表达鸡马立克氏病病毒gB基因重组鸡痘病毒的遗传稳定性及生物安全性评价

为了评价表达鸡马立克氏病病毒gB基因重组鸡痘病毒(rFPV-gB/R)的遗传稳定性,我们将纯化后的重组病毒在CEF单层上连续传30代,引起细胞病变的速度和形态均未发生明显变化;覆盖含X-Gal的琼脂引起的空斑均为蓝色;间接免疫荧光实验证明rFPV-gB/R中的gB基因始终能稳定表达;序列测定结果表明,重组病毒在细胞上连续传30代、在SPF鸡上连续传5代后,gB基因序列没有发生任何变化;以0、10、20、30代重组病毒制成冻干疫苗进行的实验室免疫效力实验表明,细胞连续传代后rFPV-gB/R仍然保持了原有的免疫原性。可见重组鸡痘病毒gB基因的结构和免疫原性都是高度稳定的。为了评价rFPV-gB/R的生物安全性,我们将rFPV-gB/R通过SPF鸡连续传5代,检测病毒在鸡体的存在部位及其消长、生长繁殖性能和毒力变化;将rFPV-gB/R免疫鸡与未免疫鸡同笼饲养,攻击FPV-102E6强毒,以检测rFPV-gB/R感染鸡的接触传染性。结果显示,rFPV-gB/R在鸡体的存在时间大约为7d,在体内仅存在于接种部位;鸡体传代后痘病毒毒力有一定程度下降,gB基因核苷酸序列未发生任何变化;同居未免疫SPF鸡在痘病毒强毒攻击后全部发痘,可见重组病毒免疫鸡没有接触传染性,能在鸡体内稳定地传代,rFPV-gB/R具有高度的生物安全性。

Genetic stability and biological safety of the recombinant fowlpox virus expressing glycoprotein B gene of serotype 1 Marek's disease virus

In order to evaluate the genetic stability of the recombinant fowlpox virus expressing glycoprotein B gene (rFPV-gB/R)of CVI 988/Rispens strain of Marek's disease virus,rFPV-gB/R was passaged in chicken embryo fibroblast (CEF)continuously for 30 times and then in specific pathogen-free(SPF)chickens for 5 times.No significant changes in plaque development were observed throughout period of passages.Protective efficacies of rFPV-gB/R remained unchanged up to passage 30.Sequence analysis of the gB gene derived from different passages showed no nucleotide changes.The rFPV-gB/R was evaluated for its stability in SPF chickens and its ability of horizontal transmission.SPF chickens were challenged with FPV strain of 102E6 30 days after rFPV-gB/R inoculation.The results showed that chickens immunized with rFPV-gB/R were protected from challenge.However the co-caged chickens that were not inoculated all developed symptoms after challenge.The virus could only be isolated at the inoculation site for 7 days after inoculation.The gB gene of rFPV-gB/R remained stable throughout 5 time passages in SPF chicken,with no nucleotide change.These results confirmed that rFPV-gB/R was stable and safe to use.