摘要
根据PCV2 ORF2基因的序列设计两条引物从PCV2的PK15细胞培养物中扩增出ORF2基因(702 bp)。将此基因片段克隆入pMD 18_T载体,经酶切、测序鉴定筛选出重组质粒pMD_ORF2。将PCV2 ORF2基因克隆入pAdeasy腺病毒载体系统的穿梭质粒pAdTrack_CMV中,获得重组穿梭质粒pAdTrack_CMV_ORF2,将重组质粒与腺病毒骨架载体共转化大肠杆菌BJ5183,通过细菌内同源重组获得重组腺病毒质粒pAdCMV_ORF2,然后用PacⅠ线性化后转染293细胞,在293细胞内包装出重组腺病毒。通过荧光显微镜观察、PCR检测和Western blot检测证明PCV2 ORF2基因在293细胞内获得表达,ORF2多肽具有良好的免疫原性。
According to the sequence of PCV2 ORF2 gene, two pairs of PCR primers were designed and ORF2 gene were amplified from PK15 cells inoculated with PMWS samples. The PCR products were cloned into pMD 18-T vector and the recombinant plasmids were obtained. Then, the ORF2 gene was subcloned into the shuttle plasmid of pAdTrack-CMV. The recombinant plasmid and adenoviras backbone DNA were cotransformated into E. coli BJ5183 and the the recombinant adenoviras genomic DNA was obtained. After transfecting 293 cell line with recombinant adenovirus the genomic DNA with the lipofectamine method, the recombinant adenovirus carrying ORF2 gene was screened under fluorescent microscopy, PCR analysis and Western blot analysis. The results demonstrated that the protein expressed by the recombinant adenoviras system could react with polyclonal antibody against PCV2 by Western blot, showly the good antigenicity.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2005年第5期348-351,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家863计划资助项目(批准号:2003AA241110)
广东省农业攻关项目(批准号:2003B21404)
