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Procambarus clarkia chitin peptide gene and encoded chitin peptide and application thereof

A technology of protocrayfish and crustacean, which is applied in the field of genetic engineering and can solve problems such as no reports of protocrayfish crustacean gene and the like.

Inactive Publication Date: 2010-11-17
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 a variety of proteins or peptides that can resist external pathogenic microorganisms, such as astaxin, crustins, etc., but in related reports, there is no Procambarus clarkii report of the chitin gene

Method used

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  • Procambarus clarkia chitin peptide gene and encoded chitin peptide and application thereof
  • Procambarus clarkia chitin peptide gene and encoded chitin peptide and application thereof
  • Procambarus clarkia chitin peptide gene and encoded chitin peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Cloning of Crayfish carapace peptide cDNA

[0069] 1) Extraction of total RNA: Total RNA was extracted by one-step method using the prior art.

[0070] 2) cDNA first-strand synthesis: 4 microliters of total RNA, plus 1 microliter of SmartF (5'-TAC GGC TGC GAGAAGACGACA GAA 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.

[0071] 3) PCR reaction: chain polymerase reaction (PCR) reagents and conditions:

[0072] First mix the following reagents together:

[0073] 5 microliters (μl) of 10xTaq DNA polymerase buffer

[0074] ●Template cDNA 1μl

[0075] ●Forward primer (10mM) 1μl

[0076] ●Reverse pr...

Embodiment 2

[0096] Example 2: Construction, expression and purification of prokaryotic recombinant expression vectors

[0097] (1) According to the sequence of Procambarus clarkii crustacean peptide (remove signal peptide sequence) and the cloning site of expression vector pPET30a (Novagen Company), design primers:

[0098] Cru-pET F:5′TAC TCA GAA TTC CAC CTT AAA CGA CCC AAG CC 3′(EcoRI)

[0099] Cru-pET R:5′TAC TCA CTC GAG CTA ATT GAT GTA AAA GTG TGC 3′(XhoI)

[0100] The present invention selects the EcoR I and XhoI restriction sites of the pET30a cloning site. Therefore, when designing primers, an EcoR I restriction site is introduced into the upstream primer, and an Xho I restriction site is introduced into the downstream primer.

[0101] (2) Gene amplification, cloning and recombinant plasmid screening

[0102] Using pMD-18T-Lec as template, carry out PCR reaction with the above primers, the amplification conditions are: 94°C, 2min pre-denaturation; 94°C, 30s, 56°C, 45s, 72°C, ...

Embodiment 3

[0113] Example 3: Recombinant expression and antibacterial activity determination of chitin in Pichia pastoris

[0114] (1) Construction of pPIC9K (Invetrogen Company) recombinant expression vector

[0115] The present invention selects the EcoR I and Not I restriction sites of the pPIC9K cloning site. Therefore, when designing the primers, the EcoR I restriction site is introduced into the upstream primer, and the Not I restriction site is introduced into the downstream primer:

[0116] Cru-pPIC F:5′TAC TCA GAA TTC CAC CTT AAA CGA CCC AAG CC 3′(EcoRI)

[0117] Cru-pPIC R:5′TAC TCA GCGGCCGC CTAs ATG ATG ATG ATG ATG ATG ATG ATG ATG ATT GAT GTA AAA GTG TGC 3′ (NotI)

[0118] Using cDNA of blood cells of Procambarus clarkii as template, the sequence of mature peptide of crustacean was amplified by conventional PCR method, and the PCR amplification product was purified. Then the PCR amplified fragments of pPIC9K and Crustin were digested with EcoR I and Not I. The diges...

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Abstract

The invention discloses a gene sequence of encoding an antibacterial peptide-chitin peptide cloned from procambarus clarkia, and provides a recombinant expression and purification method thereof. The invention further discloses an amino acid sequence of the antibacterial peptide-chitin peptide cloned from the procambarus clarkia. The invention further provides the uses of the gene sequence and the chitin peptide. In the invention, a yeast expression vector can be prepared by virtue of the procambarus clarkia chitin peptide gene of the invention; an engineering strain can be obtained by transfecting Pichia stipitis; and recombinant protein with antibacterial activity can be obtained by induction expression. The recombinant chitin peptide obtained by the invention can be applied to an antibacterial feed additive, food preservation, animal and plant gene transformation and drug development.

Description

technical field [0001] The present invention relates to a kind of crust peptide gene and its coded protein and its application, in particular to a kind of Procambarus clarkii crust peptide (Crustin) gene and its coded crust peptide and the gene in the preparation of recombinant protein with antibacterial activity. The invention belongs to the technical field of genetic engineering. Background technique [0002] It is well known that although invertebrates do not have adaptive immunity, they have strong innate immune defense responses, including cellular and humoral immunity, and rely on these defense responses 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; among them, antimicrobial peptides are peptides or proteins produced in various organisms that can inhibit or kill pathogenic microorganisms. Important humoral immune effector ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63C07K14/435A01H5/00
Inventor 王金星赵小凡
Owner SHANDONG UNIV
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