信号肽对流感病毒血凝素在昆虫细胞-杆状病毒表达系统中分泌性表达的影响  

Effects of signal peptide on secretory expression of influenza virus hemagglutinin in insect cell-baculovirus expression system

作　　者：秦紫瑶 王旭 姚启元 刘含菲 罗剑 郑眉 Qin Ziyao;Wang Xu;Yao Qiyuan;Liu Hanfei;Luo Jian;Zheng Mei(No.4 Research Laboratory,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China)

机构地区：[1]上海生物制品研究所有限责任公司第四研究室,上海200051

出　　处：《国际生物制品学杂志》2024年第6期365-370,共6页International Journal of Biologicals

摘　　要：目的比较不同信号肽序列对流感病毒血凝素(hemagglutinin,HA)在Sf9昆虫细胞中分泌性表达的影响。方法以H1N1亚型流感病毒A/Victoria/2570/2019(IVR-215)的HA为靶标,选取全长HA基因序列,在其5′端分别连接天然信号肽、蜂毒素信号肽、糖蛋白67信号肽(glycoprotein 67 signal peptide,Gp67sp)、几丁质酶信号肽、人肝素结合蛋白信号肽,分别克隆至pFastBac1转移质粒基因组;通过Bac-to-Bac系统构建5种重组杆状病毒,并通过杆状病毒滴度测定试剂盒检测病毒滴度;将重组杆状病毒以不同的感染复数感染Sf9细胞,在感染后不同时间收获细胞上清液,通过免疫印迹法检测蛋白表达水平,优化HA分泌性表达的条件。结果收获的5种重组杆状病毒的滴度均大于106 PFU/ml。Sf9细胞能够正确表达HA,相对分子质量为80000左右。Gp67sp介导的HA表达量较高;当重组杆状病毒AcMNPV-Gp67sp-HA以感染复数5.0或10.0感染Sf9细胞96 h时,HA的表达量最高,血凝效价为256血凝单位/50μl。结论研究选择的外源信号肽均能引导流感病毒HA在Sf9细胞中的分泌性表达,其中Gp67sp更加有利于HA的高效表达。Objective To compare the effects of different signal peptide sequences on secretory expression of influenza virus hemagglutinin(HA)in Sf9 insect cells.Methods The HA of H1N1 subtype influenza virus A/Victoria/2570/2019(IVR-215)was used as a target.The full-length HA gene sequence was selected and naïve signal peptide,honeybee melittin signal peptide,glycoprotein 67 signal peptide(Gp67sp),chitinase signal peptide,and human azurocidin gene signal peptide were connected to the 5´terminal of the gene,respectively.The genes were cloned into the genome of the pFastBac1 transfer plasmid,and five recombinant baculoviruses were constructed by the Bac-to-Bac system.The viral titers were detected by BacPAKTM qPCR titration kit.The recombinant baculoviruses were used to infect Sf9 cells at different multiplicities of infection,and the cell supernatants were harvested at different time points after infection,and the protein expression levels were detected by immunoblotting to optimize the conditions for HA secretory expression.Results The titers of the five recombinant baculoviruses harvested were all>106 PFU/ml,and the Sf9 cells were able to correctly express and secrete HA,with a relative molecular mass of about 80000.The amount of HA expressed was the highest when mediated by Gp67sp.The highest expression of HA was achieved when the recombinant baculovirus AcMNPV-Gp67sp-HA infected Sf9 cells for 96 h with multiplicity of infection 5.0 or 10.0,and the hemagglutination efficiency was 256 hemaqqlutinetion units/50μl.Sf9 cells for 96 h,the highest secretory expression of HA was observed with a hemagglutination potency of 256/50μl.Conclusion All signal peptides tested effectively guide the secretory expression of influenza virus HA protein in Sf9 cells,among which Gp67sp is particularly advantageous for the efficient secretory expression of HA.

关 键 词：血凝素糖蛋白类 流感病毒 信号肽 分泌表达 昆虫细胞-杆状病毒表达系统 

分 类 号：Q812[生物学—生物工程]