摘要
目的探索含有流感病毒血凝素茎部α螺旋区(HAα)和6个串联重复M2蛋白胞外区域(6M2e)的融合蛋白的表达和制备方法,并对其在小鼠体内的免疫效果进行评价。方法采用去除高变区的鞭毛蛋白序列,将HAα和6M2e插入到原高变区位点,构建重组质粒pET28a-FljBΔD2D3-HAα-6M2e。在大肠埃希菌中诱导表达后进行纯化和鉴定,并评估其细胞毒性。最后将制备的融合蛋白免疫BALB/c小鼠,采用ELISA方法检测小鼠血清特异性抗体效价,实时荧光PCR法检测小鼠IL-2、IL-4、IL-6、IFN-γ、TNF-ɑ的mRNA水平,CCK-8法检测脾细胞的增殖。结果成功构建重组质粒pET28a-FljBΔD2D3-HAα-6M2e,蛋白表达最适条件为1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)37℃诱导10 h,通过镍柱纯化获得高纯度的无毒融合蛋白。免疫小鼠后抗M2e-IgG和抗HAα-IgG的抗体ELISA效价均达到1∶102400,且与对照组相比差异有统计学意义(t=0.33,P=0.774;t=7.60,P=0.001)。各类细胞因子的mRNA水平升高,其中实验组IFN-γ转录水平为38.74±3.65,蛋白水平为(66.03±3.31)pg/mL,高于对照组(t=21.33,P=0.003;t=10.21,P<0.01)。结论成功制备了高纯度的融合蛋白FljBΔD2D3-HAα-6M2e,能有效刺激小鼠产生抗M2e-IgG和抗HAα-IgG,具有良好的免疫效果,为流感疫苗候选物研发奠定了基础。
Objective To explore the expression and preparation method of the fusion protein containing influenza virus hemagglutininα-helical stem spiral area(HAα)and 6 tandem repeat M2 protein extracellular regions(6M2e),and to evaluate its immunological effect in mice.Methods Recombinant plasmid pET28a-FljBΔD2D3-HAα-6M2e was constructed by knockdown of flagellin in hypervariable regions and incorporation of HAαand 6M2e in these regions.The recombinant protein was expressed in Escherichia coli.After purification and identification,the cytotoxicity of fusion protein was evaluated.Then,the fusion protein immunized into BALB/c mice.The serum specific antibody titers were determined by ELISA,and the mRNA levels of IL-2,IL-4,IL-6,IFN-γand TNF-αin mice were tested by real-time fluorescent PCR.The proliferation of splenic cells was detected by CCK-8.Results The recombinant plasmid pET28a-FljBΔD2D3-HAα-6M2e was constructed successfully.The optimal condition for expression of fusion protein was induced with isopropyl-thio-beta-D-galactopyranoside(IPTG)at a final concentration of 1 mmol/L at 37℃for 10 h.The high-purified non-toxic fusion protein was obtained by nickel column.After immunizing mice,anti-M2e-IgG and anti-HAα-IgG antibody both reached 1∶102400 by ELISA,and were significantly different with the control group(t=0.33,P=0.774;t=7.60,P=0.001).The mRNA transcription levels of various cytokines increased significantly.In the experimental group,the transcription level and protein level of IFN-γwere 38.74±3.65 and(66.03±3.31)pg/mL,which significantly increased compared with those in the control group(t=21.33,P=0.003;t=10.21,P<0.001).Conclusions The high-purified fusion protein FljBΔD2D3-HAα-6M2e is constructed successfully,and can effectively stimulate mice to produce anti M2e-IgG and anti HAα-IgG.The good immune effect lays the foundation for development of influenza vaccine candidate.
作者
项婷婷
马淑敏
何泳愉
路鑫怡
郭潮潭
杨灿
杨新燕
沃恩康
Xiang Tingting;Ma Shumin;He Yongyu;Lu Xinyi;Guo Chaotan;Yang Can;Yang Xinyan;Wo Enkang(School of Laboratory Medicine and Bioengineering,Hangzhou Medical College,Hangzhou 310013,China)
出处
《国际流行病学传染病学杂志》
CAS
2023年第1期31-38,共8页
International Journal of Epidemiology and Infectious Disease
基金
浙江省自然科学基金(LQY19H190001、LQQ20H190001)
浙江省医药卫生科技计划(2021KY637、2021KY636、2022KY099、2021KY133)
浙江省教育厅一般科研项目(Y202249202)。
关键词
正黏病毒科
流感病毒
疫苗
融合蛋白
免疫原性
Orthomyxoviridae
Influenza virus
Vaccine
Fusion protein
Immunogenicity
