摘要
为了检测表达鸡传染性喉气管炎病毒糖蛋白gB基因重组鸡痘病毒 (rFPV -ILTVgB)的稳定性 ,我们对重组病毒进行 6代和 16代克隆纯化 ,对纯化后的病毒通过转瓶连续培养传 2 0代。结果发现第 6代纯化病毒 (rFPV -ILTVgB -C6)在传代过程中有白斑出现 ,说明病毒纯度不够 ;经过 16代纯化的病毒 (rFPV -ILTVgB -C16)对细胞的嗜性加强 ,病毒的效价进一步升高 ,2 0代传代后蓝斑率仍然为 10 0 % ,PCR的检测进一步证明插入的外源基因能在病毒的长期传代过程中稳定地遗传。抽取病毒细胞传代过程中的第 5 ,10 ,15 ,2 0代次的病毒翅膀内侧无血管处皮下接种 2周龄SPF鸡 ,2 0天后分为两组 ,分别用传染性喉气管炎病毒WG株和鸡痘病毒 10 2株攻击 ,结果不同代次的重组病毒均可以使免疫鸡抵抗传染性喉气管炎病毒和鸡痘病毒强毒的攻击。鸡体内病毒传代试验表明 ,重组病毒在接种部位仅存在 6天 ,之后就检测不到病毒 ,重组病毒通过SPF鸡连续传代 ,不存在病毒返祖的可能。由此可以得出一个结论 ,rFPV -ILTVgB在结构上和免疫原性上是稳定的 ,无论是在体外传代 ,还是在体内均可以稳定遗传 ,不存在因为接种重组疫苗而给免疫鸡群带来安全威胁的可能 ,完全可以作为疫苗毒株进行商品化开发。
In order to evaluate the inherent stability of Recombinant Fowl poxvirus (rFPV-ILTVgB)expressing glycoprotein B (gB)gene of Infectious Laryngotracheitis virus(ILTV). The rFPV-ILTVgB-C6 and rFPV-ILTVgB-C16 (which was obtained by six and sixteen passages of plague purification) was passaged in rotating bottle culture for 20 times. The purity of the virus was detected by blue plaque assays. The results showed that rFPV-ILTVgB-C16 is stable, with 100% plaque blue, while rFPV-ILTVgB-C6 has white plaques in the following passage. PCR detecting results showed that gB gene of ILTV was stable in rFPV-ILTVgB-C16 after 20 times passaging, and the infectivity of rFPV-ILTVgB to chicken embryo fibroblast cells increased. In order to test the immune potency of rFPV-ILTVgB-C16, the viruses from 5th,10th,15th and 20th passage were chosen to inoculate 14-days-old SPF chickens through subcutaneous route under the wing of chicken (SPF) at the dose of 5 10 6 PFU/chicken 9 respectively. All groups of the chickens were challenged with ILTV strain WG and FPV strain 102 at the 20th day after inoculation, the results showed that all chickens were protected form challenge, indicating that the molecular structure and immunogenicity of the recombinant virus is stable. The in vitro virus isolation experiment showed that the recombinant virus could survive at the inoculation site for only six days. It further confirmed that rFPV-ILTVgB is safe.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2001年第6期426-429,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家 8 63计划资助 ( 10 1- 0 5 - 0 3 - 1)
