共表达鸡IL-6和H5亚型禽流感病毒HA基因重组鸡痘病毒的遗传稳定性研究  

Genetic stability of recombinant fowlpox virus co-expressing cytokine IL-6 genes and HA gene of H5 subtype avian influenza virus

作　　者：陈素娟[1] 丁彦红[1] 钱程[1] 柴茂[1] 彭大新[1] 刘秀梵[1] 

机构地区：[1]扬州大学兽医学院农业部畜禽传染病重点开放实验室/禽类预防医学教育部重点实验室,江苏扬州225009

出　　处：《中国预防兽医学报》2012年第5期405-408,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：公益性行业科研专项(201003012);国家863计划(2011AA10A209);教育部创新团队(IRT0978);江苏高校优势学科建设工程;青蓝工程资助

摘　　要：为评价共表达鸡IL-6和H5亚型禽流感病毒HA基因重组鸡痘病毒(rFPV-AIH5AIL6)的遗传稳定性,本研究将其接种CEF,绘制生长曲线。结果显示,该重组病毒与亲本病毒的生长速度及蚀斑大小一致;在CEF连续培养20代,取第0代、5代、10代、20代重组病毒PCR扩增HA和IL-6基因进行鉴定,显示HA基因无核苷酸突变,IL-6基因在第20代时有1个氨基酸发生突变,但不影响该蛋白的功能区;间接免疫荧光试验证明所选代次病毒均能够表达HA蛋白,RT-PCR试验表明各代次病毒均可以表达鸡IL-6基因;通过蓝斑鉴定重组病毒纯度,结果显示所有蚀斑均为蓝斑。因此,该重组鸡痘病毒具有良好的遗传稳定性,符合兽医生物制品的生产要求。In this study, we investigated the genetic stability of the recombinant fowlpox virus （rFPV） co-expressing cytokine IL6 genes and HA gene of H5 subtype avian influenza virus （rFPV-AIH5AIL6） for evaluating its possibility for product. The rFPV was grown in chicken embryo fibroblast （CEF） and harvested at different time points. The growth curve and sizes of plaque diameters of rFPV were similar as that of wild type strain. The rFPV-AIH5A1L6 was then passaged in CEF for the 20 passages. The 5th, 10th, 20th passages of rFPV-AIH5AIL6 were selected for PCR amplification, sequence, and determination of genes expression. Sequencing analysis of HA gene and IL-6 gene showed that there was no any mutation in HA gene after CEF passage, while there was one amino acid mutant at the stop code in IL-6 gene at the 20th passages of rFPV-AIH5AIL6, but the mutation did not affect activity of the protein. The expression of IL-6 and HA gene in vitro were confirmed by RT-PCR and indirect immunofluorescence assay. The plaques of rFPV-AIH5AIL6 fi＇om selected four passages were all blue after the agar cover containing X-gal. These data indicate that rFPV-AIH5AIL6 has a good genetic stability.

关 键 词：禽流感病毒 HA基因 鸡IL-6 重组鸡痘病毒 遗传稳定性 

分 类 号：S852.4[农业科学—基础兽医学]