Studies On The Recombination And Application Of Hydroltyic Enzyme From Phage LSB-1

In October, 2010, several countries had reported the“Super- bug”, a bacteria which has an antibiotic-resistant " gene" known as NDM-1. This type of resistant bacteria can cause fatal urinary tract infections and pneumonia. Referred to by some medical professionals as one of the world’s most pressing public health challenges, a superbug is an antibiotic resistant strain of bacteria, the prevalence of which is on the rise throughout the world.This event give us a chance to consider the disaster of resistant bacteria again. Study and explores the new anti-bacteria technology is a urgent duty in the field of medine.Bacteriophage, takes bacterium’s virus, can specificity infection and decomposition the bacterium. With rich biological characteristic, the bacteriophage could present us colourful imaging. It is the important biological model to design the new drugs.The hydroltyic enzyme is phage’s essential lysis ingredient. Observe and explore its structure and the function has the vital significance in the developing biology enzymeIn this study, the phage LSB-1, which was isolated from sewage samples, has been used as test obiect. The methods of microbiology, molecular biology and biological information have been used to observe and measure the new bacteriophage’s shapes, structure. The gene and the protein structure of endo-N belong to hydrolisis enzyme system also have been studied. The research procedure and results as follows.1. The biological nature and molecular characteristic of phage LSB-1: Measured by electron microscopy, a step growth curve and the nuclease cuts techniques, the results have showed that LSB-1, a reference coliphage strain, was classified as a member of the Podoviridae family with a cystic form (50±5 nm diameter) and short tail (60±5 nm long). The 5min adsorption rate is 94.3% , the incubation period is 23min and the average release quantity is 80. The double stranded DNA was about 30 kilobase pairs in length. 2. Determination of endo-N: With the methods of shotgun entire genome team sequence technique, the Biological information analysis , 15 major potential ORFs were identified. The gp17 protein is endo-N, has the function of adsorption and digestion .3. Structure analysis of endo-N:With the methods of the Biological information analysis, the gp17 protein was composed of 33.10% Alpha helix (Hh), 25.25% Extended strand (Ee) and 30.29% Random coil (Cc). The endo-N 3-dimensional structure model predicted two domains: Domain A with hydrolase activity connected by an intervening unstructured sequence to Domain B. The later domain may have a host-anchoring function.4.Gp17 gene clone: The gp17 have been increases and insert into the carrier of phage T7. The PCR confirmed the recorganization. Afte recorganized the gene of gp17, the phage T7 increased the lysis function to E.coli 8401.