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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (5): 66-72    DOI: 10.13523/j.cb.20180509
技术与方法     
共表达构建呈现人白细胞介素-13抗原肽的Qβ噬菌体病毒样颗粒 *
白红妹,黄惟巍,刘存宝,孙文佳,杨旭,马雁冰()
中国医学科学院/北京协和医学院 医学生物学研究所 昆明 650118
Co-expression Construction of Qβ Phage Virus-like Particles Presenting Human Interleukin-13 Antigenic Peptide
Hong-mei BAI,Wei-wei HUANG,Cun-bao LIU,Wen-jia SUN,Xu YANG,Yan-bing MA()
Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College,Kunming 650118,China
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摘要：

目的:构建呈现人白细胞介素-13(interleukin-13,IL-13)抗原肽的Qβ噬菌体病毒样颗粒(virus-like particles, VLPs)疫苗。方法:将人 IL-13 抗原肽经基因重组插入Qβ噬菌体衣壳蛋白(CP)的C端。在BL21细菌中,经IPTG诱导CP及C端接有IL-13 抗原肽的CP(CP /IL-13)同时表达。以硫酸铵沉淀及蔗糖密度梯度离心进行VLPs纯化及分析嵌合VLPs的存在,以HPLC分析VLPs纯度,以电镜观察颗粒形态。小鼠经皮下免疫VLPs后采集血清,以ELISA检测人 IL-13特异性IgG抗体水平。结果:重组蛋白CP与CP /IL-13获得成功表达,两者在密度梯度离心中有一致的、与QβVLPs相同的沉降行为,而CP /IL-13单独无Qβ颗粒行为。经纯化获得了高纯度颗粒,嵌合颗粒与Qβ颗粒形态相似。此外,该VLPs疫苗诱导小鼠产生了IL-13特异的抗体应答。结论:利用共表达策略可成功构建呈现人 IL-13 抗原表位的嵌合VLPs,为以主动免疫方式调控IL-13在疾病中的病理作用,提供了具有临床应用潜能的疫苗形式。
关键词： 白细胞介素-13;  病毒样颗粒;  疫苗    
Abstract:

Objective:To construct Qβ phage virus-like particles (VLPs) vaccines presenting human interleukin-13 (IL-13) antigen peptide.Methods:The human IL-13 antigen peptide was genetically recombined into the C terminus of the Qβ phage capsid protein (CP). In BL21 bacteria, the native CP and the recombinant CP with C terminal fused with IL-13 antigen peptide (CP / IL-13) were induced simultaneously by IPTG. VLPs were purified by ammonium sulfate precipitation and sucrose density gradient centrifugation, and the presence of chimeric VLPs was analyzed. The purity of VLPs was analyzed by HPLC and the morphology of the particles was observed by electron microscopy. The mice were subcutaneously immunized with VLPs and sera were collected for detection of human IL-13-specific IgG antibodies by ELISA. RESULTS: The recombinant protein CP and CP / IL-13 were successfully expressed. Both of them appeared in the same fractions of collected samples from density gradient centrifugation and had the same sedimentation behavior as the native Qβ VLPs, while CP / IL-13 alone had no Qβ particle behavior. After purification, high purity particles were obtained. The chimeric particles were similar in shape to Qβ particles. In addition, the VLPs vaccine induced mice to develop an IL-13-specific antibody response.Conclusion:The co-expression strategy can successfully construct chimeric VLPs presenting human IL-13 epitopes, and provide a vaccine form with potentials for clinical application antagonizing the pathological effects of IL-13 in severe human diseases.
Key words: Interleukin-13    Virus-like particles    Vaccine
收稿日期: 2018-01-14 出版日期: 2018-06-05
ZTFLH:  Q819  
基金资助: * 云南省应用基础研究计划重点项目(2016FA049);国家自然科学基金面上项目资助项目(81773270)
通讯作者: 马雁冰     E-mail: may@imbcams.com.cn
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白红妹
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引用本文:

白红妹,黄惟巍,刘存宝,孙文佳,杨旭,马雁冰. 共表达构建呈现人白细胞介素-13抗原肽的Qβ噬菌体病毒样颗粒 *[J]. 中国生物工程杂志, 2018, 38(5): 66-72.

Hong-mei BAI,Wei-wei HUANG,Cun-bao LIU,Wen-jia SUN,Xu YANG,Yan-bing MA. Co-expression Construction of Qβ Phage Virus-like Particles Presenting Human Interleukin-13 Antigenic Peptide. China Biotechnology, 2018, 38(5): 66-72.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180509    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I5/66

名称 寡核苷酸序列(5'→3')
IL-13抗原肽 正链GATCCGTTCCGCCGTCTACCGCTCTGCGTGAACTGATCGAAGAACTGGTTAACATCACCCAGG
负链AATTCCTGGGTGATGTTAACCAGTTCTTCGATCAGTTCACGCAGAGCGGTAGACGGCGGAACG
启动子1-CP 正链CATGCCATGGCAAAATTAGAGACTGTTA
负链ATCCCTGCAGTTAATACGCTGGGTTCAGCTGA
启动子2- CP/X 正链GAGCATATGGCAAAATTAGAGA
负链AAGGGTACCTTAGAATTCGCCACCGGA
启动子1- CP/X 正链CATGCCATGGCAAAATTAGAGACTGTTA
负链AAGCTGCAGTTAGAATTCGCCACCGGA
启动子2- CP 正链GAGCATATGGCAAAATTAGAGA
负链AAGGGTACCTTAATACGCTGGGTTCAGCTGA
表1  人 IL-13抗原肽正负链核苷酸序列及PCR扩增引物
图1  pRSFDuet1质粒图谱
图2  重组蛋白的表达及密度梯度离心
图3  重组蛋白的HPLC凝胶过滤层析分析及电镜观察
图4  ELISA 检测小鼠血清中 IL-13 抗体水平
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