【摘要】 设计、合成FMDV非结构蛋白 3B基因的PCR引物 ,并在引物 5’端和 3’端分别加入含BamHI和HindⅢ限制性酶切位点序列。以FMDV基因组RNA为模板 ,利用RT_PCR技术扩增 3B基因 ,得到的基因片段经BamHI/HindⅢ双酶切后与转座载体pFASTBACHTa连接 ,转化宿主菌DH10BAC ,在含庆大霉素、卡那霉素、X_gal、IPTG的LB平板上 37℃培养 2 4h ,提取白色菌落 ,从中提取重组穿梭载体Bacmid 3B ,与脂质体共同转染昆虫细胞sf9,得到重组杆状病毒 ,病毒经传代扩增后感染High 5细胞 ,表达目的蛋白———FMDV非结构蛋白 3B。SDS_PAGE及WesternBlot结果显示 ,本研究成功构建了Bacmid₃B重组表达载体 ,并在昆虫细胞中表达了NSP₃B蛋白 ,该表达产物可与FMDV感染的猪、牛血清产生免疫反应。更多还原

【Abstract】 The FMDV non_structural protein 3B primers were designed and synthesized.The 5’ end and 3’ end of primers were added the sequence of restriction endonucleases BamH I and HindⅢ respectively.The 3B gene coding region was obtained from the viral genome RNA by RT_PCR.The amplified fragment was cloned into baculovirus transfer vector pFASTIBAC HTa.The recombinant vector was transformated into DH10BAC and cultured in LB yolutes vowtuiing GM,Ka,X_gal and ITPG.The white colonies were selected and Bacmid₃B recombinant vectors were used for transfecting Sf9 cells mediated with CELLFECTIN reagent.After three to four times amplification of the viral stocks,the NPS₃B was expressed in High 5 cells.The SDS_PAGE result showed that recombinant eukaryotic vector was constructed and the NSP₃B was expressed in insect cells successfully.The specafic electrophoretic band in SDS_PAGE（16KD） was noted and authenticated by Western Blotting.更多还原