昆虫杆状病毒系统表达人流感多表位基因盒的研究  

Expression of human influenza multi epitope genes by insect baculovirus system

作　　者：李凯 丁焱 庄忻雨 田明尧 曹亮 朱光泽[4] 金宁一 LI Kai;DING Yan;ZHUANG Xin-yu;TIAN Ming-yao;CAO Liang;ZHU Guang-ze;JIN Ning-yi(Animal Husbandry and Veterinary Station,Hongqiao Town,Taixing City,Taizhou City,Jiangsu 225400,China;Changchun Veterinary Institute,Chinese Academy of Agricultural Sciences;Jilin Medical College;Laboratory Department,Affiliated Hospital of Changchun University of Traditional Chinese Medicine)

机构地区：[1]泰兴市虹桥镇畜牧兽医站,江苏泰兴225400 [2]中国农业科学院长春兽医研究所 [3]吉林医药学院 [4]长春中医药大学附属医院检验科

出　　处：《中国病原生物学杂志》2023年第4期390-394,共5页Journal of Pathogen Biology

基　　金：长春市科技发展计划项目(No.21ZGY30);吉林省教育厅科学技术研究项目(No.JJKH20210482KJ)。

摘　　要：目的通过昆虫-杆状病毒表达系统表达人流感病毒多表位基因盒,为进一步研发通用型流感疫苗提供实验依据。方法将甲型流感病毒H1N1颈部区(345aa-566aa)和H1/H3/B亚型HA2片段经4GS Linker串联M2e及ferritin蛋白构成的HA2-M2e-ferritin(简称HMf),参照A/Jilin/JYT-01/2018(H1N1)毒株的全基因组序列,将其HA2基因和HMf基因序列按照昆虫细胞密码子偏好性进行优化合成,经两次双酶切后分别克隆至pFastBacTM双载体中。热激法将重组质粒转化入DH10BacTM大肠埃希菌感受态,含目的基因的pFastBacDual-HA2-HMf转移载体与DH10Bac的Bacmid质粒重组,构建重组转移质粒rBacmid-HA2-HMf。在脂质体介导下,用rB-HA2-HMf转染Sf9细胞进行重组杆状病毒的拯救。通过限制性酶双酶切、测序、重组Bacmid PCR、Western blot和间接免疫荧光法(IFA)鉴定目的蛋白的表达。用重组杆状病毒感染悬浮sf9细胞,收取细胞悬液,经超速离心、蔗糖密度梯度离心获得重组蛋白,利用透射电镜对重组蛋白进行观察分析。结果HA2和HMf基因共表达,将HA2全长和HMf成功插入pFastBacTMDual双顺载体,包装并拯救出携带人流感多表位基因的重组杆状病毒rBDV-HA2-HMf。重组转移质粒pFastBacDual-HA2/HMf经限制性双酶切鉴定,重组穿梭质粒rBacmid-HA2/HMf经菌液PCR鉴定、杆状病毒共表达人流感多表位基因盒经Western blot和IFA鉴定成功,透射电镜下观察到重组蛋白形成的约20 nm的类病毒样颗粒。结论成功构建重组杆状病毒,其表达的流感多重保守抗原具有反应原性,为进一步研发通用型流感疫苗奠定了实验依据。Objective To express human influenza virus multi-epitope ge ne box through insect baculovirus expression system,so as to provide scientific basis for further development of universal influenza vaccine.Methods The H1N1 neck region(345aa-566aa)and H1/H3/B subtype HA2 fragments of influenza A virus were optimized and synthesized according to the codon preference of insect cells by the sequence of HA2-M2e ferritin(HMf)gene composed of M2e and Ferritin proteins linked by 4GS Linker,and then cloned into pFastBacTM dual vector after twice double digestion;The heat shock method was used to transform the recombinant plasmid into the competent state of DH10BacTM E.coli.The pFastBacDual HA2-HMf transfer vector containing the target gene was recombined with the Bacmid plasmid of E.coli DH10Bac to construct the recombinant transfer plasmid rBacmid HA2-HMf.RB-HA2-HMf was transfected into Sf9 cells by liposome to rescue the recombinant baculovirus.The expression of the target protein was identified by restriction double enzyme digestion,sequencing,recombinant Bacmid PCR,Western Blot and indirect immunofluorescence assay(IFA).The recombinant baculovirus infected the suspended sf9 cells,collected the cell suspension,obtained the recombinant protein by ultracentrifugation and sucrose density gradient centrifugation,and analyzed the recombinant protein by electron microscope.Results HA2 and HMf genes were co expressed.The full length of HA2 and HMf were successfully inserted into the vector using pFastBacTM Dual bicistronic vector,and the recombinant baculovirus rBDV-HA2-HMf carrying human influenza multi epitope genes was packaged and rescued.The recombinant transfer plasmid pFastBacDual HA2/HMf was digested by restriction double enzymes,the recombinant shuttle plasmid rBacmid-HA2/HMf was identified by bacterial solution PCR,and the baculovirus co epitope gene box of human influenza was identified successfully by Western blot and IFA.The virus like particles of about 20 nm could be seen under TEM.Conclusion The results indicate th

关 键 词：流感病毒 重组杆状病毒 颈部区结构域 多重保守抗原 疫苗 

分 类 号：R373.1[医药卫生—病原生物学]