手足口病新型疫苗的初步研究
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摘要
手足口病(Hand,foot and mouth disease,HFMD)是由肠道病毒感染引起的一种病毒性疾病,主要病原体为新肠道病毒71型(Enterovirus 71,EV71)和柯萨奇A16型(Coxsackie A16,CA16),EV71感染常常引发严重的中枢神经系统疾病并发症,CA16是引起心肌炎和心包炎等疾病的主要病原体。目前尚无有效治疗HFMD的药物,疫苗成为防控HFMD暴发最有效的手段之一,因此,研发安全、有效的HFMD疫苗势在必行。
本文采用反转录聚合酶链反应(RT-PCR)克隆EV71(中国安徽阜阳株Genbank----FJ607337)VP1和CA16(中国山东株Genbank----GQ253390)VP1基因,构建重组pFastBac HT A-EV71 VP1、pFastBac HT A-CA16 VP1和pFastBac HT B-Mel-EV71 VP1-CA16 VP1质粒,将各重组质粒转化至感受态细胞(DH10),获得各重组Bacmid DNA转染至昆虫细胞(Sf9),应用杆状病毒昆虫表达系统(Bacterium to Baculovirus,Bac-to-Bac)表达或分泌表达各重组蛋白,采用Ni2+离子亲和层析柱纯化各重组病毒表达产物;依据昆虫细胞偏爱密码子优化EV71 P1和EV71 3CD基因克隆至表达载体pFastBac Dual多角体蛋白启动子pPh和pP10上,构建重组pFastBac Dual EV71 P1-3CD质粒,将重组质粒与Bacmid同源重组,转染至Sf9,在Bac-to-Bac系统中利用病毒编码蛋白酶3CD切割核衣壳病毒结构蛋白P1,组装并用蔗糖密度梯度离心纯化EV71病毒样颗粒(Virus Like Particles,VLPs);经免疫细胞化学法证实各目的蛋白在Sf9中的表达;将制备的重组单价蛋白疫苗,腹腔、滴鼻免疫Balb/c小鼠,检测免疫应答水平,取得如下结果:
1.所克隆的各DNA片段均与Genbank中的相应DNA序列一致,重组EV71 VP1、CA16 VP1蛋白在Sf9中均获得表达,得到纯化重组蛋白浓度分别为0.318mg/mL和0.412mg/mL,纯度均>90%。
2.重组EV71 VP1和CA16 VP1单价蛋白最佳抗原免疫剂量均为20μg;腹腔注射组中,小鼠血清特异性IgG抗体效价分别为875.43和1588.95,中和抗体效价分别为282.84和250.00,抗体亚型分布均以IgG1和IgG2b为主;CD4+/CD8+比值与对照组差异显著(P<0.05),小鼠T淋巴细胞增殖能力增强,脾细胞IL-4分泌数量>IFN-γ分泌数量(P<0.05);鼻腔和小肠灌洗液sIgA抗体与PBS组差异不显著(p>0.05);重组单价蛋白疫苗均可激发一定水平的体液免疫和细胞免疫,不能诱导粘膜免疫应答,以体液免疫应答为主。
3.滴鼻免疫EV71 VP1组和CA16 VP1组血清IgG效价分别为278.54和211.66,鼻腔灌洗液sIgA抗体分别为64.31和54.22,小肠灌洗液sIgA抗体分别为15.98和13.55,脾细胞中sIgA-ASCs数量增多,重组单价蛋白疫苗可诱导体液免疫应答和一定水平的粘膜免疫应答。
4.利用Bac-to-Bac系统分泌表达了EV71 VP1-CA16 VP1融合蛋白,得到纯化重组蛋白浓度为0.516mg/mL,纯度>90%。
5.经昆虫密码子优化的EV71 P1和EV71 3CD基因,利用Bac-to-Bac系统成功组装了EV71 VLPs,纯化了EV71 VLPs的浓度为0.817mg/mL,纯度>90%。
结论:制备了重组EV71 VP1和CA16 VP1单价蛋白疫苗,在它们免疫的小鼠模型中,免疫两次均能够产生较好的体液免疫应答,也能产生一定的细胞免疫和粘膜免疫应答;构建、表达和纯化了重组EV71 VP1-CA16 VP1二价融合蛋白和EV71 VLPs。因此,本实验结果为HFMD疫苗的深入研究奠定了基础。
Hand,foot and mouth disease (HFMD) is a viral disease,caused by intestinal infection. The main pathogens are Enterovirus 71 (EV71) and Coxsackie A 16 (CA16), central nervous system complications often were caused by EV71, myocarditis and pericarditis were caused by CA16. There were no drugs to treat HFMD,vaccines were the most effective means of prevention and control, It was imperative to develop safe and effective vaccines.
In this study, EV71 (Anhui Fuyang strain,China,Genbank----FJ607337) VP1 and CA16 (Shandong strain,China,Genbank----GQ253390) VP1 genes were amplified by reverse transcription polymerase chain reaction (RT-PCR), were constructed recombinant plasmid pFastBac HT A-EV71 VP1,pFastBac HT A-CA16 VP1 and pFastBac HT B-Mel-EV71 VP1-CA16 VP1,all recombinant plasmids were transformed to E.coli DH10. Each recombinant Bacmid DNA was obtained and then transfected into insect cells (Sf9), the recombinant protein were expressed in insect baculovirus expression system (Bac-to-Bac).
