| 刘郁夫 1,2，郝文茜 2，冯美莹 1，欧阳征亮 3，陈瑞爱 2,3.H7 亚型禽流感病毒 HA 蛋白在Sf9 中的表达与纯化[J].广东农业科学,2023,50(10):149-156 |
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H7 亚型禽流感病毒 HA 蛋白在Sf9 中的表达与纯化 |
| Expression and Purification of H7 Subtype Avain Influenza Viruses HA Protein in Sf9 |
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| DOI：10.16768/j.issn.1004-874X.2023.10.016 |
| 中文关键词: H7 亚型禽流感病毒 血凝素蛋白（HA） 昆虫细胞 杆状病毒 基因表达 蛋白纯化 |
| 英文关键词: H7 subtype avain influenza viruses hemagglutinin (HA) insect cell baculovirus gene expression protein purification |
| 基金项目: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） |
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| 中文摘要: |
| 【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 Western blots 试验鉴定重组病毒是否拯救成功；再通过 SYBR green 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 10 MOI 的比例接种 Sf9，孵育 72 h，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 0.268 mg/mL，鸡红细胞凝集效价为 4 log2。【结论】本试验在 Sf9 中成功表达并纯化 H7 亚型 AIV HA 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。 |
| 英文摘要: |
| 【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. |
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