寨卡病毒DNA疫苗新型佐剂的筛选
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摘要
目的以卡波姆971p辅以国产白喉、百日咳、破伤风灭活疫苗及Poly(I:C)配制成新型佐剂,与DNA疫苗pVAX-Zika-E联合免疫BALB/c小鼠,观察新型卡波姆佐剂对DNA疫苗抗原的适应性及免疫应答能力。方法 PCR扩增ZIKA E蛋白基因,连接到pVAX-1载体上,构建表达ZIKA E蛋白的DNA疫苗。将重组质粒转染至HEK293细胞,采用Western blot检测目的蛋白的表达。用配制的佐剂与表达ZIKA E蛋白的DNA疫苗联合免疫BALB/c小鼠。通过淋巴细胞增殖试验和特异性抗体检测验证新型佐剂的增强免疫效果。结果 Western blot结果显示所构建的DNA疫苗pVAX-ZIKA-E表达56×10~3目的蛋白。用重组疫苗做动物试验,免疫35 d时佐剂组淋巴细胞增殖刺激组刺激指数是无佐剂对照组的1.30～1.55倍,特异抗体水平(A_(450)值)是无佐剂组对照组的1.27～1.50倍。结论 pVAX-ZIKA-E DNA疫苗辅以新型卡波姆佐剂免疫小鼠可增强机体对该疫苗的免疫应答能力。
Objective In order to explore the ability of a new carbopol adjuvant to adapt to antigens in a DNA vaccine and its ability to trigger an immune response, this study used carbopol 971 p supplemented with a domestic inactivated diphtheria, pertussis, and tetanus vaccine and Poly(I:C) to prepare new adjuvants and combine them with a DNA vaccine expressing the Zika E protein to immunize BALB/c mice. Methods The Zika E gene was amplified using PCR and ligated into a pVAX-1 vector to construct a DNA vaccine designated pVAX-ZIKA-E. The recombinant plasmid was transfected into HEK293 cells, and the target protein expressed by the recombinant DNA vaccine was verified as correct with Western blotting. BALB/c mice were immunized with the DNA vaccine combined with the formulated adjuvant, and the immunogenicity of the vaccine was evaluated based on lymphocyte proliferation assays and specific antibody detection. Results Western blotting indicated that the constructed Zika DNA vaccine pVAX-ZIKA-E expressed 56×10~3 of the target protein. After 35 days of immunization with the recombinant vaccine, the specific stimulation index of the adjuvant group was 1.30 to 1.55 times that in the non-adjuvant control group(P<0.05). The specific antibody titer of the group given the adjuvant was 1.27-1.50 times higher than that in the group not given the adjuvant(P<0.05). Conclusion A Zika DNA vaccine, pVAX-ZIKA-E, was successfully constructed. Immunization of mice with the pVAX-ZIKA-E DNA vaccine combined with a new carbopol adjuvant enhanced the body's immune response to the vaccine.
Objective In order to explore the ability of a new carbopol adjuvant to adapt to antigens in a DNA vaccine and its ability to trigger an immune response, this study used carbopol 971 p supplemented with a domestic inactivated diphtheria, pertussis, and tetanus vaccine and Poly(I:C) to prepare new adjuvants and combine them with a DNA vaccine expressing the Zika E protein to immunize BALB/c mice. Methods The Zika E gene was amplified using PCR and ligated into a pVAX-1 vector to construct a DNA vaccine designated pVAX-ZIKA-E. The recombinant plasmid was transfected into HEK293 cells, and the target protein expressed by the recombinant DNA vaccine was verified as correct with Western blotting. BALB/c mice were immunized with the DNA vaccine combined with the formulated adjuvant, and the immunogenicity of the vaccine was evaluated based on lymphocyte proliferation assays and specific antibody detection. Results Western blotting indicated that the constructed Zika DNA vaccine pVAX-ZIKA-E expressed 56×10~3 of the target protein. After 35 days of immunization with the recombinant vaccine, the specific stimulation index of the adjuvant group was 1.30 to 1.55 times that in the non-adjuvant control group(P<0.05). The specific antibody titer of the group given the adjuvant was 1.27-1.50 times higher than that in the group not given the adjuvant(P<0.05). Conclusion A Zika DNA vaccine, pVAX-ZIKA-E, was successfully constructed. Immunization of mice with the pVAX-ZIKA-E DNA vaccine combined with a new carbopol adjuvant enhanced the body's immune response to the vaccine.
