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Preparation and application of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) with sterilizing activity

A technique for identifying proteins, Chlamys farreri, applied in the field of molecular biology

Inactive Publication Date: 2010-01-06
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently, the structure and function of peptidoglycan recognition proteins have been studied to some extent in insects and mammals, but little research has been done in aquaculture animals

Method used

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  • Preparation and application of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) with sterilizing activity
  • Preparation and application of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) with sterilizing activity
  • Preparation and application of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) with sterilizing activity

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Experimental program
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Effect test

experiment example 1

[0020] The in vitro prokaryotic recombinant expression of the Chlamys farreri peptidoglycan recognition protein Cf-PGRP gene coding region comprises the following steps:

[0021] 1. Construction of recombinant vector

[0022] The recombinant vector used in the present invention is the pET prokaryotic expression system of Novagen Company.

[0023] By PCR technique, gene-specific primers P1 (5'-GGATCCATTACAGCTGATTACCTGATAAC-3') and P2 (5'-GCGGCCGCTTAAGGGCATCCCGGAC-3') with specific restriction sites of BamH I and Not I added at the 5' end were used to amplify the comb A fragment of the coding region of the peptidoglycan recognition protein Cf-PGRP of the scallop scallop. The reaction conditions were as follows: 94°C pre-denaturation for 5 minutes, and then the following cycles: 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, a total of 30 cycles, and finally 72°C extension for 10 minutes. The amplified fragment was purified and r...

Embodiment 2

[0031] Low concentration of Cf-PGRP protein can inhibit the reproduction of Gram-positive bacteria, while high concentration can kill it. This protein also has a certain antibacterial effect on some Gram-negative bacteria. It has potential applications in the development of broad-spectrum antibacterial drugs, immune enhancers and feed additives.

[0032] Growth inhibition experiment on Micrococcus luteus, Bacillus subtilis and Escherichia coli: Take 36 sterile test tubes and arrange them in three rows, add 1mL of SOB liquid medium and 10μL of logarithmic growth phase bacterial suspension to each tube, and then add to the test tube Add recombinant protein and Tris-HCl control solution with a final concentration of 50 μg / mL and 80 μg / mL, incubate at 220 rpm at 37°C, and observe OD at 0h, 3h, 6h, 9h, and 22h 600 Three repetitions were set up for each sample, and the average value was plotted according to the three measured results. Experiments have shown that low concentration ...

Embodiment 3

[0033] Implementation Example 3: Determination of the minimum inhibitory concentrations of Micrococcus luteus, Bacillus subtilis and Escherichia coli.

[0034] Bacteria were cultured at 37°C to OD600=0.5, diluted 1×10 4 Incubate with the recombinant protein and Tris-HCl control solution with a final concentration range of 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL for 30 min, Pour into the SOB plate for culture, and after the plate is cultured at room temperature for 18 hours, the minimum protein concentration that does not allow any visible colony growth is the minimum inhibitory concentration (MIC). Three replicates are set for each sample. Results: The minimum inhibitory concentrations of the recombinant protein against Micrococcus luteus, Bacillus subtilis and Escherichia coli were 2 μg / mL, 2 μg / mL and 4 μg / mL, respectively.

Description of drawings:

[0035] The figure shows the inhibitory effect of recombinant peptidoglycan recognition pr...

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Abstract

The invention relates to an invitro recombinant expression technology and functional identification of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) gene. Cf-PGRP is provided with an amino acid sequence in a SEQ ID NO.1. The Cf-PGRP is obtained by the invitro recombinant expression technology, and the recombinant protein has broad sterilizing effect, obvious killing effect on Gram positive bacteria and the certain antibacterial effect on Gram negative bacteria and potential application value in aspects of developing broad antibacterial medicines, immune reinforcing agent, feed additive, and the like.

Description

Technical field: [0001] The invention belongs to the technical field of molecular biology, and relates to the in vitro recombinant expression technology of the gene coding region of the scallop peptidoglycan recognition protein (Cf-PGRP), the separation and purification of the active recombinant protein, and the effect of the recombinant protein on Bacillus subtilis, Microcyst garcinia Research on the bactericidal and antibacterial effects of coccus and Escherichia coli; it also involves the application value of the recombinant protein as a new type of antibacterial drug, immune enhancer, and feed additive production. Background technique: [0002] Non-recognition is the initial step of innate immunity and a fundamental problem in immunology research. Invertebrates lack acquired immunity, therefore, the non-recognition of innate immunity is of crucial significance to invertebrates. At the same time, due to the wide variety of microorganisms and frequent mutations, the compl...

Claims

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Application Information

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IPC IPC(8): C07K14/705C12N15/70C12P21/02A61K38/17A61P31/04A61P37/04A23L1/305A23K1/16C12R1/19
Inventor 宋林生王婉赵建民邱丽梅王玲玲苏建国姚雪梅盖云超
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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