摘要
本研究以鸡传染性法氏囊病超强毒Gx株(野毒株)基因组为模板,用蛋白酶K法提取病毒基因组核酸dsRNA,在cDNA克隆的5'端上游引入了T7启动子序列,采用Long-accurateRT-PCR(LA-PCR)一步法扩增并克隆了病毒基因组A节段与B节段全长cDNA。序列测定结果表明,基因组A节段全长共3267个核苷酸,包括5'及3'端的非编码区和两个部分重叠的开放阅读框,基因组B节段全长共2843个核苷酸,包括5'及3'端的非编码区和一个开放阅读框。将IBDV-Gx株的A节段全长基因组及B节段全长基因组分别克隆入pMD18-T载体,构建成pMD-A、pMD-B两个带有T7启动子的重组质粒。两个重组质粒线性化后,进行体外转录,然后共电转染于鸡胚成纤维细胞37℃培养72h并传代。收获的细胞传代培养物分别用RT-PCR、间接免疫荧光、蚀斑试验等方法进行鉴定,结果用RT-PCR扩增出了VP3及VP5基因片段,间接免疫荧光检测到了特异性的荧光抗体,蚀斑试验结果表明蚀斑形成单位为3×103PFU/mL。
In this study, isolated dsRNA in proteinese K from the vv-IBDV designated Gx strain and amplified full-length cDNA of segment A and B by long-accurate reverse-transcription polymerase chain reaction （LA-PCR） were cloned into PMD18-T vector and sequenced. The cloned segment A contains 3 267 nucleotides in full-length and includes two partially over lapping Open Reading Frames（ORF1 and ORF2） flanked by 5＇ and 3＇ noncoding regions（NCR）; the cloned segment B contains 2 843 nucleotides in full-length and includes one Open Reading Frame flanked by 5＇ and 3＇ noncoding regions （NCR）. A T7 promoter was introduced immediately upstream of 5＇-end of segment A and B. After segment A and B cloned into T vector, we constructed two regrouped plasmids with T7 promoter, named pMD-A and pMD-B. The linearized DNA was used to produce in vitro transcripts with vitro transcripts according to the manufacturer＇s instructions, The products was co-transfected in the CEF cells and incubated at 37℃ 72 h and passed passages in CEF cells. We examined the product by RT-PCR, IFA and plaque assay, the results of PCR showed that VP3 and VP5 were amplified; the results of IFA showed that regrouped DNA was developed; the results of plaque assaying suggested that plaque forming unit was 3×10^3 PFW/mL.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2006年第4期369-374,共6页
Chinese Journal of Preventive Veterinary Medicine
关键词
鸡传染性法氏囊病病毒
Gx株全长cDNA
感染性分子克隆
infectious bursal disease virus
full-length genomic cDNA clone of Gx strain
infectious molecular clone
