噬菌体Bp7蛋白gp38的克隆、表达及其受体识别活性分析

doi:10.11843/j.issn.0366-6964.2018.11.018

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (11): 2461-2467.doi: 10.11843/j.issn.0366-6964.2018.11.018

噬菌体Bp7蛋白gp38的克隆、表达及其受体识别活性分析

麻海澜, 孙虎芝, 任慧英*, 张灿*

青岛农业大学动物医学院, 青岛 266109

收稿日期:2018-03-19 出版日期:2018-11-23 发布日期:2018-11-23
通讯作者: 张灿,副教授,E-mail:cleverflame@163.com;任慧英,教授,E-mail:renren0228@sina.com
作者简介:麻海澜(1996-),女,山东临沂人,本科,主要从事微生物与免疫研究,E-mail:17854256346@163.com
基金资助:
国家自然科学基金（31600149）；国家级大学生科技创新项目（201710435008）；青岛农业大学大学生科技创新项目

Cloning, Expression and Receptor-Recognizing Activity of Bacteriophage Bp7 Protein gp38

MA Hai-lan, SUN Hu-zhi, REN Hui-ying*, ZHANG Can*

College of Veterinary Science, Qingdao Agricultural University, Qingdao 266109, China

Received:2018-03-19 Online:2018-11-23 Published:2018-11-23

摘要/Abstract

摘要：

为了研究噬菌体Bp7蛋白gp38在噬菌体吸附宿主菌过程中的作用，PCR扩增gp38基因，对gp38蛋白氨基酸序列特征进行分析和同源性比对，构建重组表达质粒pColdTF-gp38，在E.coli BL21中诱导表达，SDS-PAGE电泳和Western blot检测重组蛋白gp38的表达；亲和层析法纯化蛋白，制备gp38多克隆抗体。免疫电镜检测gp38在噬菌体Bp7表面的定位，蛋白竞争试验和抗体阻断试验测定gp38及其抗体对噬菌体Bp7吸附效率的影响。序列分析结果表明，gp38蛋白氨基酸序列与T4噬菌体无同源性，与噬菌体T2、T6有同源性，C端序列保守区与高变区间隔排列，呈现马赛克特征；构建的重组质粒pColdTF-gp38在E.coli BL21实现可溶性表达，制备的gp38多克隆抗体效价达1∶16；免疫电镜检测结果表明，gp38蛋白抗体能够与长尾丝结合，引起噬菌体Bp7的聚集；蛋白竞争试验和抗体阻断试验结果表明gp38蛋白及其抗体均能完全抑制噬菌体Bp7对宿主菌E.coli K12的吸附。以上研究结果表明，gp38是噬菌体Bp7的尾丝蛋白，位于长尾丝末端，作为受体识别蛋白在噬菌体Bp7吸附宿主菌过程中具有关键作用，这为进一步阐明宽宿主谱噬菌体Bp7识别受体的机制提供了理论基础。

Abstract:

To identify the role of protein gp38 in the adsorption process of phage Bp7, gp38 gene was amplified by PCR, and the amino acid sequence was characterized. The recombinant plasmid pColdTF-gp38 was constructed and expressed in E. coli BL21, then identified by SDS-PAGE and Western blot. Protein gp38 was purified by affinity chromatography to prepare polyclonal antibody. Immunoelectron microscopy assay was used to locate gp38 on phage Bp7, protein competition assay and antibody blocking assay were used to determine gp38 and its antibody effects on phage Bp7 adsorption efficiency. Amino acid sequence analysis of phage Bp7 protein gp38 showed that there was no homology with T4 bacteriophage, but shared homology with phage T2 and T6. The conserved regions and variable regions of gp38 C-terminal sequence were arranged at intervals, showing a mosaic characteristics; the recombined plasmid pColdTF-gp38 was constructed and expressed in E. coli BL21, The gp38 polyclonal antibody was prepared with a 1:16 titer. The immunoelectron microscopy showed that the gp38 antibody could bind to the long tail fiber and cause the aggregation of phage Bp7; protein competition test and antibody blocking test showed that gp38 and its antibody could completely inhibit the absorption of phage Bp7 to host strain E. coli K12. All of results indicated that gp38 is the tail fiber protein of phage Bp7, located at the end of the long tail fiber, and played a key role as the receptor recognition protein in phage Bp7 adsorption process, which further provided the theoretical foundation to elucidate the receptor recognition mechanism of phage Bp7.

中图分类号:

Q939.48

麻海澜, 孙虎芝, 任慧英, 张灿. 噬菌体Bp7蛋白gp38的克隆、表达及其受体识别活性分析[J]. 畜牧兽医学报, 2018, 49(11): 2461-2467.

MA Hai-lan, SUN Hu-zhi, REN Hui-ying, ZHANG Can. Cloning, Expression and Receptor-Recognizing Activity of Bacteriophage Bp7 Protein gp38[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(11): 2461-2467.

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噬菌体Bp7蛋白gp38的克隆、表达及其受体识别活性分析

Cloning, Expression and Receptor-Recognizing Activity of Bacteriophage Bp7 Protein gp38

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