大肠杆菌噬菌体Bp4尾部蛋白在宿主识别中的作用研究

A study on the role of tail proteins of E.coli phage Bp4 in host recognition

为了研究大肠杆菌噬菌体Bp4尾部蛋白中的假定蛋白gp10、尾丝蛋白gp11和尾刺蛋白gp13在噬菌体吸附宿主菌中的作用,本研究经PCR扩增gp10、gp11和gp13基因,克隆至原核表达质粒pcoldTF中,构建重组质粒pcoldTF-gp10、pcoldTF-gp11、pcoldTF-gp13,并分别在E.coli BL21(DE3)中经IPTG诱导后经SDS-PAGE鉴定,结果显示各重组蛋白均获得了表达。将获得的各尾部蛋白纯化后分别4次免疫家兔,获得的各重组蛋白的多克隆抗体(anti-gp10、anti-gp11、anti-gp13)经琼脂双向扩散试验测定效价,分别为1:4、1:8、1:8。将各尾部蛋白与O;血清型大肠杆菌孵育10 min后,加入噬菌体Bp4增殖液进行竞争吸附试验,5 min后利用双层平板法检测并计数噬菌斑,计算噬菌体对大肠杆菌的相对吸附率;分别将各多克隆抗体与噬菌体孵育5 min以阻断噬菌体对大肠杆菌的吸附,再加入大肠杆菌5 min后采用上述双层平板法检测并计数大肠杆菌的噬菌斑,计算噬菌体对大肠杆菌的相对吸附率。尾部蛋白竞争吸附试验结果显示,gp13组中噬菌体对大肠杆菌的相对吸附率为0,与对照组差异极显著(P<0.01);而gp10组和gp11组中噬菌体对大肠杆菌的相对吸附率均为100%,均与对照组无差异,表明gp13能有效抑制噬菌体Bp4对宿主菌的吸附,而gp10和gp11则无此抑制作用。抗体阻断吸附试验结果显示,anti-gp11和anti-gp13组中噬菌体对大肠杆菌的相对吸附率均为0,与对照组差异均极显著(P<0.05);而antigp10组与对照组中噬菌体对宿主菌的相对吸附率均为100%,表明anti-gp13和anti-gp11均能够完全阻断噬菌体对宿主菌的吸附,而anti-gp10对其的吸附无阻断作用。上述结果首次证实大肠杆菌尾部蛋白gp13是大肠杆菌噬菌体Bp4吸附宿主菌的关键蛋白,本实验为研究噬菌体Bp4的感染机制及拓宽该噬菌体的宿主谱奠定了基础。

In order to study the role of hypothetical protein gp10,tail filament protein gp11 and tail thorn protein gp13 in the tail protein of coliphage Bp4 in phage adsorption of host bacteria,the genes of gp10,gp11 and gp13 were amplified by PCR and cloned into prokaryotic expression plasmid pcoldtf,and the recombinant plasmids pcoldtf-gp10,pcoldtf-gp11 and pcoldtf-gp13were constructed.The recombinant proteins were induced by IPTG in E.coli BL21(DE3)and identified by SDS-PAGE.The polyclonal antibodies(anti-gp10,anti-gp11,anti-gp13)of each recombinant protein were determined by double immunodiffusion test.The results showed that the titer of anti-gp10,anti-gp11,anti-gp13 were 1:4,1:8,and 1:8,respectively.After incubating each tail protein with O;serotype E.coli for 10 minutes,phage Bp4 proliferation solution was added for competitive adsorption test After 5 minutes,the plaque was detected and counted by double-layer plaque assay,and the relative adsorption rate of phag to E.coli was calculated.Each antiserum was incubated with phage for 5 minutes to block the adsorption of phage to E.coli After adding E.coli for 5 minutes,the plaque of E.coli was detected and counted by the above double-layer plaque assay,and th relative adsorption rate of phage to E.coli was calculated.The results of tail protein competitive adsorption test showed that th relative adsorption rate of phage to E.coli in gp13 group was 0,which was significantly different from that in the control group(P<0.05).The relative adsorption rate of phage to E.coli in gp10 group and gp11 group was 100%,which had no difference with the control group,indicating that gp13 could effectively inhibit the adsorption of phage bp4 to host bacteria,while gp10 and gp11had no such inhibitory effect;The results of antibody blocking adsorption test showed that the relative adsorption rate of phage to E.coli in anti-gp11 and anti-gp13 groups was 0,which was significantly different from that in the control group(P<0.05).Th relative adsorption rate of phage to host bacteria in anti-gp10 group and control group was 100%.The results showed that both anti gp13 and anti-gp11 could completely block the adsorption of phage to host bacteria,while anti-gp10 had no blocking effect on it adsorption.This study confirmed for the first time that E.coli tail protein gp13 is the key protein of coliphage Bp4 in the process o adsorbing the host,which lays a foundation for further study of the infection mechanism of phage Bp4 and broadening the hos spectrum of the phage.