| Antibiotics are specially effective drugs that have been used to deal withpathogenic infection. Microorganism and viruses have developed numerousresistance mechanisms that enable them to evade the drugs of antimicrobialsand antivirals. With rapid development of resistance, microbes and viruseshave produced an evolutionary response to the selective pressure ofantimicrobials and multiple antibiotic resistance. So it is urgent for us to findnew approaches to treat infections of bacteria.The idea that bacteriophage could be used as a biological agent to controlbacterial infections was increasingly attented since it was discovered in theearly part of the 20th century. Phage is a kind of virus that infects bacteria. Thelast stage of infection is releasing phage particle from a sensitively bacterialhost by degradeing they cell wall of host.Many studies have shown that lysishave three common features:1.All double strand DNA bacteriophages canproduce a holin and at least one peptidoglycan hydrolase,or "lysin"which arecapable of degrading they bacterial cell wall.2.Most of phage have three typesof genes which respectively encode lysozymes, and N-acetylmuramoyl-L-alanine amidase, and endopeptidases.3.By compared with sequence, lysinfamily members have consistent chimaeric structure, consisting of a usuallywell-conserved catalytic domain of N-terminal and a largely divergentspecificity or binding domain of C-terminal.4. High-affinity binding isdirected towards species-or strain-specific cell-wall carbohydrates that areoften essential for viability. Although bacteriophage by their very nature have been used to killbacteria for many decades, they not were widely applied in clinic.The mainreason is that each kind of bacteriophage has it's specific host range ofbacteria.lysin, added to sensitive organisms in the absence of bacteriophage,lyses the cell wall, producing a phenomenon which was known as "lysis fromwithout".The high-affinity binding and species-or strain-specific of lysinimply that it could not do any harm to good bactieria living in human body.Thus lytic enzyme, as a new type of antimicrobial drug,has some advantages. The lysin Bacillus anthracis of consists of 233 amino acid.The bondbetween N-acetyl muramic acid and L-alanine.of cell wall can be cleaved by it.The lysin gene of Bacillus anthracis bacteriophage,which obtained by PCRamplification, was cloned into the Escherichia coli exepression vector pET22bwhich had been digested by EcoR I and Nde I.The recombinant pET22b-γlysinvector was transformed DH5αand verified to be correctly constructed byPCR, sequencing and enzyme digestion .The recombinant lysin protein washighly expressed in E.coli BL21(DE3), the expression level reached 1000mg/L and the OD600 reach 13 through 9-10 hours culture.Furthermore theexpression level reached 15 g/L, reaching 40% total protein content, through7-9 hours fermentention in the 5 L fermentor. The bacterial pellet from fermentention was resuspended by ultrasonicbuffer with the ratio of 1:10.After ultrasonication,the lysate was centrifuged toremove remained bacterial debris, and the clear supernatant was diluted andapplied to a Streamline SP column which had been equilibrated with 50mmol/L ammonium acetate buffer, The recombinant lysin protein was elutedin a linear gradient from 0 to 0.6mol/L ammonium acetate further(pH6.6),nextly purified by Sepharose SP HP column which had been equilibrated with50 mmol/L ammonium acetate buffer, eluted as in a Streamline SP purification,subseqently the protein was applied to a Sephacryl S-100 column and therecombinant γlysin was finally obtained with purity of higher than 95 per centby determined by gel scan. The final yield of purified recombinant proteinwas19.1 per cent, with a greater-than-350-fold increase in specific activity. The bacteria were grown overnight in rocking bottle. Divided intoidentical volume,the centrifuged pellet of bacteria was dilute with PBS.Purifed lysin was serial diluted by universal buffer and added to sample ofbacteria in the same volume. The value of OD600 was monitored for eachsample dury the course of the experiment. The activity of lysin for each strainwas evaluated by the initial velocity of lysis.After incubation in a water bath at37℃for 20 min, according to the standard protein with 1mg/mL,the OD600was measured for each dilution, and the reciprocal of the highest dilution thatwas nearest to half of the control value was defined as the activity of lysin inunits/mL.(i.e., from an OD600 of 1.0 to 0.5).The pure enzyme has been shownactive to Bacillus anthracis,and not to E.coli,Bacillus subtilis and Bacilluscereus. Its specific activity was about 1400 u/mg. The stability of enzyme was measured,the course of study was same asabove.After lysin was exposed for 10 min at 65℃, for 30min at 50℃, for 50days at 37℃, for 50 days at 30℃, for 6 months at 4℃, and for 6 months at37℃,the activity of lysin was tested at a pH range of 2 to 13 in a universalbuffer:in 0 to 200 mmol/L NaCl, in 0 to 20 mmol/L MgCl2, in 0 to 20 mmol/L... |
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