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羊口疮病毒榆林株B2LF1L串联基因在昆虫杆状病毒系统的表达
冯 平1,2, 李云章1, 孙丰廷,等3
1.内蒙古农业大学 兽医学院;2.榆林学院 生命科学学院;3.武汉中拓康明生物科技有限公司
摘要:
【目的】克隆分析羊口疮病毒(ORFV)榆林株(ORFV-Yulin)的F1L基因序列,通过昆虫杆状病毒表达B2LF1L串联基因。【方法】采用PCR方法扩增ORFV-Yulin株F1L全长基因,测序后对其进行序列分析和遗传进化分析。扩增ORFV-Yulin株B2L融合基因(rB2L)和 F1L融合基因(cF1L),利用碱基linker连接,通过重叠PCR方法扩增获得ORFV rB2L-linker-cF1L重组基因(rB2LcF1L),将其与昆虫杆状病毒表达载体pBac5连接构建表达载体pBac-rB2LcF1L。将pBac-rB2LcF1L包装出杆状病毒,侵染sf9细胞,通过SDS-PAGE、Western-blotting以及质谱鉴定等方法验证目的基因rB2LcF1L的表达。【结果】克隆获得了1 029 bp的F1L全长基因,该基因高度保守,与FJ-MH2015株的序列相似性最高,核苷酸和氨基酸的相似性分别为99.0%和99.4%。系统进化分析表明,ORFV-Yulin株与XP(KU199840.1)、FJ-MH2015(KM675409.1)以及Shanxi(HQ221964.1)株同处于一个分支。PCR扩增获得了855 bp的cF1L基因、1 180 bp的rB2L基因及1 983 bp的rB2LcF1L基因,成功构建了pBac-rB2LcF1L载体,并包装出相应的杆状病毒。B2LF1L串联基因通过昆虫杆状病毒表达系统获得成功表达,表达产物的分子质量为80 ku,且有部分重组蛋白能分泌到细胞培养基中。【结论】ORFV-Yulin株的B2LF1L串联基因在昆虫杆状病毒系统表达成功。
关键词:  羊口疮病毒  F1L基因  F1L+B2L串联表达  昆虫杆状病毒  基因表达
DOI:
分类号:
基金项目:陕西省科技厅农业科技创新与攻关项目(2016NY-120);陕西省科技厅重大统筹项目(2014KTDZ02-01);陕西省教育厅专项科研计划项目(14JK1860);榆林学院重点项目(14YK41)
Co-expression of B2L and F1L genes of Orf virus strain ORFV-Yulin in baculovirus system
FENG Ping,LI Yunzhang,SUN Fengting,et al
Abstract:
【Objective】This study cloned and analyzed the sequence of F1L gene of Orf virus (ORFV) strain Yulin (ORFV-Yulin) and its co-expressed B2L and F1L genes in baculovirus system.【Method】The full-length gene of F1L of ORFV-Yulin was amplified by PCR,and the sequenced gene was analyzed by genetic evolution.The fused gene of B2L (rB2L) and F1L (cF1L) of ORFV-Yulin was amplified by PCR and the ORFV B2L-linker-cF1L recombinant gene (rB2LcF1L) was amplified by overlapping PCR method with linker.The baculovirus expression vector pBac-rB2LcF1L was constructed with pBac5 and rB2LcF1L to get baculovirus expressing rB2LcF1L gene after the infection of sf9 cells.The expressed product was identified by SDS-PAGE,mass spectrometry and western blot.【Result】The length of F1L gene obtained by cloning was 1 029 bp.The gene was highly conserved and had the highest similarity with FL MH2015 strain with the nucleotide and amino acid similarity of 99.0% and 99.4%,respectively.The phylogenetic analysis showed that the ORFV Yulin strain belonged to a branch with XP (KU199840.1),FJ MH2015 (KM675409.1) and Shanxi (HQ221964.1).The lengths of cF1L,rB2L,and rB2LcF1L obtained by PCR were 855 bp,1 180 bp and 1 983 bp,respectively.The pBac rB2LcF1L vector was successfully constructed,followed by packaging the corresponding baculovirus.The tandem gene of rB2L and cF1Lwas successfully expressed as 80 ku protein by baculovirus expression system and some recombinant proteins secreted into cell culture medium.【Conclusion】The recombinant B2L and F1L gene of ORFV-Yulin was successfully expressed in baculovirus expression system.
Key words:  Orf virus (ORFV)  F1L gene  co-expression of B2L and F1L  baculovirus  gene expression