Streptomyces Griseus Rx-17 Lysozyme Gene Library Construction And Screening

The Streptomyces gresius RX-17 producted high activityN,O-diacetylmuramidase, which possessed not only β-1,4-N,6-O-diacetylmuramidaseactivity but also β-1,4-N,6-O-diacetylmuramidase activity. Three components R1, R2 and R3 were purified by ion-exchange and near homogeneity by SDS-PAGE electrophoresis . It can be used to elucidate the fine structure of Staphylococcus aureus as well as Streptococcus mutants. The construction of Streptomyces gresius RX-17 gene library aims to selected the three lysome gene for futher study.According to shotgun method, Sau3AI cutted the genome of S.gresius incompletely, then 2-10kb fragment was linked with the linear plasmid pUC19 digested by BamHI and CIAP completely. Then the recombination plasmid transformed into competence cell of E.coli DH5a.. According to a complementary principle, the recombinants were obtained. After ampliative cultivation, the recombination plasmids were extracted to identify. Through gene amplification wecan get the gene library of Streptomyces gresius.Two strains were selected containing lysome activity through low temperature inducement at 25 ℃. Furthermore, PCR can amplify 700bp similar to R2 gene. SDS-PAGE can be detected 24kD which was coherent to R2. According to the N-terminal of R1 another pair of primers were designed, and we obtained 1.3kb for further sequencing.