Antifungal gene, polypeptide and recombinant protein as well as preparation method and application thereof

A recombinant protein and antifungal technology, applied in the directions of antifungal agents, peptide/protein components, botanical equipment and methods, etc., can solve the problems of ecological destruction of antibiotics, food and drug residues, diseases, etc., and achieve the effect of broad application prospects.

Active Publication Date: 2020-04-17
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to make up for the deficiencies of the prior art, the present invention provides an antifungal gene, polypeptide, recombinant protein and its preparation method and application, which provide a solution to the current problems of serious aquaculture diseases, ecological damage caused by antibiotic abuse, and food and drug residues. a new approach

Method used

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  • Antifungal gene, polypeptide and recombinant protein as well as preparation method and application thereof
  • Antifungal gene, polypeptide and recombinant protein as well as preparation method and application thereof
  • Antifungal gene, polypeptide and recombinant protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A method for preparing the full-length nucleotide sequence of cDNA of antifungal gene (Clam philippines antifungal Rpmacin gene) includes the following steps:

[0035] (1) cDNA synthesis

[0036] ① Extraction of total RNA from Philippine clams: Trizol reagent method (Invitrogen) was used to extract total RNA from blood cells of Philippine clams, 1.2% agarose gel electrophoresis to determine the integrity of total RNA, and Nanodrop2000 to detect total RNA concentration;

[0037] ②Total RNA purification: The total RNA is treated with RQ1 DNA enzyme (Promega, USA) to remove DNA contamination and complete RNA purification;

[0038] ③Reverse transcription to obtain cDNA: 25 μL reverse transcription reaction system contains 2 μg total RNA, 200 UM-MLV and 0.5 μMoligo dT primers, react at 42 ℃ for 1 h to reverse transcription to synthesize cDNA, and then at 95 ℃ for 5 min. Inactivated.

[0039] (2) Generate the full-length cDNA of the antifungal gene: design primers and construct a gene...

Embodiment 2

[0048] A preparation method of antifungal recombinant protein (Clam philippines antifungal Rpmacin recombinant protein) includes the following steps:

[0049] (1) Obtaining recombinant expression vector

[0050] ①Connect the T vector: design primers at both ends of the sequence fragment encoding the mature peptide in the antifungal Rpmacin gene cDNA sequence of the clam clam Philippine, and add the BamHI restriction site to the upstream primer and the HindШ restriction site to the downstream primer, and then proceed PCR amplification, PCR product 2 is obtained, PCR product 2 is purified and recovered by agarose gel electrophoresis, ligated into PMD19T vector, transformed into E. coli competent trans 5α, and selected a single clone for colony PCR verification and sequencing to confirm the sequence is correct Sex

[0051] ②Construction of recombinant expression vector: The plasmid is extracted from the strain confirmed by sequencing, the target fragment is recovered after double enzym...

Embodiment 3

[0063] The activity test of a fungal recombinant protein (Rpmacin antifungal recombinant protein of clam clam philippines) includes the following aspects:

[0064] (1) Sterilization kinetics

[0065] Yeast is cultivated in the medium until the exponential growth phase, and the sterilization kinetics is tested. After harvesting the yeast by centrifugation, it was washed 3 times with PBS buffer (pH 7.4), resuspended, and incubated with the Rpmacin recombinant protein of 1×MIC concentration at 37°C for 2×10 7 Yeast cells, the control group replaced the Rpmacin recombinant protein with PBS, and tested them at 0, 10, 30, 60, 160, 400, and 1000 min after incubation. Colony forming units (CFU) are used to express the number of viable yeasts, and according to the number of live bacteria The logarithm of the number plots the kinetic curve of sterilization over time. The experiment was repeated 3 times.

[0066] The result is image 3 As shown, compared with the control group, when the yeast...

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Abstract

The invention provides an antifungal gene, polypeptide and recombinant protein as well as a preparation method and an application thereof, the antifungal gene is a Ruditapes philippinarum antifungal Rpmacin gene, and a nucleotide sequence of the Rpmacin gene is shown as SEQ ID NO.1; the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 2; the recombinant protein contains the antifungal gene and expresses the antifungal polypeptide. The antifungal gene, polypeptide and recombinant protein provided by the invention can be applied to production of antibacterial drugs, vaccines or feed additives for vegetables, livestock, poultry and aquatic products, and have broad application prospects. The gene, polypeptide and recombinant protein as well as the preparation method and the application thereof can provide a basis for further researching the immune defense mechanism of Ruditapes philippinarum, and lay a foundation for disease control, genetic breeding and feed additive research of Ruditapes philippinarum.

Description

Technical field [0001] The invention belongs to the technical field of biological genetic engineering, and relates to an antifungal shellfish polypeptide and a preparation method and application thereof, in particular to an antifungal gene, a polypeptide, a recombinant protein, and a preparation method and application thereof. Background technique [0002] Antimicrobial peptides (AMPs) are a class of endogenous small molecule peptide active substances, which are directly involved in killing and removing infectious pathogenic microorganisms. They are important effector molecules of invertebrate innate immunity, because they can be used as traditional antibiotics. Alternatives are getting more and more attention. Because it is an endogenous small molecule peptide, AMPs have a series of advantages such as pollution-free, antigen-free, strong pertinence, heat stability, and broad-spectrum antibacterial. They have important application prospects in the research and development of dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/70C07K14/435A61K38/17A61P31/10A23K20/195A23K20/147A23K50/80A01N47/44A01P3/00
CPCA01N47/44A61K38/00A23K20/147A23K20/195A23K50/80A61P31/10C07K14/43504C12N15/1096C12N15/70C12Q2531/113Y02A40/818
Inventor 杨顶珑薛蕊赵建民
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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