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(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。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B2L和F1L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 |
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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) |
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Co-expression of B2L and F1L genes of Orf virus strain ORFV-Yulin in baculovirus system |
FENG Ping,LI Yunzhang,SUN Fengting,et al
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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 cF1Lwas 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 |