Optimization codon of EV71 P1 and EV71 3CD in insect cells were cloned into pFastBac Dual polyhedrin promoter pPh and Pp10, pFastBac Dual EV71 P1-3CD and Bacmid were constructed by homologous recombination, Bacmid DNA was transfected into Sf9. Structural protein capsid P1 was cut by virus-encoded protease 3CD in Bac-to-Bac, EV71 virus like particles (VLPs) were assembled and purified by sucrose density gradient centrifugation. The expressed protein was detected by immunocytochemistry. mice were immunized by intraperitoneal and intranasal immunization, recombinant protein vaccine were assessed. The results showed as follows:
1.The cloned DNA genes were consistent with DNA sequence in Genbank. EV71 VP1 and CA16 VP1 protein were expressed in Sf9, their concentration were 0.318mg/mLand 0.412 mg/mL,respectively, and their purity>90%.
2.The best antigen doses of recombinant EV71 VP1 and CA16 VP1 protein were 20μg. Their specific IgG antibody titers were 875.43 and 1588.95, neutralizing antibody titers were 282.84 and 250.00, antibody subtype distribution were mainly IgG1 and IgG2b,CD4+/CD8+ were significantly different from control groups(P<0.05), T lymphocyte proliferation were enhanced, the number of cytokines IL-4 were more than IFN-γin spleen cells (p <0.05),sIgA antibodies in nasal and intestinal lavage with PBS were no significant differences (p>0.05) in intraperitoneal injection groups.They were induced humoral and cellular, no mucosal immune response, so mainly caused humoral immune response.
3.Intranasal immuned groups , their serum IgG titers were 278.54 and 211.66,respectively. sIgA antibodies in nasal lavage were 64.31 and 54.22,respectively. sIgA antibodies in intestinal lavage were 15.98 and 13.55,respectively. sIgA-ASCs numbers in spleen cells were increased, recombinant protein vaccines induced humoral and mucosal immune response.
4.EV71 VP1-CA16 VP1 fusion protein was expressed in Bac-to-Bac, the concentration of purified protein was 0.516mg/mL, its purity > 90%.
5.EV71 3CD and EV71 P1 were optimized by insect preferred codon, and EV71 VLPs was assembled successfully in Bac-to-Bac, the concentration of purified EV71 VLPs was 0.817mg/mL, its purity>90%.
Conclusion: EV71 VP1 and CA16 VP1 protein vaccines were preparated, they were able to induce better humoral immune response, also induced certain cellular and mucosal immune response. The recombinant EV71 VP1-CA16 VP1 protein and EV71 VLPs were constructed,expressed and purified.
本文采用反转录聚合酶链反应(RT-PCR)克隆EV71(中国安徽阜阳株Genbank----FJ607337)VP1和CA16(中国山东株Genbank----GQ253390)VP1基因,构建重组pFastBac HT A-EV71 VP1、pFastBac HT A-CA16 VP1和pFastBac HT B-Mel-EV71 VP1-CA16 VP1质粒,将各重组质粒转化至感受态细胞(DH10),获得各重组Bacmid DNA转染至昆虫细胞(Sf9),应用杆状病毒昆虫表达系统(Bacterium to Baculovirus,Bac-to-Bac)表达或分泌表达各重组蛋白,采用Ni2+离子亲和层析柱纯化各重组病毒表达产物;依据昆虫细胞偏爱密码子优化EV71 P1和EV71 3CD基因克隆至表达载体pFastBac Dual多角体蛋白启动子pPh和pP10上,构建重组pFastBac Dual EV71 P1-3CD质粒,将重组质粒与Bacmid同源重组,转染至Sf9,在Bac-to-Bac系统中利用病毒编码蛋白酶3CD切割核衣壳病毒结构蛋白P1,组装并用蔗糖密度梯度离心纯化EV71病毒样颗粒(Virus Like Particles,VLPs);经免疫细胞化学法证实各目的蛋白在Sf9中的表达;将制备的重组单价蛋白疫苗,腹腔、滴鼻免疫Balb/c小鼠,检测免疫应答水平,取得如下结果:
1.所克隆的各DNA片段均与Genbank中的相应DNA序列一致,重组EV71 VP1、CA16 VP1蛋白在Sf9中均获得表达,得到纯化重组蛋白浓度分别为0.318mg/mL和0.412mg/mL,纯度均>90%。
2.重组EV71 VP1和CA16 VP1单价蛋白最佳抗原免疫剂量均为20μg;腹腔注射组中,小鼠血清特异性IgG抗体效价分别为875.43和1588.95,中和抗体效价分别为282.84和250.00,抗体亚型分布均以IgG1和IgG2b为主;CD4+/CD8+比值与对照组差异显著(P<0.05),小鼠T淋巴细胞增殖能力增强,脾细胞IL-4分泌数量>IFN-γ分泌数量(P<0.05);鼻腔和小肠灌洗液sIgA抗体与PBS组差异不显著(p>0.05);重组单价蛋白疫苗均可激发一定水平的体液免疫和细胞免疫,不能诱导粘膜免疫应答,以体液免疫应答为主。
3.滴鼻免疫EV71 VP1组和CA16 VP1组血清IgG效价分别为278.54和211.66,鼻腔灌洗液sIgA抗体分别为64.31和54.22,小肠灌洗液sIgA抗体分别为15.98和13.55,脾细胞中sIgA-ASCs数量增多,重组单价蛋白疫苗可诱导体液免疫应答和一定水平的粘膜免疫应答。
4.利用Bac-to-Bac系统分泌表达了EV71 VP1-CA16 VP1融合蛋白,得到纯化重组蛋白浓度为0.516mg/mL,纯度>90%。
