Expression of coxsackievirus and adenovirus receptor (CAR)-Fc fusion protein in Pichia pastoris and characterization of its anti-coxsackievirus activity.
Expression of coxsackievirus and adenovirus receptor (CAR)-Fc fusion protein in Pichia pastoris and characterization of its anti-coxsackievirus activity.
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DOI：
10.1016/j.jbiotec.2013.01.015
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发表时间：
2013-04
影响因子：
4.1
通讯作者：
Kebin Zhang;Hua Yu;W. Xie;Zihui Xu;Shiwen Zhou;Chunji Huang;Halei Sheng;Xiaomei He;Junzhi Xiong;G. Qian
中科院分区：
文献类型：
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作者：
Kebin Zhang;Hua Yu;W. Xie;Zihui Xu;Shiwen Zhou;Chunji Huang;Halei Sheng;Xiaomei He;Junzhi Xiong;G. Qian
柯萨奇病毒和腺病毒受体（汽车）是介导柯萨奇病毒或腺病毒感染的常见细胞受体。受体陷阱疗法是利用可溶性病毒受体阻断病毒的粘附和内化，用于抑制病毒感染。本研究构建了pPIC 3. 5 K/CAR-Fc表达质粒，为在毕赤酵母中经济、规模化生产CAR-Fc融合蛋白奠定了基础。根据宿主密码子使用偏好性对融合蛋白的编码序列进行了优化。通过1%甲醇诱导96 h，CAR-Fc蛋白占总细胞蛋白的量高达10%，蛋白纯化后纯度高达96%。接下来，病毒下拉测定证明了CAR-Fc与柯萨奇病毒的结合活性。MTT法、免疫荧光染色法和病毒中和后实时荧光定量PCR分析表明，CAR-Fc在体外能显著阻断柯萨奇病毒B3的感染。在柯萨奇病毒B3感染的小鼠模型中，CAR-Fc治疗降低了死亡率、心肌水肿、病毒载量和炎症，表明了体内显著的病毒阻断作用。结果表明，P. pastoris表达系统可用于生产大量具有生物活性的CAR-Fc，用于进一步的临床目的。
Coxsackievirus and adenovirus receptors (CARs) are the common cellular receptors which mediate coxsackievirus or adenovirus infection. Receptor trap therapy, which uses soluble viral receptors to block the attachment and internalization of virus, has been developed for the inhibition of virus infection. In this study, we have constructed a pPIC3.5 K/CAR-Fc expression plasmid for the economical and scale-up production of CAR-Fc fusion protein inPichia pastoris. The coding sequence of the fusion protein was optimized according to the host codon usage bias. The amount of the CAR-Fc protein to total cell protein was up to 10% by 1% methanol induction for 96 h and the purity was up to 96% after protein purification. Next, the virus pull-down assay demonstrated the binding activity of the CAR-Fc to coxsackievirus. The analyses of MTT assay, immunofluorescence staining and quantitative real-time PCR after virus neutralization assay revealed that CAR-Fc could significantly block coxsackievirus B3 infectionin vitro. In coxsackievirus B3 infected mouse models, CAR-Fc treatment reduced mortality, myocardial edema, viral loads and inflammation, suggesting the significant virus blocking effectin vivo. Our results indicated that theP. pastorisexpression system could be used to produce large quantities of bioactive CAR-Fc for further clinical purpose.