引文
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[13] 黄燕霞, 张萍. 寨卡病毒感染动物模型研究进展[J]. 热带医学杂志, 2018, 18(2): 260-3.
[14] Langellotti CA, Pappalardo JS, Quattrocchi V, et al. Induction of specific cytotoxic activity for bovine herpesvirus-1 by DNA immunization with different adjuvants[J]. Antiviral Res, 2011, 90(3): 134-42.
[15] Larocca RA, Abbink P, Peron JP, et al. Vaccine protection against Zika virus from Brazil[J]. Nature, 2016, 536(7617): 474-8.
[16] 周洋, 耿兴超, 汪巨峰, 等. 疫苗佐剂最新研究进展[J]. 中国新药杂志, 2013, 22(1): 34-42.
[17] 罗清琼, 陈万涛, 胡水清, 等. 口腔鳞状细胞癌细胞TLR3表达及其配体poly(I:C)的诱导凋亡作用[J]. 上海交通大学学报(医学版), 2011, 31(1): 5-8.
[2] Duffy MR, Chen TH, Hancock WT, et al. Zika virus outbreak on Yap Island, Federated States of Micronesia[J]. N Engl Journ Med, 2009, 360(24): 2536-43.
[3] Cao-Lormeau VM, Roche C, Teissier A, et al. Zika virus, French polynesia, South pacific, 2013[J]. Emerg Infect Dis, 2014, 20(6): 1085-86.
[4] Zanluca C, Melo VC, Mosimann AL, et al. First report of autochthonous transmission of Zika virus in Brazil[J]. Mem Inst Oswaldo Cruz, 2015,110(4): 569-72.
[5] 谢甜, 罗陆婷, 尹庆庆, 等. 蚊寨卡病毒核酸检测体系的建立与初步应用[J]. 中国病原生物学杂志, 2017, 12(9): 811-7, 824.
[6] Messina JP, Kraemer MU, Brady OJ, et al. Mapping global environmental suitability for Zika virus[J]. Elife, 2016(5): e15272
[7] Hamel R, Liégeois F, Wichit S, et al. Zika virus: epidemiology, clinical features and host-virus interactions[J]. Microbes Infect, 2016,18(7-8): 441-9.
[8] van Hemert F, Berkhout B. Nucleotide composition of the Zika virus RNA genome and its codon usage[J]. Virol J, 2016(13): 95.
[9] Chen Y, Maguire T, Hileman RE, et al. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate[J]. Nat Med, 1997, 3(8): 866-71.
[10] Lin HH, Yip BS, Huang LM, et al. Zika virus structural biology and progress in vaccine development[J]. Biotechnol Adv, 2018, 36(1): 47-53.
[11] Abbasi J. Zika vaccine enters clinical Trials[J]. JAMA, 2016, 316(12): 1249.
[12] Khusro A, Aarti C, Barbabosa-Pilego A, et al. Outbreak of Zika virus pathogenesis and quest of its vaccine development: Where do we stand now?[J]. Microb Pathog, 2018(116): 289-95
[13] 黄燕霞, 张萍. 寨卡病毒感染动物模型研究进展[J]. 热带医学杂志, 2018, 18(2): 260-3.
[14] Langellotti CA, Pappalardo JS, Quattrocchi V, et al. Induction of specific cytotoxic activity for bovine herpesvirus-1 by DNA immunization with different adjuvants[J]. Antiviral Res, 2011, 90(3): 134-42.
[15] Larocca RA, Abbink P, Peron JP, et al. Vaccine protection against Zika virus from Brazil[J]. Nature, 2016, 536(7617): 474-8.
[16] 周洋, 耿兴超, 汪巨峰, 等. 疫苗佐剂最新研究进展[J]. 中国新药杂志, 2013, 22(1): 34-42.
[17] 罗清琼, 陈万涛, 胡水清, 等. 口腔鳞状细胞癌细胞TLR3表达及其配体poly(I:C)的诱导凋亡作用[J]. 上海交通大学学报(医学版), 2011, 31(1): 5-8.
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