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Expression method of lactococcus lactis of porcine streptococcus phage catenase

A technology of Lactococcus lactis and Streptococcus suis, applied in the field of biology

Inactive Publication Date: 2011-09-14
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the lyase can be expressed in large quantities by the E. coli expression system, the expression product needs to be further purified before it can be applied to animals.

Method used

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  • Expression method of lactococcus lactis of porcine streptococcus phage catenase
  • Expression method of lactococcus lactis of porcine streptococcus phage catenase
  • Expression method of lactococcus lactis of porcine streptococcus phage catenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1. Purification of phage, preparation of DNA and expression of lyase:

[0057] Step 1, take the phage SMP that grows into densely connected plaques on the plate, add 5mL SM (phage dilution, every liter contains 5.8g NaCl, 2g MgSO 4 .7H 2 O, 50mL 1M Tris-Cl, pH 7.5, 5mL 2% gelatin), shake on a shaker at 4°C for 3 hours, collect the SM solution and centrifuge at 4000g in a sterile centrifuge tube for 10 minutes to remove cell debris. Add pancreatic DNaseI and RNaseI to the supernatant to a final concentration of 1 μg / mL, and incubate at 37°C for 30 minutes. Add NaCl to a final concentration of 1 mol / L, and after 1 hour of ice-bathing, centrifuge at 11,000 g at 4°C for 10 minutes to collect the supernatant. Add PEG 8000 to the supernatant to a final concentration of 10% and stir at room temperature to dissolve, then ice-bath for 1 hour to precipitate the phage particles. Centrifuge at 12000g at 4°C for 10 minutes, discard the supernatant, and resuspend the precipitated ...

Embodiment 2

[0065] Western-blot immunotransfer: SDS-PAGE gel, NC membrane (nitrocellulose membrane) and filter paper obtained by SDS electrophoresis in the same manner as in Example 1 were equilibrated in transfer buffer for 30 min. Stack in order from anode to cathode: filter paper, PVDF membrane, gel, filter paper, turn on the power, and transfer the membrane with V.

[0066] After the membrane transfer, the membrane was washed with TTBS for 5 min×3 times to remove Tris and glycine. Put the membrane into a heat-sealable plastic bag, add blocking solution 0.1mi / cm according to the membrane area 2 , blocked overnight at 4°C. Wash the membrane with TTBS for 10 min×3 times; add mouse LySMP polyclonal antibody diluted with blocking solution, incubate at 37°C for 1 hour; wash the membrane with TTBS for 10 min×3 times, add goat anti-mouse secondary antibody working solution (1:100), and incubate at 37°C Shake and incubate for 2h; wash the membrane with TTBS for 10min×3. Add TMB chromogenic so...

Embodiment 3

[0068] Resuspend SS2- H, adjusted to OD 600 is 1.0. The prepared bacterial suspension was added to a 96-well plate, 100 μL per well, 6 wells per gradient. Add 100 μL of lys crude extract to each well of bacterial suspension, and do 3 replicates for each gradient. In the control group, 100 μL of the crude extract of the L9000 strain containing the empty plasmid pNZ8148 induced by isoconditions was added to each well, and each gradient was replicated three times. After shaking and reacting at 37°C for 30 minutes, determine the pH condition that makes the bacterial turbidity decrease the most. The optimal reaction buffer was determined to be 20mM pH 7.2 PBS buffer.

[0069] Resuspend the SS2-H grown to the logarithmic growth phase with 0.01M PBS buffer pH7.2, adjust to OD 600 is 1.0. The prepared bacterial suspension was added to a 96-well plate, 100 μL per well, and each bacterial strain was added to 6 wells. Add 100 μL of lys crude extract to each well of bacterial suspe...

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Abstract

The invention discloses an expression method of lactococcus lactis of porcine streptococcus phage catenase in the technical field of biology. A pyrolyzed gene of porcine streptococcus virulent phage SMP is augmented by utilizing polymerase chain reaction (PCR) to obtain a target fragment which is connected with a pNZ8148 vector so as to obtain a recombined expression plasmid; lactococcus lactis L9000 expression is adopted so s to obtain a fusion protein the molecular weight of which is 52kDa, so as to obtain activated catenase which has a pyrolyzed actioin on multiple clinically separated porcine streptococcus in vitro.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to a method for expressing a Streptococcus suis phage lyase in Lactococcus lactis. Background technique [0002] Streptococcus suis is a zoonotic infection that can cause meningitis, septicemia, arthritis, endocarditis, pneumonia in piglets and meningitis in humans. Streptococcus suis type 2 is the most prevalent and the most pathogenic to pigs, causing huge economic losses to the pig industry and posing a serious threat to the lives of relevant practitioners in terms of public health. The main treatment for S. suis is antibiotic therapy. However, with the widespread use of antibacterial drugs, the spread of bacterial drug-resistant strains has been further exacerbated. The lyase is an enzyme encoded by the phage genome that can hydrolyze the bacterial cell wall. Phage lytic enzymes, like bacteriophages, only attack bacteria, not animal cells, and have high specificity so ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/74C12N15/66
Inventor 孙建和方圆子严亚贤陆承平王琰
Owner SHANGHAI JIAO TONG UNIV
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