Meretrix lysozyme gene and coding protein and application thereof

A technology of encoding protein and lysozyme, which is applied in the field of molecular biology, can solve the problems of obtaining lysozyme gene, etc., and achieve the effect of high antibacterial activity

Inactive Publication Date: 2010-12-22
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Meretrix lysozyme gene and coding protein and application thereof
  • Meretrix lysozyme gene and coding protein and application thereof
  • Meretrix lysozyme gene and coding protein and application thereof

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Embodiment 1

[0015] A cloned clam lysozyme gene has the sequence shown in SEQ ID NO.1.

[0016] The cDNA sequence cloning of clam lysozyme among the present invention comprises the following steps:

[0017] a) extraction of clam total RNA and purification of mRNA;

[0018] b) Clam cDNA library construction;

[0019] c) Large-scale determination of the EST sequence of the clam cDNA library;

[0020] d) Homology analysis of clam EST sequence and screening of lysozyme gene fragment;

[0021] e) The full sequence of clam lysozyme obtained by RACE amplification and the verification of the full sequence.

[0022] The specific operation is as follows:

[0023] 1. Extraction of clam total RNA and purification of mRNA: Total RNA was extracted from clam larvae using Trizol reagent from Invitrogen Company, and mRNA was purified using Oligotex mRNA purification kit from QIAGENE Company.

[0024] 2. Clam cDNA library construction: using Stratagene company cDNA Synthesis Kit and The Synthesis Kit...

Embodiment 2

[0068] Embodiment 2. Obtaining of clam lysozyme recombinant protein

[0069] The specific operation is as follows:

[0070] According to the cDNA sequence corresponding to SEQ ID NO.2, specific primers LYBamH I-F and LYSa1 I-R containing restriction endonuclease BamH I and SalI restriction sites were designed to amplify the gene fragment encoding the mature peptide of lysozyme by PCR , and then clone it into the pGEX-4T-1 expression vector by enzyme digestion, transform Escherichia coli BL21, after sequencing to confirm that the expression frame is correct, inoculate the positive clone into LB medium containing ampicillin (100mg / ml), 37°C Shake culture to 0.D. 600 = 0.4-0.6, add IPTG to a final concentration of 1 mM and induce for 4 hours to collect the cells by centrifugation. The bacteria were treated with ultrasonic wave 200W for 30-60 minutes under ice bath conditions (1 second each time, 1 second interval). Centrifuge to remove the supernatant, wash the precipitate (co...

Embodiment 3

[0078] Embodiment 3. In vitro bacteriostatic experiment of clam lysozyme recombinant protein

[0079] The bacteriolytic activity of the recombinant protein was detected by detecting the size of the inhibition zone against Micrococcus luteus (Gram-positive bacteria) and Pseudomonas aeruginosa (Gram-negative bacteria).

[0080] The specific operation is as follows:

[0081] The classic Oxford cup antibacterial test procedure was adopted. Add Micrococcus luteus and Pseudomonas aeruginosa to the undissolved solid medium, mix well, and then pour it onto the plate. The concentration of bacteria is controlled at 10 5 -10 6 CFU / μl medium. After the medium solidified, put 6 Oxford cups on the surface of the plate. The solution added to the Oxford cup is as follows:

[0082] Oxford cup No. 1: 100 μl lysozyme (commercial egg white lysozyme) with a concentration of 0.3 μg / μl

[0083] Oxford cup No. 2: 100 μl of lysozyme (commercial egg white lysozyme) with a concentration of 0.3 μg / ...

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Abstract

The invention relates to molecular biology, in particular to a meretrix lysozyme gene and a coding protein and application thereof. The meretrix lysozyme gene is whole cDNA series 552bp of the meretrix lysozyme which is amplified in the meretrix by using a cDNA library and an RACE technique, wherein a whole reading frame is 441bp; a 5' non-coding region is 15bp; a 3' non-coding region is 96bp; the whole coding protein contains 146 amino acids; 1 to 15 of a coding sequence is a signal peptide sequence; and a mature peptide contains 131 amino acids including 14 cysteine. An expression primer is designed according to the obtained sequence, and after the overall length of the expression primer is amplified, the expression primer is subjected to enzyme cutting to connect into an expression vector pGEX-4T-1; and then a recombinant plasmid is converted into Escherichia coli BL21, and subjected to IPTG inducing, GSTrap FF affinity column purifying and thrombin enzyme cutting GST to form recombinant lysozyme. The lysozyme has wide medicinal and industrial value and wide application prospect, and can be used as a bactericide, an immune potentiator of shellfish growth, an additive of medicaments, feeds, foods and the like, and the like.

Description

technical field [0001] The invention relates to molecular biology, in particular to a clam lysozyme gene, its encoded protein and its application. Background technique [0002] Lysozyme can catalyze the hydrolysis of the β-1,4-glucosidic bond between N-acetylglucan and N-acetylmuramic acid in the peptidoglycan of bacterial cell walls, thereby leading to the lysis of cells and achieving the purpose of sterilization. The found lysozymes are divided into different types. In animals, there are C type (chicken egg white lysozyme), G type (goose egg white lysozyme) and type I (invertebrate type), which are widely distributed. Lysozyme plays a role in the immune defense system of organisms, especially for shellfish lacking acquired immunity, it plays a pivotal role in resisting the invasion of foreign bacteria. In addition, there are also reports that lysozyme can be used as a digestive enzyme for shellfish, and shellfish can obtain the nutrients they need by decomposing filter-fe...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/36A01N63/02A01P1/00A61K38/47
CPCY02A50/30
Inventor 刘保忠岳欣郇聘王鸿霞
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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