| 猪链球菌蛋白表面展示系统的构建及展示蛋白的初步观察 |
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| 引用本文： | 黄萌萌,刘冉,陈平,朱金鲁,卫东,谢芳,李刚,刘思国,张跃灵.猪链球菌蛋白表面展示系统的构建及展示蛋白的初步观察[J].中国预防兽医学报,2020(2):105-113. |
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| 作者姓名： | 黄萌萌 刘冉 陈平 朱金鲁 卫东 谢芳 李刚 刘思国 张跃灵 |
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| 作者单位： | 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室 |
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| 基金项目： | 国家重点研发计划(2017YFD0500203);国家自然科学基金(31772757、31672575);黑龙江省自然科学基金(C2017075、C2017078)。 |
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| 摘 要： | 为构建猪链球菌(Streptococcus suis)蛋白表面展示系统,本研究通过序列分析,确定猪链球菌的LPxTG蛋白及其信号肽(SP)和胞壁锚定基序(CWA),通过PCR扩增Peno-SP、GFP、CWA的DNA片段并融合,构建强启动子Peno控制表达编码SP-GFP-CWA融合蛋白的DNA片段,将该重组DNA片段连接pSET2载体,获得蛋白表面展示质粒,转化猪链球菌,构建得到以GFP为报告蛋白的猪链球菌蛋白表面展示系统。结果显示,利用猪链球菌的10个LPxTG蛋白及其SP和CWA序列,构建了10个含有Peno-SP-GFP-CWA融合片段的重组pSET2表面蛋白展示质粒pSsPSD1至pSsPSD10,分别转化猪链球菌05ZYH33,PCR鉴定显示其中7个转化猪链球菌。采用western blot初步检测其展示蛋白,结果显示,7个转化阳性菌株均能有效表达GFP蛋白,以成熟GFP条带为指标,均表现出了一定的外源GFP表面展示水平,分别命名为SsPSD1、SsPSD2、SsPSD4、SsPSD7-SsPSD10,其中SsPSD1、SsPSD4、SsPSD8和SsPSD9表面展示水平相对较好,在猪链球菌表面展示外源蛋白方面具有很好的潜力。本研究首次尝试建立猪链球菌蛋白表面展示系统,为猪链球菌表面递呈外源蛋白或抗原提供了新的策略。
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| 关 键 词： | 猪链球菌 蛋白表面展示 LPxTG 信号肽 胞壁锚定基序 |
Construction of Streptococcus suis protein surface display system and preliminary observation of the displayed foreign proteins |
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| HUANG Meng-meng,LIU Ran,CHEN Ping,ZHU Jin-lu,WEI Dong,XIE Fang,LI Gang,LIU Si-guo,ZHANG Yue-ling.Construction of Streptococcus suis protein surface display system and preliminary observation of the displayed foreign proteins[J].Chinese Journal of Preventive Veterinary Medicine,2020(2):105-113. |
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| Authors: | HUANG Meng-meng LIU Ran CHEN Ping ZHU Jin-lu WEI Dong XIE Fang LI Gang LIU Si-guo ZHANG Yue-ling |
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| Institution: | (State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China) |
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| Abstract: | In order to develop a protein surface display system for Streptococcus suis, the LPxTG proteins and their signal peptides(SP) and cell wall anchoring motifs(CWA) were identified from S. suis by sequence analysis. By amplification and fusion of the DNA fragments of Peno-SP, GFP and CWA, the recombinant DNA fragment encoding the SP-GFP-CWA fusion protein controlled by a strong promoter Peno was constructed. The recombinant DNA fragments were ligated into the vector pSET2,resulting in the protein surface displaying plasmids. The plasmids were transformed into S. suis to obtain the protein surface display systems, with GFP as the reporter protein. The surface displayed proteins were preliminarily observed by western blots. The results showed that 10 LPxTG proteins and their SPs and CWAs were identified from S. suis and 10 pSET2 surface protein display plasmids containing Peno-SP-GFP-CWA fusion fragments were respectively constructed, namely pSsPSD1 to pSsPSD10. Seven of them were successfully transformed into S. suis. Western blot and preliminary quantitative analysis showed that all seven transformation-positive strains effectively express GFP protein. Using mature GFP band as an indicator, all of them showed certain levels of exogenous GFP surface display. This indicated that the protein surface display system of S. suis was successfully constructed. They were named as SsPSD1, SsPSD2, SsPSD4, SsPSD7-SsPSD10, respectively. The surface display of SsPSD1, SsPSD4, SsPSD8 and SsPSD9 showed higher surface display levels and have good potential in displaying exogenous proteins on the surface of S. suis. This study is the first attempt to establish protein surface display systems for S. suis. It provides a new strategy for presenting foreign proteins or antigens on the surface of S. suis. |
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| Keywords: | Streptococcus suis protein surface display LPxTG signal peptide cell well anchor motif |
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