MZTP02噬菌体tmp基因的鉴定:与尾巴装配相关的蛋白质

Identification of tmp Gene from Lysogenic Bacteriophage MZTP02:A Protein Associated with Phage Tail Packaging

苏云金芽孢杆菌(Bacillus thuringiensis)广泛用于生物防治,但溶原性噬菌体的随机爆发严重影响了它的生产。一种新的苏云金芽孢杆菌溶原性噬菌体被分离出来并进行了基因组测序。TMP(tape measure protein)蛋白质具有协助和控制噬菌体尾巴装配的功能,是噬菌体侵染期间协助其吸附宿主的一种必需蛋白质。使用PCR方法从MZTP02噬菌体基因组中扩增出tmp基因后,连接到pQE30质粒并用M15菌株进行表达,获得了相对分子质量为35 000的蛋白质条带;该蛋白质经Ni-NTA纯化后,作为抗原注入新西兰家兔制备获得多克隆抗体;Western blot分析发现了一条与原核表达大小一致的蛋白质表达带。噬菌体悬浮液混合TMP血清抗体后测定双层平板的滴度,在噬菌体的侵染复数(m.o.i)小于1的情况下,噬菌体的滴度与血清抗体的质量浓度呈反比关系。当一定滴度的噬菌体悬液分别混合25μg/mL和250μg/mL的血清质量浓度时,滴度分别为1×105pfu/mL和2×103pfu/mL。这一结果表明TMP血清抗体能够与TMP蛋白质相结合,从而导致了噬菌体滴度的降低。

Bacillus thuringiensis is widely used for pest control.However,the fermentation production of this bacterium was severely influenced by the ramdom outbreak of lysogenic phage from B.thuringiensis. In the present study,a novel bacteriophage MZTP02 was isolated from industrial B.thuringiensis strain MZ1 and sequenced for tmp gene encoding tail tape measure protein.By assisting and control the tail packaging of bacteriophage,the protein is an essential component to help phage attach to the host for suc-cessful infection.By PCR,tmp gene was amplified from the genome DNA of MZTP02 and cloned into pQE30 and expressed in M1 5 .The TMP protein of 35 000 was obtained and purified by Ni-NTA column. Using the purified TMP protein as the immunogen,polyclonal serum was raised in the New Zealand rab-bit.Western blot analysis of TMP proteins expressed in prokaryotic cells demonstrated a polypeptide with size of 35 000 which was in agreement with the predicted size of this protein.Results of plaque formation of phage after TMP antiserum treatment showed a negative relationship to the concentration of antiserum when the concentration of multiplicity of infection （m.o.i）was less than 1 ,e.g.the titers were 1 ×1 05 pfu/mL and 2 ×1 03 pfu/mL when a degnized amounts of phage were mixed with TMP antiserum with con-centration of 25 μg/mL and 250 μg/mL respectively.This result indicated that TMP antiserum can com-bine with TMP protein,and then depress the phage titer.