结核分枝杆菌基因Rv2660c、Rv2460c、Rv3875、Rv3804c细胞表位融合蛋白的表达及其免疫原性评价
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摘要
目的评价结核分枝杆菌基因Rv2660c、Rv2460c、Rv3875和Rv3804c的细胞表位融合蛋白的免疫原性,为研制新型多阶段结核疫苗提供可靠的靶抗原。方法将Rv2660c、Rv2460c、Rv3875和Rv3804c基因中的细胞表位串联构成融合抗原基因(命名为msv),克隆到原核表达载体pEASY-Blunt E1中,诱导表达后采用亲和层析纯化表达产物,经SDS-PAGE和Western blot鉴定后,用纯化的融合抗原蛋白免疫小鼠,ELISA法检测免疫小鼠的特异性抗体滴度;分离免疫小鼠的脾淋巴细胞,采用淋巴细胞增殖法检测其免疫原性。结果成功构建了能表达融合抗原基因msv的原核表达质粒;SDS-PAGE和Western blot结果表明,表达产物亲和层析纯化后,获得相对分子质量为41.3×10~3的目的蛋白;ELISA检测免疫小鼠血清抗体滴度,结果表明特异性抗体效价约为1∶81 920;淋巴细胞增殖实验结果表明,抗原致敏的淋巴细胞经融合蛋白msv刺激后明显增殖。结论成功表达并纯化出融合蛋白msv,该融合蛋白可诱导小鼠特异性抗体表达和刺激细胞免疫应答,可作为结核疫苗的抗原组分。
Objective To analyse the immunogenicity of a fusion protein containing cell epitopes of Mycobacterium tuberculosis genes Rv2660c, Rv2460c, Rv3875 and Rv3804, and to evaluate the feasibility of using it as a novel target antigen for developing multi-stage TB vaccines. Methods Cell epitopes of Rv2660c, Rv2460c, Rv3875 and Rv3804c were fused in series to form a new antigen gene(named msv). Then msv was cloned into the prokaryotic expression vector pEASY-Blunt E1. The fusion protein msv was expressed by pEASY-Blunt E1 under the induction of isopropyl-β-d-thiogalactoside(IPTG). Purified the protein by affinity chromatography and identified the protein by SDS-PAGE and Western blot. To evaluate the immunogenicity of the protein, the mice were immunized with the purified fusion protein, and the titer of the antibody in mice serum was evaluated by ELISA. Besides, splenocytes of immunized mice were separated and splenocytes proliferation was determined under the stimulation of the protein. Results The prokaryotic expression plasmid carrying msv gene was constructed successfully and msv protein could be expressed by the plasmid under the induction of IPTG. SDS-PAGE and Western blot results confirmed that a purified protein(relative molecular mass was 41.3×10~3) was obtained. ELISA result indicated that the titer of the antibody in msv immunized mice serum was about 1∶81 920.The spleen lymphocyte proliferation assay showed that after immunization with msv protein, significant proliferation of antigen-sensitized lymphocytes was observed. Conclusion The fusion protein msv was successfully expressed and purified, which can induce humoral and cellular immunity in mice. It may be used as an antigen component for the development of TB vaccine in the future.
Objective To analyse the immunogenicity of a fusion protein containing cell epitopes of Mycobacterium tuberculosis genes Rv2660c, Rv2460c, Rv3875 and Rv3804, and to evaluate the feasibility of using it as a novel target antigen for developing multi-stage TB vaccines. Methods Cell epitopes of Rv2660c, Rv2460c, Rv3875 and Rv3804c were fused in series to form a new antigen gene(named msv). Then msv was cloned into the prokaryotic expression vector pEASY-Blunt E1. The fusion protein msv was expressed by pEASY-Blunt E1 under the induction of isopropyl-β-d-thiogalactoside(IPTG). Purified the protein by affinity chromatography and identified the protein by SDS-PAGE and Western blot. To evaluate the immunogenicity of the protein, the mice were immunized with the purified fusion protein, and the titer of the antibody in mice serum was evaluated by ELISA. Besides, splenocytes of immunized mice were separated and splenocytes proliferation was determined under the stimulation of the protein. Results The prokaryotic expression plasmid carrying msv gene was constructed successfully and msv protein could be expressed by the plasmid under the induction of IPTG. SDS-PAGE and Western blot results confirmed that a purified protein(relative molecular mass was 41.3×10~3) was obtained. ELISA result indicated that the titer of the antibody in msv immunized mice serum was about 1∶81 920.The spleen lymphocyte proliferation assay showed that after immunization with msv protein, significant proliferation of antigen-sensitized lymphocytes was observed. Conclusion The fusion protein msv was successfully expressed and purified, which can induce humoral and cellular immunity in mice. It may be used as an antigen component for the development of TB vaccine in the future.
