Procambrus clarkii type i lysozyme gene, lysozyme coded by lysozyme gene and application of lysozyme

A technology of Procambarus clarkii and lysozyme, which is applied in the field of genetic engineering, can solve problems such as the absence of Procambarus clarkii lysozyme gene

Inactive Publication Date: 2011-01-12
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Many studies have shown that in arthropods such as shrimp, there are various proteins or peptides that can resist external pathogenic mi

Method used

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  • Procambrus clarkii type i lysozyme gene, lysozyme coded by lysozyme gene and application of lysozyme
  • Procambrus clarkii type i lysozyme gene, lysozyme coded by lysozyme gene and application of lysozyme
  • Procambrus clarkii type i lysozyme gene, lysozyme coded by lysozyme gene and application of lysozyme

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Experimental program
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Effect test

Embodiment 1

[0077] Embodiment 1: the cloning of cDNA of lysozyme gene cDNA of Crayfish clarkii type I

[0078] 1) Extraction of total RNA: Total RNA was extracted by one-step method using the prior art.

[0079] 2) cDNA first-strand synthesis: 4 microliters of total RNA, plus 1 microliter of SmartF (5'-TAC GGC TGC GAG AAG ACG ACAGAA GGG-3') and 1 microliter of Oligoanchor R (5'-GAC CAC GCG TAT CGA TGT CGA CT 16 (A / C / G)-3'), react at 72°C for 5 minutes, then add 4 microliters of 5-fold Buffer, 1.25 microliters of dNTP, 0.625 microliters of RNase inhibitor, 1 microliter of MMLV reverse transcriptase, RNase-free 12.875 microliters of sterilized water were reacted at 42°C for 60 minutes, and the reaction was terminated at 70°C for 10 minutes.

[0080] 3) PCR reaction: chain polymerase reaction (PCR) reagents and conditions:

[0081] First mix the following reagents together:

[0082] 5 microliters (μl) of 10xTaq DNA polymerase buffer

[0083] ·Template cDNA 1μl

[0084] ·Forward primer ...

Embodiment 2

[0104] Example 2: Construction, expression and purification of prokaryotic recombinant expression vectors

[0105] (1) According to the sequence of Procambarus clarkii lysozyme (remove the signal peptide sequence) and the cloning site of the expression vector pPET30a (Novagen Company), the present invention has selected the BamH I and Xho I restriction sites of the pET30a cloning site, Therefore, when designing the primers, a BamH I restriction site was introduced into the upstream primer, and an Xho I restriction site was introduced into the downstream primer.

[0106] (2) Gene amplification, cloning and recombinant plasmid screening

[0107] Using pMD-18T-Lysi as a template, the PCR reaction was carried out with the designed expression primers. The amplification conditions were: 94°C, 2min pre-denaturation; 94°C, 30s, 56°C, 45s, 72°C, 45s, 35 cycles; 72 ℃ extension 10min.

[0108] 2% agarose gel electrophoresis PCR product detection:

[0109] The PCR product was subjected...

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PUM

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Abstract

The invention discloses a procambrus clarkii type i lysozyme gene of which nucleotide sequence is shown in the SEQ ID NO. 1 and provides a recombinant expression and purification method thereof. The invention also provides a lysozyme polypeptide coded by the procambrus clarkii type i lysozyme gene and applications of the gene sequence and the lysozyme, wherein the amino acid sequence of the lysozyme polypeptide is shown in SEQ ID NO. 2.; and the lysozyme of the invention has inhibitory activity to both Gram-positive bacteria and Gram-negative bacteria. The lysozyme gene of the invention can be transferred in crops through the gene engineering method to obtain crops with antibacterial ability. The recombinant lysozyme obtained in the invention can be used in antibacterial feed additives and the fields of food preservation, gene transformation of animals and plants and drug development.

Description

technical field [0001] The present invention relates to a kind of i-type lysozyme gene and its coded protein and its application, especially to a kind of Procambarus clarkii i-type lysozyme (i-type Lysozyme) gene and its coded lysozyme and said gene in preparation The application of the recombinant protein with antibacterial activity belongs to the technical field of genetic engineering. Background technique [0002] Because invertebrates do not have adaptive immunity, they rely on innate immune defense responses, which include cellular and humoral immunity, to clear or eliminate pathogens. The main effector molecules of humoral immunity include antimicrobial peptides, phenol oxidase-dependent melanization system and apoptosis-related gene system, etc.; among them, antimicrobial peptides are polypeptides produced by organisms in the defense response against pathogenic microorganisms, which can inhibit or It is an important humoral immune effector molecule in invertebrates t...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/36A01H5/00
Inventor 王金星赵小凡
Owner SHANDONG UNIV
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