羊口疮病毒榆林株B2L和F1L串联基因在昆虫杆状病毒系统的表达  

Co-expression of B2L and F1L genes of Orf virus strain ORFV-Yulin in baculovirus system

作　　者：冯平[1,2] 李云章[1] 孙丰廷 屈雷[2] 闫海龙[2] FENG Ping;LI Yunzhang;SUN Fengting;QU Lei;YAN Hailong(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia 010018,China;College of Life Sciences,Yulin University,Yulin,Shaanxi 719000,Chin;Combio-Pioneer Co.,L TD.,Wuhan,Hubei 430040,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]榆林学院生命科学学院,陕西榆林719000 [3]武汉中拓康明生物科技有限公司,湖北武汉430040

出　　处：《西北农林科技大学学报（自然科学版）》2018年第6期1-8,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：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)

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B2L和F1L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(r B2L)和F1L融合基因(c F1L),利用碱基linker连接,通过重叠PCR方法扩增获得ORFV r B2L-linker-c F1L重组基因(r B2Lc F1L),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r B2Lc F1L的表达。【结果】克隆获得了1 029bp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855bp的c F1L基因、1 180bp的r B2L基因及1 983bp的r B2Lc F1L基因,成功构建了pBacrB2LcF1L载体,并包装出相应的杆状病毒。B2L和F1L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80ku,且有部分重组蛋白能分泌到细胞培养基中。【结论】ORFV-Yulin株的B2L和F1L串联基因在昆虫杆状病毒系统表达成功。【Objective】This study cloned and analyzed the sequence of F1 Lgene of Orf virus（ORFV）strain Yulin（ORFV-Yulin）and its co-expressed B2 Land F1 Lgenes in baculovirus system.【Method】The full-length gene of F1 Lof ORFV-Yulin was amplified by PCR,and the sequenced gene was analyzed by genetic evolution.The fused gene of B2 L（r B2 L）and F1 L（cF1 L）of ORFV-Yulin was amplified by PCR and the ORFV B2 L-linker-cF1 Lrecombinant gene（r B2 LcF1 L）was amplified by overlapping PCR method with linker.The baculovirus expression vector pBac-rB2 LcF1 Lwas constructed with pBac5 and r B2 LcF1 Lto get baculovirus expressing r B2 LcF1 Lgene after the infection of sf9 cells.The expressed product was identified by SDS-PAGE,mass spectrometry and western blot.【Result】The length of F1 Lgene 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 cF1 L,r B2 L,and r B2 LcF1 Lobtained by PCR were 855 bp,1 180 bp and 1 983 bp,respectively.The pBac-rB2 LcF1 Lvector was successfully constructed,followed by packaging the corresponding baculovirus.The tandem gene of r B2 Land cF1 L was successfully expressed as 80 ku protein by baculovirus expression system and some recombinant proteins secreted into cell culture medium.【Conclusion】The recombinant B2 Land F1 Lgene of ORFV-Yulin was successfully expressed in baculovirus expression system.

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 

分 类 号：S852.654[农业科学—基础兽医学] Q784[农业科学—兽医学]