Cloning, Expression, Anti-bacteria Effects Analysis And Optimizing Design Prediction Of Bacteriophage LSB-1Lyase Gene Gp17in E.Coli

Bacteriophages are related with antimicrobial research since discovered. In1928,penicillin was discovered by Fleming as antibiotics, because of the significant effect ofantibiotic treatment, and bacteriophage research was not mature, phage research onanti-bacteria had been on hold. Nelson et al obtained the purified phage lytic enzymes byrecombinant expression and study as research materials in2001. In recent years, along witha wide range of drug-resistant and the severe pressure of environmental protection, as wellas the antibacterial development cycle is much longer than the period of bacteria becametolerant to the antibacterial, all of these make phage related research become hotspot inantimicrobial research. Natural antibacterial effect of phage and artificially modified bymolecular biology techniques built up a new direction of antibacterial development inenvironmental cleanup, water purification, food safety and clinical application. Europe hasalso set up PhagoBurn program which was devoted to phage therapy in E. coli andPseudomonas aeruginosa infection after burnt in2013.Phages, as viruses of bacteria, which can recognize and lyse the host bacteriumspecifically, attract more and more attention by its sterilization effect. Lyase, which is usedby the phage to get into the bacteria or lyase bacteria, draws more and more attention ofresearchers.We obtained a new E. coli phage named LSB-1from the sewage of hospital in theprevious study. In the preliminary research we found the gene gp17was related to the lysisof bacteria, then sequenced and submitted it to the GenBank (GenBank number:KC425724.1). In our research we constructed the recombinant plasmid, expressed andpurified the recombinant protein, and we tested its effective bacteria spectrum by the KBmethod, which was compared with the result by recombinant phage T7-LSB-gp17obtainedby phage display technology. To improve the activity of enzyme further up, we constructedserious of mutants, and examined the effects by AutoDock. The contents and results of our study include the following aspects:1. Cloning, expression and purification of sialidase: we constructed the recombinantplasmid by recombinant technology and expressed in E. coli BL21(DE3), and tested itsconcentration by BCA method. Finally we obtained the gene expression product of gp17,the molecular weight of which was78kDa and the concentration of which was2.38mg/ml.2. Antibacterial activity of the recombinant protein was determined by the antibacterialassay: we tested many strains including E.coli and Staphylococcus, found that therecombinant sialidase had good bacteriostasis to the host strain EIEC8401, obviousbacteriostatic cycle was about2cm in diameter, and this phenomenon didn’t occur in anyother strains, which showed good specificity. Compared with the result of recombinantphage T7-LSB-gp17obtained by phage display technology, we found obvious difference,which might be caused by structure change.3. Mutants was designed by the methods of domain expressed only and mutants innonconservative sites, and analysed by AutoDock, we found that the domain only (mutant2)and mutants in124(mutant6) and167(mutant7) from Asn to Ser showed better bingdingability to the substance, and found bioinformatics difference in mutants and original.In summary, we obtained the purified protein by cloning, expression, purification; wefound the protein had good specificity to the host strain EIEC8401. Compared with theresults of recombinant phage T7-LSB-gp17obtained by phage display technology, wefound obvious difference. By constructing a series of mutants, we found the domain only(mutant2) and mutants in124(mutant6) and167(mutant7) from Asn to Ser showed goodbinding capacity than the original protein, which could be examined by experiment. Theresults of this study also laid foundation in function and mechanisms research of thesialidase.