结核杆菌 D29 噬菌体几丁质酶基因的表达及功能研究

噬菌体一般通过表达内溶素来降解宿主菌细胞壁上的肽聚糖 . 用 PCR 技术从结核杆菌 D29 噬菌体基因组中克隆了 gene10 ，并使其在大肠杆菌中得到了高效表达，蛋白质 C 端带有 6×His. 用镍柱亲和纯化了大肠杆菌表达的 gp10 蛋白可溶性部分 . 活性测定表明， gp10 不但具有几丁质酶活性，还具有溶菌酶活性，是一种双功能的酶 . 耻垢杆菌经 gp10 作用后，其生长受到抑制，扫描电镜观察发现部分耻垢杆菌被降解 . 说明与其他种类噬菌体降解细胞壁的方式不同， D29 噬菌体可能利用 gp10 的溶菌酶活性使结核杆菌细胞壁降解 . 这有助于揭示结核杆菌噬菌体与其宿主的相互作用机制，是关于噬菌体几丁质酶的首次报道 .

Expression and Function Analysis of Mycobacteriophage D29 Chitinase Gene

Endolysins or lysins are cell-wall-hydrolysing enzymes synthesized during late gene expression in the lytic cycle of bacteriophage mutiplication and enable the release of progeny virions from infected cells through degradation of the bacterial peptidoglycan. The gene10 was amplified from purified DNA of Mycobacteriophage D29 by using PCR and was expressed with 6×His tag in E.coli. The soluble portion of recombinant protein was purified by Ni-NTA column and the activity was detected. The results showed that gp10 is a bi-functional enzyme, has chitinase and lysozyme activities. The cell wall degradation was observed by SEM.