Production method for streptococcus specific bacteriophage lyase
A technology of phage lyase and production method, which is applied in the production field of streptococcus-specific phage lyase, can solve problems such as the high-efficiency expression of streptococcus phage lyase PlyC and the like, and achieves a product with strong activity, remarkable effect and low price. Effect
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Embodiment 1
[0033] 1) Gene PlyCA and PlyCB of the streptococcal phage lyase PlyC described in the DNA sequence of PlyCA and PlyCB in the amplified sequence listing:
[0034] 2) constructing recombinant expression vectors of PlyCA and PlyCB;
[0035] 3) Transforming the recombinant expression vectors into host cells respectively to obtain engineering bacteria expressing streptococcal phage lyase PlyC heavy chain PlyCA and light chain PlyCB;
[0036] 4) fermenting and culturing engineering bacteria expressing heavy chain PlyCA and light chain PlyCB of streptococcal phage lyase PlyC to obtain recombinant gene expression products;
[0037] 5) Purify, isolate, renature, and induce expression to obtain streptococcal phage lyase PlyC.
Embodiment 2
[0038] Example 2: PlyC sequence cloning, vector connection and plasmid naming
[0039] Design primers according to the sequences of PlyCA and PlyCB, which are characterized in that the upstream and downstream primers have Nde I and Xho I restriction sites respectively, and the reactants are sequentially added and amplified according to the following order, conditions and ratios: the reaction volume is 25 μL, where Contains 0.5μL template DNA, 2.5μL 10×buffer, 2.5mmol / L MgCl 2 2μL, 2.5mmol / L dNTP 2μL, 50μmol / L P1 1μL, 50μmol / L P2 1μL, ddH 2 O 14.5 μL, Taq enzyme (5U / μL) 1.5 μL. PCR parameters: pre-denaturation at 95°C for 5 minutes; 30 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 60s; finally, extension at 72°C for 10 minutes. During the amplification, no template was added and only ddH was added 2 O blank control. After the reaction, 5 μL of the amplified product was identified by 1.2% agarose gel electrophoresis.
[0040] Enzyme digestion, purification and cloning...
Embodiment 3
[0072] Example 3: High-efficiency expression of PlyCA and PlyCB of PlyC protein in engineering bacteria and purification and renaturation of products
[0073] Recombinant bacteria BL21(DE3) / pET-32a(+)-PlyCA and BL21(DE3) / pET-32a(+)-PlyCB were inoculated into LB culture medium containing ampicillin (100 μg / mL), 37°C, 250r / After culturing overnight with shaking for 1 min, transfer to 100mL LB medium at a ratio of 1:50, shake culture until the A600 value at 600nm reaches 1.0-1.5, add IPTG to a final concentration of 1 mmol / L, induce expression for 4 hours, and detect by SDS-PAGE Express the situation. According to the results of electrophoresis, a monoclonal strain was selected as the experimental strain. The strain was inoculated with shaking culture to A 600 When the value is 0.8-1.2, it is used as a seed liquid, and is inserted into a fermentation medium with a 5% (v / v) inoculation amount for fermentation. A fed-batch high-density fermentation test was carried out in a sel...
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