Role of Tail Fiber Protein in Attaching Riemerella anatipestifer to Phage
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摘要:
目的 以鸭疫里默氏菌（ Riemerella anatipestifer ）噬菌体CRP2为对象，研究尾丝蛋白（Tail fiber protein，TFP）对噬菌体吸附功能的影响。 方法 克隆ORF70假定的TFP基因，构建重组表达载体pET28a（+）-TFP，在大肠杆菌E.Coli BL21（DE3）中诱导表达，通过Ni-NTA亲和层析纯化。酶联免疫吸附试验（ELISA）测定重组蛋白与CRP2噬菌体的宿主菌外膜蛋白（Outer membrane protein, OMP）的结合作用。竞争吸附试验测定重组蛋白对CRP2噬菌体吸附率的影响。 结果 构建了pET28a（+）-TFP重组表达质粒，在大肠杆菌中得到了可溶性表达。纯化的重组蛋白可以与宿主菌的OMP结合，也可以抑制CRP2对其宿主菌的吸附。 结论 ORF70编码的TFP是噬菌体CRP2的一种受体结合蛋白，其对应的受体是宿主菌的OMP。 Abstract:Objective Role of tail fiber protein (TFP) plays in adhering Riemerella anatipestifer by CRP2 phage was investigated. Method The putative TFP gene of ORF70 was cloned. The recombinant expression vector pET28a(+)-TFP was constructed and induced to express in E. coli BL21(DE3). The recombinant protein was purified by Ni-NTA affinity chromatography. ELISA was used to determine the binding effect of the recombinant protein with the outer membrane protein (OMP) of host bacteria. A competitive adsorption test was performed to determine the adhering effect of the CRP2 protein. Result The recombinant plasmids of pET28a(+)-TFP was successfully constructed and expressed in E. coli. The purified recombinant protein rTFP could bind to the OMP of or suppress the CRP2 adsorption to the host bacteria. Conclusion The TFP encoded by ORF70, one of the receptor binding proteins of CRP2 phage, was confirmed to adhere to the host bacteria OMP facilitating the subsequent bactericidal effect. -
Key words:
- Riemerella anatipestifer /
- phage /
- tail fiber protein /
- adsorption
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图 1 重组蛋白的SDS-PAGE分析
M：蛋白Marker；1：TFP未诱导；2：TFP裂解液上清；3：TFP裂解液沉淀；4：纯化的rTFP。
Figure 1. SDS-PAGE of recombinant protein
M: Protein molecular weight marker; 1: TFP expression without induction; 2: Expression of TFP lysate supernatant; 3: Expression of TFP lysate precipitate; 4: Purified protein of rTFP.
图 2 重组蛋白的Western blotting分析
1：rTFP；M：蛋白Marker。
Figure 2. Recombinant protein identified by western blot
1: Recombinant rTFP; M: Protein molecular weight marker.
图 3 重组蛋白与OMP的ELISA结果
OMP+rTFP：OMP与rTFP的作用结果；OMP+PBS：OMP与PBS的作用结果。***：两组数据差异极显著（P<0.01）。
Figure 3. ELISA results on recombinant protein and OMP
OMP+rTFP: Interaction between OMP and rTFP; OMP+PBS: Interaction between OMP and PBS. ***: extremely significant difference between two data sets（ P <0.01）.
图 4 重组蛋白对噬菌体CRP2吸附率的影响
Figure 4. Effect of recombinant proteins on CRP2 adsorption rate
图 5 噬菌体CRP2、CRP5、CRP12、CRP19尾丝蛋白氨基酸序列的比较
图形上的黑色矩形框表示序列相似性最高，红色次之，绿色更低。黑点表示氨基酸缺失。
Figure 5. Amino acid sequences of TFPs in CRP2, CRP5, CRP12, and CRP19
Black rectangle box indicates highest sequence similarity; red, intermediate; and green, lowest. Black dot indicates missing amino acid.
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