5.经昆虫密码子优化的EV71 P1和EV71 3CD基因,利用Bac-to-Bac系统成功组装了EV71 VLPs,纯化了EV71 VLPs的浓度为0.817mg/mL,纯度>90%。
结论:制备了重组EV71 VP1和CA16 VP1单价蛋白疫苗,在它们免疫的小鼠模型中,免疫两次均能够产生较好的体液免疫应答,也能产生一定的细胞免疫和粘膜免疫应答;构建、表达和纯化了重组EV71 VP1-CA16 VP1二价融合蛋白和EV71 VLPs。因此,本实验结果为HFMD疫苗的深入研究奠定了基础。
Hand,foot and mouth disease (HFMD) is a viral disease,caused by intestinal infection. The main pathogens are Enterovirus 71 (EV71) and Coxsackie A 16 (CA16), central nervous system complications often were caused by EV71, myocarditis and pericarditis were caused by CA16. There were no drugs to treat HFMD,vaccines were the most effective means of prevention and control, It was imperative to develop safe and effective vaccines.
In this study, EV71 (Anhui Fuyang strain,China,Genbank----FJ607337) VP1 and CA16 (Shandong strain,China,Genbank----GQ253390) VP1 genes were amplified by reverse transcription polymerase chain reaction (RT-PCR), were constructed recombinant plasmid pFastBac HT A-EV71 VP1,pFastBac HT A-CA16 VP1 and pFastBac HT B-Mel-EV71 VP1-CA16 VP1,all recombinant plasmids were transformed to E.coli DH10. Each recombinant Bacmid DNA was obtained and then transfected into insect cells (Sf9), the recombinant protein were expressed in insect baculovirus expression system (Bac-to-Bac).
Optimization codon of EV71 P1 and EV71 3CD in insect cells were cloned into pFastBac Dual polyhedrin promoter pPh and Pp10, pFastBac Dual EV71 P1-3CD and Bacmid were constructed by homologous recombination, Bacmid DNA was transfected into Sf9. Structural protein capsid P1 was cut by virus-encoded protease 3CD in Bac-to-Bac, EV71 virus like particles (VLPs) were assembled and purified by sucrose density gradient centrifugation. The expressed protein was detected by immunocytochemistry. mice were immunized by intraperitoneal and intranasal immunization, recombinant protein vaccine were assessed. The results showed as follows:
1.The cloned DNA genes were consistent with DNA sequence in Genbank. EV71 VP1 and CA16 VP1 protein were expressed in Sf9, their concentration were 0.318mg/mLand 0.412 mg/mL,respectively, and their purity>90%.
2.The best antigen doses of recombinant EV71 VP1 and CA16 VP1 protein were 20μg. Their specific IgG antibody titers were 875.43 and 1588.95, neutralizing antibody titers were 282.84 and 250.00, antibody subtype distribution were mainly IgG1 and IgG2b,CD4+/CD8+ were significantly different from control groups(P<0.05), T lymphocyte proliferation were enhanced, the number of cytokines IL-4 were more than IFN-γin spleen cells (p <0.05),sIgA antibodies in nasal and intestinal lavage with PBS were no significant differences (p>0.05) in intraperitoneal injection groups.They were induced humoral and cellular, no mucosal immune response, so mainly caused humoral immune response.
3.Intranasal immuned groups , their serum IgG titers were 278.54 and 211.66,respectively. sIgA antibodies in nasal lavage were 64.31 and 54.22,respectively. sIgA antibodies in intestinal lavage were 15.98 and 13.55,respectively. sIgA-ASCs numbers in spleen cells were increased, recombinant protein vaccines induced humoral and mucosal immune response.
4.EV71 VP1-CA16 VP1 fusion protein was expressed in Bac-to-Bac, the concentration of purified protein was 0.516mg/mL, its purity > 90%.
5.EV71 3CD and EV71 P1 were optimized by insect preferred codon, and EV71 VLPs was assembled successfully in Bac-to-Bac, the concentration of purified EV71 VLPs was 0.817mg/mL, its purity>90%.
Conclusion: EV71 VP1 and CA16 VP1 protein vaccines were preparated, they were able to induce better humoral immune response, also induced certain cellular and mucosal immune response. The recombinant EV71 VP1-CA16 VP1 protein and EV71 VLPs were constructed,expressed and purified.
引文
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