV分离株和细胞适应株的VP2基因并测序,将其与参考毒株进行同源性和进化树分析,同时对二者在VP2高变区及七肽区的推导氨基酸序列进行对比分析.[结果]IBDV分离株X1对10日龄SPF鸡胚的ELD50为10-8 mL-1,经驯化获得了细胞适应株C1.对2株病毒的VP2基因进行克隆并鉴定,测序分析得IBDV X1株在第222,256,294和299位上具有A、I、I、S4个特征性氨基酸,与IBDV超强毒株(vvIBDV) HK46 VP2基因氨基酸序列的同源性高达99.3％,确定X1株为IBDV超强毒株.同源性及进化树分析结果显示,C1株与IBDV减毒株CEF94亲缘关系较近,同源性较高,为99.6％,确定C1株为IBDV减毒株.在VP2高变区及七肽区推导氨基酸比对中显示,C1株第330位精氨酸R取代了X1株在330位的丝氨酸S;第222位氨基酸由A变成P;决定病毒毒力的位点第256和284位分别由I、A变为V和T.[结论]成功驯化了1株vvIBDV使其适应细胞,初步揭示vvIBDV在适应细胞、从超强毒力向弱毒力转化的过程中,VP2高变区及七肽区氨基酸序列有明显变化.%[Objective] A IBDV strain isolated from the diseased flocks in Henan was cultivated to adapt to immortalized cell DF-1 and its VP2 gene was cloned and analyzed by bioinformatics.The VP2 hypervariable region and heptapeptide region of the IBDV isolate strain and the cell-adapted strain were compared.[Method] The ELD50 of IBDV isolate X1 on 10 days old SPF chicken embryos was determined.The strain X1 was adapted to the immortalized cells DF-1 through SPF embryo,CEF and DF-1.According to the IBDV VP2 gene sequence,a pair of specific primers were designed and used to clone and sequence VP2 genes of the IBDV isolate and the cell-adapted strain by RT-PCR.The homology and phylogenetic tree analysis were conducted and the VP2 hypervariable and heptapeptide regions were deduced.[Result] The ELD50 of IBDV isolate X1 was 10-8 mL-1 for 10 days old SPF chicken embryo,and the cell-adapted strain C1 was obtained by acclimation.The VP2 genes of the two viruses were cloned and identified.The IBDV strain X1 had the characteristic amino acids A,I,I and S at positions 222,256,294 and 299,and the amino acid sequence homology of VP2 gene with vvIBDV HK46 was 99.3％.Based on above information,the IBDV isolate X1 was determined as vvIBDV.The homology and phylogenetic tree analysis showed that strain C1 was close to the attenuated strain CEF94 with homology of 99.6 ％ and the strain C1 was confirmed as attenuated IBDV.In the VP2 hypervariable region and heptapeptide region,the position 330 arginine R atthe cell-adapted strain C1 replaced the strain X1 at position 330 serine S,the 222 amino acid changed from A to P,and the virulence determining sites 256 and 284 were changed from I and A to V and T.[Conclusion] This study successfully domesticated a cell-adapted strain vvIBDV,preliminarily revealed that it had obvious changes of amino acid sequences in hypervariable region and heptapeptide region of VP2 during adapting cells and the transforming virulence from strong to weak.
