食品科学 ›› 2021, Vol. 42 ›› Issue (2): 66-73.doi: 10.7506/spkx1002-6630-20191031-359

• 生物工程 • 上一篇    下一篇

沙门氏菌噬菌体LPST144尾纤维gp38的序列分析及其结合活性

杨其乐,丁一峰,张宇,聂若男,李亚萌,王佳,王小红   

  1. (华中农业大学食品科学技术学院,湖北 武汉 430070)
  • 出版日期:2021-01-18 发布日期:2021-01-27
  • 基金资助:
    国家自然科学基金面上项目(31772054);“十三五”国家重点研发计划重点专项(2017YFC1600100)

Sequence Analysis and Binding Activity of Salmonella Phage LPST144 Tail Fiber gp38

YANG Qile, DING Yifeng, ZHANG Yu, NIE Ruonan, LI Yameng, WANG Jia, WANG Xiaohong   

  1. (College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China)
  • Online:2021-01-18 Published:2021-01-27

摘要: 对1 株沙门氏菌短尾噬菌体LPST144的尾纤维进行序列结构分析和表达纯化,成功验证其尾纤维基因orf38表达产物gp38的特异性受体结合活性,从而得到一个潜在的沙门氏菌检测探针。首先采用生物信息学的方法分析LPST144噬菌体尾纤维gp38的氨基酸序列、理化性质、遗传进化关系和C末端二级结构,结果表明:LPST144的尾纤维包含646 个氨基酸残基,N端具有2 个保守结构域,且与T7噬菌体相似度较高;而C末端序列差异极大,存在大量的β-折叠结构,与T7噬菌体尾纤维C末端二级结构类似。gp38具有噬菌体受体结合蛋白的多个特点:具有模块化的性质、序列相似性低且C末端富含β-折叠结构。进一步将尾纤维基因orf38进行异源表达、纯化,采用全菌包被的酶联免疫吸附法检测其特异性结合宿主鼠伤寒沙门氏菌ATCC13311的能力,结果表明实验组的吸光度为0.94±0.02,阴性对照组吸光度为0.17±0.01,对宿主菌具有较强的结合能力;且gp38重组蛋白与实验中受试的其他6 株不同血清型的沙门氏菌均能结合;采用大肠杆菌T10、沙门氏菌外膜蛋白、金黄色葡萄球菌6538及磷酸盐缓冲液代替宿主菌作为对照,吸光度分别为0.58±0.03、0.56±0.01、0.59±0.03和0.53±0.005,实验组与对照组结果具有显著差异,表明尾纤维gp38具有特异性结合宿主鼠伤寒沙门氏菌ATCC13311以及实验中其他受试沙门氏菌的能力。本研究可为开发基于噬菌体受体结合蛋白为分子探针建立沙门氏菌检测方法奠定实验基础。

关键词: 鼠伤寒沙门氏菌;噬菌体尾纤维;序列分析;表达纯化;特异性结合能力

Abstract: In this study, the tail fiber gene orf38 of Salmonella Podoviridae LPST144 was sequenced and expressed and the product (gp38) was purified, whose specific receptor binding activity was successfully verified, yielding a potential probe for Salmonella detection. Bioinformatics was used to analyze the amino acid sequence, physicochemical properties, genetic evolution relationship and C-terminal secondary structures of LPST144 tail fiber gp38. Results showed that the tail fiber gp38 contained 646 amino acid residues, and two conserved regions with high similarity to T7 existed at its N-terminal. However, the C-terminal sequence was extremely different, and there were large numbers of β-sheet structures similar to the C-terminal secondary structures of T7 tail fiber. gp38 had many features of phage receptor binding proteins such as modular properties, low sequence similarity and β-sheet-rich C-terminal. The ability of the recombinant protein to specifically bind to the host Salmonella typhimurium (ATCC13311) was detected by whole bacterial cell coating ELISA. Results showed that the absorbance value of the experimental group was 0.94 ± 0.02, as compared to 0.17 ± 0.01 for the negative control, indicating strong binding ability to the host cells. The recombinant gp38 could also be combined with six other serotypes of Salmonella tested. When Escherichia coli (T10), Salmonella outer membrane protein, Staphylococcus aureus (6538) and PBS buffer were used instead of the host cells, the absorbance values were 0.58 ± 0.03, 0.56 ± 0.01, 0.59 ± 0.03 and 0.53 ± 0.005, respectively. The observed significant difference between the experimental and control groups indicated that the tail fiber gp38 has the ability to specifically bind to the host S. typhimurium (ATCC13311) and the other serotypes of Salmonella. This study can lay an experimental foundation for the development of Salmonella detection methods using phage receptor binding proteins as a molecular probe.

Key words: Salmonella typhimurium; phage tail fiber; sequence analysis; expression and purification; specific binding ability

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