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

Inactive Publication Date: 2008-10-01
ZHEJIANG SHUREN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report on the high expression of st

Method used

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  • Production method for streptococcus specific bacteriophage lyase
  • Production method for streptococcus specific bacteriophage lyase
  • Production method for streptococcus specific bacteriophage lyase

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention provides a producing method of streptococcus specific phage lyase. The lyase is composed of a heavy chain (about 50kDa/per chain) )PlyCA and a light chain (about 8kDa/per chain) according to the ratio of 1:8, its gene name is PlyC, having following nucleotide sequences: DNA sequences of PlyCA(sequence 1) and PlyCB(sequence 3) in sequence table code amino acid sequence (sequence 2) of PlyCA and amino acid sequence (sequence 4) of PlyCB in the sequence table. The invention adopts coli expression system, constructing recombinant plasmids containing lyase heavy chain gene PlyCA and light chain PlyCB respectively, transforming to BL21(DE3) cell, fermenting culture engineering bacteria, making PlyCA and PlyCB highly express. Streptococcus phage lyase PlyC is obtained by purifying and renaturing expression production. Compared with the conventional method, the method of the invention has low production cost, high product renaturation yield, strength product activity, safety production method, meeting mass production.

Description

technical field [0001] The invention relates to a production method of streptococcus specific phage lyase. Background technique [0002] Bacteria of the genus Streptococcus are Gram-positive bacteria widely distributed in nature, including a wide variety of pathogenic bacteria and commensal bacteria that inhabit various hosts, including humans, horses, pigs, and cattle. In the host, streptococci usually colonize the mucosal surfaces of the mouth, nostrils, and pharynx. However, in some cases, they can also inhabit the skin, heart or muscle tissue. Streptococcus can be divided into several subgroups: according to their cell wall antigenicity, they are divided into 18 families such as A, B, and C; according to their ability to dissolve red blood cells, they are divided into type A (incomplete hemolysis), type B (complete hemolysis ) and type C (non-hemolytic). Human pathogenic streptococci include Streptococcus pyogenes (S.Pyogenes), Streptococcus pneumoniae (S.Pneumoniae) ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N15/70C12N9/88
Inventor 陈蔚青张德勇陆胤陈虹柯薇张建芬胡文浪朱成钢
Owner ZHEJIANG SHUREN UNIV
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