摘要
利用RT-PCR、同源重组等技术构建了含有猪流行性腹泻病毒(PEDV)S基因上3个片段的重组腺病毒质粒pAd-S498～638,pAd-S1～638,pAd-S498～1384,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化分别获得了重组腺病毒rAd-S498～638,rAd-S1～638,rAd-S498～1384。重组腺病毒于HEK-293A细胞连续传代至20代效价稳定,TCID50分别为每毫升10-6.29、10-5.75、10-6.5。应用猪流行性腹泻病毒阳性血清进行间接免疫荧光抗体试验,在3个腺病毒感染的HEK-293A细胞的胞质见有清晰荧光。将3个重组腺病毒分别免疫小鼠,结果均诱导产生较强的体液免疫应答。结果表明这3个重组腺病毒对S基因的3个片段均可成功表达,并具有较好的免疫原性,为PEDV重组腺病毒活载体疫苗的研究奠定了基础。
Three fragments were amplified by RT-PCR from the spike protein(S)gene of porcine epidemic diarrhea disease virus,and cloned to pshuttle-CMV.And three recombinant plasmids replication-defective human adenovirus serotype 5,pAd-S498-638,pAd-S1-638,and pAd-S498-1 384,were constructed by using the method of homologous recombination in E.coli BJ5183.These three recombinant plasmids were linearized with PacI and transfected to HEK-293A cells,and three recombinant adenovirus rAd-S498-638,rAd-S1-638 and rAd-S498-1 384 were obtained and purified in HEK-293A cells for three times with plaque purification assay.These recombinant adenoviruses could be stably passaged in HEK-293A cells with TCID50 of 10-6.29,10-6.5 and 10-5.75/mL respectively.The expression of S protein was detected in the HEK-293A cells infected with the recombinant viruses by IFA with antibody against PEDV.Mice immunized with these recombinant adenoviruses produced PEDV-specific ELISA antibody.The results indicated that the recombinant adenoviruses could express these three target proteins of PEDV and can be used for develop ment of the new PEDV vaccine.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2008年第10期1128-1132,共5页
Chinese Journal of Veterinary Science
关键词
猪流行性腹泻病毒
重组腺病毒
S基因
免疫原性
Porcine epidemic diarrhea disease virus(PEDV)
recombinant adenovirus
S gene
immunogenicity
