摘要
根据已发表的犬瘟热病毒(CDV)Onderstepoort株的序列设计两对引物,以犬瘟热病毒云南株感染的Vero细胞收获病毒提取的总RNA为模板,RT-PCR扩增出H基因的939 bp(H基因前段,H1)和920 bp(H基因后段,H2)片段,分别将其定向克隆于PET-28a(+)中,将重组质粒转化宿主菌BL21(DE3)plysS,在35℃1、.0 mmol/L IPTG诱导下获得良好表达,经SDS-PAGE鉴定,表达的融合蛋白为34 ku,与预期大小一致。免疫印迹试验显示,该重组蛋白可被CDV Onderstepoort株多克隆抗体识别,表明该重组蛋白具有抗原性。
Two pairs of primers were designed and synthesized based on the Haemgglutinin（H） protein gene sequerce of the Onderstepoort strain of Canine distemper virus （CDV）. The templates were produced from the reverse transcription reaction, of which total RNA was isolated from vero cell infected Yunnan isolate of CDV. The H1 and H2 gene fragment were amplified by polymerase chain reaction （PCR）. The 939 bp and 920 bp fragment of the tN2R products digested with BamHl and PstI were cloned into the expression plasmid vector PET-28a（＋）, respectively. The recombinant were transformed into the BL2I （DE3） plysS and induced to express by 1.0 mmol/L IPTG at 35℃. The expressed products of 34 ku were identified by SDS-PAGE and Western blot with CDV Onderstepoort antiserum. The results revealed that the expressed fusion protein in vitro had the critical antigenit epitopes of H gene of CDV.
出处
《动物医学进展》
CSCD
2008年第1期9-12,共4页
Progress In Veterinary Medicine
关键词
犬瘟热病毒
H基因
克隆与表达
免疫印迹
Canine distemper virus
H gene
cloning and expression
Western blot