引文
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[11] YIHAO D,HONGYUN H,MAODAN T.Latency-associated protein Rv2660c of Mycobacterium tuberculosis augments expression of proinflammatory cytokines in human macrophages by interacting with TLR2.Infect Dis (Lond),2015,47(3):168-177.
[12] AAGAARD C,HOANG T,DIETRICH J,et al.A multistage tuberculosis vaccine that confers efficient protection before and after exposure.Nat Med,2011,17(2):189-194.
[13] LUABEYA AK,KAGINA BM,TAMERIS MD,et al.First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected healthy adults.Vaccine,2015,33(33):4130-4140.
[14] 康月茜,张春燕,穆柳青,等.结核分枝杆菌Rv2460c基因的克隆,表达及编码产物的免疫特性分析.中国免疫学杂志,2015,31(6):790-794.
[15] 许崇利,杨梅,许崇波.大肠杆菌表达系统的影响因素.中国动物检疫,2010,27(8):66-68.
[16] 马建伟,李春明,冯思源,等.牛源多杀性巴氏杆菌融合基因pm0979-PlpE的表达及其融合蛋白的免疫效果.中国兽医科学,2015,45(10):1053-1057.
[2] 范琴,鲍朗.结核疫苗优势抗原ESAT6、CFP10的分子生物学基础及应用研究进展.生物医学工程学杂志,2012,29(2):392-396.
[3] KARBALAEI MZB,SOLEIMANPOUR S,REZAEE SA.Antigen 85 complex as a powerful Mycobacterium tuberculosis immunogene:biology,immune-pathogenicity,applications in diagnosis,and vaccine design.Microb Pathog,2017,112:20-29 [2018-10-10].https://doi.org/10.1016/j.micpath.2017.08.040.
[4] HOUGHTON J,CORTES T,SCHUBERT O,et al.A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced duringstarvation and infection.PLoS One,2013,8(12):e80047 [2018-10-20].https://doi.org/10.1371/journal.pone.0080047.
[5] SHERRID AM,RUSTAD TR,CANGELOSI GA,et al.Characterization of a Clp protease gene regulator and the reaeration response in Mycobacterium tuberculosis.PLoS One,2010,5(7):e11622[2018-10-20].https://doi.org/10.1371/journal.pone.0011622.
[6] World Health Organisation.Global tuberculosis report 2017.(2018-09-18) [2018-10-20].https://www.who.int/tb/publications/global_report/en/.
[7] PENN-NICHOLSON A,TAMERIS M,SMIT E,et al.Safety and immunogenicity of the novel tuberculosis vaccine ID93+GLA-SE in BCG-vaccinated healthy adults.Lancet Respir Med,2018,6(4):287-298.
[8] BALDWIN SL,REESE VA,HUANG PW,et al.Protection and long-lived immunity induced by the ID93/GLA-SE vaccine candidate against a clinical Mycobacterium tuberculosis isolate.Clin Vaccine Immunol,2015,23(2):137-147.
[9] 徐正中,胡婷,刘泽,等.用小鼠模型分析可溶性表达结核分枝杆菌Ag85A的免疫原性.微生物学报,2016,56(5):804-813.
[10] SHAKOURI M,MOAZZENI SM,GHANEI M,et al.A novel dendritic cell-targeted lentiviral vector,encoding Ag85A-ESAT6 fusion gene of Mycobacterium tuberculosis,could elicit potent cell-mediated immune responses in mice.Mol Immunol,2016,75:101-111[2018-10-20].https://doi.org/10.1016/j.molimm.2016.04.014.
[11] YIHAO D,HONGYUN H,MAODAN T.Latency-associated protein Rv2660c of Mycobacterium tuberculosis augments expression of proinflammatory cytokines in human macrophages by interacting with TLR2.Infect Dis (Lond),2015,47(3):168-177.
[12] AAGAARD C,HOANG T,DIETRICH J,et al.A multistage tuberculosis vaccine that confers efficient protection before and after exposure.Nat Med,2011,17(2):189-194.
[13] LUABEYA AK,KAGINA BM,TAMERIS MD,et al.First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected healthy adults.Vaccine,2015,33(33):4130-4140.
[14] 康月茜,张春燕,穆柳青,等.结核分枝杆菌Rv2460c基因的克隆,表达及编码产物的免疫特性分析.中国免疫学杂志,2015,31(6):790-794.
[15] 许崇利,杨梅,许崇波.大肠杆菌表达系统的影响因素.中国动物检疫,2010,27(8):66-68.
[16] 马建伟,李春明,冯思源,等.牛源多杀性巴氏杆菌融合基因pm0979-PlpE的表达及其融合蛋白的免疫效果.中国兽医科学,2015,45(10):1053-1057.