摘要
旨在构建能够表达禽网状内皮组织增生病病毒(reticuloendotheliosis virus,REV)env基因的重组马立克病毒(Marek′s disease virus,MDV),并对其生物学特性进行初步研究。以REV SNV株DNA为模板,PCR扩增其env基因,克隆进真核表达载体pcDNA3.1(+);以其为模板,PCR扩增整个REVenv基因真核表达盒并与卡那霉素抗性基因分别克隆进pUC18载体;以其为模板,利用含有MDV SC9-1株meq基因位点两侧同源臂的引物经PCR扩增REVenv真核表达盒以及卡那霉素抗性基因,利用Red E/T同源重组技术插入SC9-1的meq位点,最终利用阿拉伯糖诱导表达flp重组酶,敲除卡那霉素抗性基因,阳性重组质粒命名为SC9-env BAC。提取SC9-env BAC质粒转染CEF细胞,拯救重组MDV,利用MDV单抗H19以及REV单抗11B118经间接免疫荧光试验鉴定阳性病毒,命名为SC9-env。分析比较SC9-env在CEF细胞上的复制水平,利用动物试验初步评价SC9-env对SPF鸡的致病性以及SC9-env对SPF鸡感染MDV、REV的免疫保护效果。结果表明,利用同源重组技术构建的重组MDV SC9-env能够稳定表达REV env蛋白,且复制水平与亲本病毒SC9-1相似;该重组毒接种SPF鸡没有明显的致病性,且对rMd5的强毒攻击提供92%的免疫保护,与SC9-1差异不显著;SC9-env显著降低REV感染SPF鸡所引起的体重减轻以及灭活苗抗体下降。构建的表达REVenv的重组MDV对感染MDV、REV的SPF鸡具有良好的免疫保护效果。
The study aimed to construct a Marek＇s disease virus（MDV）recombinant expressing the env gene of Reticuloendotheliosis virus（REV）,and explore its biocharacteristics preliminarily.Firstly,envgene was amplified from the DNA of REV SNV strain and cloned into eukaryotic expression vector pcDNA3.1（＋）.The env gene expression cassette and the kanamycin resistance gene（kan^r）amplified from the recombinant plasmid were then cloned into pUC18 vector,respectively.Using the primers containing homologous arms on both sides of the meq gene of MDV strain SC9-1,the REVenv gene expression cassette and the kanr gene were amplified and integrated into the site of meq gene in the genome via Red E/T homologous recombination system.The kanr gene was further knocked out by the Arab sugar induced flg recombinant enzyme to obtain the recombinant BAC plasmid,naming SC9-env BAC.The recombinant MDV strain SC9-env was rescued after transfecting the SC9-env BAC plasmid into CEF cells,and identified by indirect immunofluorescence（IFA）using the MDV monoclonal antibody H19 and the REV monoclonal antibody 11 B118.Replication of SC9-env on CEF cells,the pathogenicity of SC9-env,and the protective efficacy of SC9-env against challenge of MDV and REV in SPF chickens were further evaluated.The results showed that the REV env protein was stably expressed by the recombinant virus SC9-env.The recombinant virus SC9-env showed similar replication capacity to the parental virus SC9-1,but had no pathogenicity to SPF chickens.In addition,SC9-env protected 92% of SPF chickens against challenge of MDV strain rMd5,which showed no significant difference with SC9-1,and eased the weight loss and the decreased inactivated antibody levels caused by REV infection.In conclusion,the constructed MDV recombinant expressing the env gene of REV possesses good protective efficacy against infection of MDV and REV in SPF chickens.
作者
周忠文
林桂芳
王好好
苏帅
崔治中
孙淑红
ZHOU Zhong-wen;LIN Gui-fang;WANG Hao-hao;SU Shuai;CUI Zhi-zhong;SUN Shu-hong(College of Veterinary Medicine,Shandong Agricultural University,Tai＇an 271018,China;Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention,Tai＇an 271018,China;Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention,Tai＇an 271018,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2018年第7期1482-1491,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
山东省科技发展计划项目(2014GNC110006)
国家自然科学基金青年基金项目(31402235)
山东省"双一流"奖补资金
