摘要
【目的】真核表达H7亚型禽流感病毒(Avian influenza virus,AIV)HA蛋白,为进一步制备H7亚型HIV亚单位疫苗,评价其免疫原性提供基础。【方法】首先根据草地贪夜蛾卵巢细胞(Spodoptera frugiperda clone 9,Sf9)的密码子偏嗜性,优化并合成H7亚型AIV的HA序列,将其克隆至穿梭质粒pFastBac1中;接着将重组HA基因的杆状病毒质粒转染至Sf9中,通过间接免疫荧光和Westernblots试验鉴定重组病毒是否拯救成功;再通过SYBRgreen染料法荧光定量PCR试验,建立基于gp64基因的杆状病毒qPCR定量方法,筛选出在Sf9中表达HA重组蛋白的最佳感染复数和孵育时间;最后通过His镍株亲和纯化HA重组蛋白。【结果】表达H7亚型AIV HA蛋白的重组杆状病毒在Sf9盲传3代后,Sf9开始出现肿胀、脱落、死亡等细胞病变,表明重组杆状病毒拯救成功;通过间接免疫荧光和Western blots试验发现,Sf9内出现可与HA重组蛋白特异性结合的绿色荧光信号,特异性结合的HA重组蛋白条带大小约70 kD左右,与预期相符,且HA重组蛋白可与H7亚型AIV阳性血清反应,表明HA重组蛋白表达成功;以构建的pUC19-gp64质粒为标准,建立基于gp64基因的杆状病毒qPCR检测方法,该方法在1×103~1×108 copy/μL间呈现良好的线性关系,标准曲线的相关系数R2=0.993;利用qPCR检测方法对杆状病毒液滴度进行定量,并通过Western blots试验筛选HA蛋白表达的最佳感染复数和孵育时间,结果显示以10MOI的比例接种Sf9,孵育72h,蛋白表达效果最好;通过His镍株亲和纯化HA重组蛋白,蛋白浓度为0.268mg/mL,鸡红细胞凝集效价为4log2。【结论】本试验在Sf9中成功表达并纯化H7亚型AIVHA蛋白,并建立与之相应的杆状病毒荧光定量PCR检测方法,可为进一步评价H7亚单位疫苗的免疫原性提供参考。
【Objective】The experiment is aimed to express the HA protein of H7 subtype avian influenza virus(AIV)in eukaryotic cells,in order to provide bass for futher preparation of H7 subunit vaccine and evaluation of their immunogenicity.【Method】First,the preferred codons of Spodoptera frugiperda clone 9(Sf9)insect cells were used to optimize and synthesize the HA sequence of H7 subtype AIV,which was cloned into the shuttle plasmid pFastBac1.Then the baculovirus plasmid of the recombinant HA gene was transfected into Sf9 insect cells,and whether the recombinant virus were successfully rescued was identified by indirect immunofluorescence and western blots assay.Later on,through the SYBR green dye fluorescent quantitative PCR test,a baculovirus qPCR quantitative method based on gp64 gene was established to screen out the optimal multiplicity of infection and incubation time for expressing HA recombinant protein in Sf9 cells.Finally,the HA recombinant protein was affinity purified by the His nickel strain.【Result】After the recombinant baculovirus expressing the H7 subtype AIV HA protein was passed on to Sf9 cells for 3 generations,the Sf9 cells began to exhibit cell lesions such as swelling,shedding,and death,indicating that the recombinant baculovirus has been rescued successfully.Through indirect immunofluorescence and western blot tests,it was found that a green fluorescence signal specifically bound to the HA recombinant protein is appeared in Sf9 cells,and the band size of the specifically bound to the HA recombinant protein was about 70 kD,which was consistent with expectations,and the HA recombinant protein can react with H7 subtype AIV positive serum,indicating successful expression of the HA recombinant protein.A baculovirus qPCR detection method was established based on gp64 gene using the constructed pUC19-gp64 plasmid as a standard,and it exhibited a good linear relationship between 1×103~1×108 copy/μL,while the correlation coefficient of standard curve R2=0.993.Additionally,it was found that the protein expression effect was the best when the Sf9 cells were inoculated with a ratio of 10 MOI and incubated for 72 h using western blots test,the HA recombinant protein was purified through affinity purification of His nickel strain,with a protein concentration of 0.268 mg/mL,the agglutination titer of chicken red blood cells is 4 log2.【Conclusion】This experiment successfully expressed the H7 subtype AIV HA protein in Sf9 insect cells and established the corresponding fluorescence quantitative PCR detection method for baculovirus,which can provide refference for further evaluation of the immunogenicity of H7 subunit vaccine.
作者
刘郁夫
郝文茜
冯美莹
欧阳征亮
陈瑞爱
LIU Yufu;HAO Wenqian;FENG Meiying;OUYANG Zhengiang;CHEN Ruiai(School of Life Sciences,Zhaoqing University,Zhaoqing 526060,China;Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology,Zhaoqing 526238,China;Key Laboratory of Manufacture Technology of Veterinary Bioproducts,Ministry of Agriculture and Rural Affairs/Guangdong Enterprise Key Laboratory of Biotechnology R&D of Veterinary Biologics/Zhaoqing Dahuanong Biology Medicine Co.,Ltd,Zhaoqing 526238,China)
出处
《广东农业科学》
CAS
2023年第10期149-156,共8页
Guangdong Agricultural Sciences
基金
广东省重点领域研发计划项目(2021B0707010009)
广东省普通高校特色创新项目(2022KTSCX148)
肇庆学院博士启动项目(21010117)。
