Protein expression and function of the vpr gene of the vpr isogenic knockout E. coli mutant

Chinese. 进一步确定vpr基因及相关蛋白的功能。在获得vpr同源基因突变株MC1061△vpr的基础上，构建了vpr基因突变株的回复株MC1061-C，并进行了噬菌体裂解试验、细胞壁吸附试验、蛋白表达和印迹试验。亲本株MC1061和回复株MC1061-C对VT2噬菌体C43b的裂解敏感，突变株MC1061△vpr抵抗噬菌体C43 d的裂解。噬菌体吸附细胞壁试验中，吸附30 min后，亲本株和回复株的上清液中分别存在6.4％和5.2％的游离噬菌体，吸附到90 min时未有大量的游离噬菌体释放。噬菌体与突变株的细胞壁吸附30 min后，上清液中有85.4％的游离噬菌体存在，表明VT2噬菌体能与亲本MC1061和回复株MC1061-C的细胞壁不可逆吸附，但不能与突变株MC1061△vpr的细胞壁发生不可逆吸附。在蛋白表达中，Western blot显示回复株和亲本株均能转印出特异性90 000蛋白主带，而突变株没有。vpr基因表达的分子量为90 000的Vpr蛋白与VT2噬菌体的裂解敏感性有关，可能是VT2噬菌体的裂解受体。

English. In order to confirm the function of vpr gene. The complement of the mutant strain MC1061△vpr was constructed by translating intact vpr into the vpr isogenic knockout E. coli mutant and identified using PCR. The lytic susceptibility of VT2 bacteriophage C43b to parent strain MC1061, mutant strain MC1061△vpr, complemented strain were detected. The cell walls of above three strains were extracted, respectively. The test of phage-adsorption to cell wall, Vpr protein expression and blotting were also carried out. One complemented strain, named MC1061-C, was obtained. The complemented strain returned the susceptibility to the lytic infection of VT2 phage with many small plaques, while the mutant was still resistant the phage. Phage-adsorption test within 30 min showed that 94.8% VT phage could adsorb on the cell walls of complemented MC1061-C, while 85.4％ VT phage could not adsorb on the cell walls of mutant MC1061△vpr. The specific peptide of 90 000 from parent and complemented strain were displayed by the Western blot using the rabbit anti-Vpr, the His-Vpr was purified by affinity chromatography. The mutant did not display this protein. All these revealed that the Vpr supported infection of VT2 phage and most likely be the receptor of VT2 phage.