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A kind of American white moth sumo‑abp‑dhc‑Cecropin A fusion protein and application thereof

A technology of sumo-abp-dhc-cecropina, American white moth, applied in the field of genetic engineering, can solve the problems of inability to realize large-scale production, high cost, complicated process, etc., and achieve obvious antifungal activity, low cost and high activity. Effect

Inactive Publication Date: 2017-08-04
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although biological extraction can provide cheap, safe and reliable antimicrobial peptide products for industrial and agricultural production, and the earliest research and discovery of antimicrobial peptides often adopts the method of biological extraction, but because the content of antimicrobial peptides in animals and plants is very small, natural extraction of antimicrobial peptides Often there are many impurities, low extraction rate, time-consuming, and complicated process, so the cost is relatively high, and large-scale production cannot be realized, which has become the biggest obstacle restricting the practical application of antimicrobial peptides
Although the method of chemical synthesis is feasible in production, it is not realistic to apply it to industrialization, especially the problems of high cost, low efficiency of long-chain peptide synthesis and unstable antibacterial activity, which limit its application and are still not as good as passed. Development Potential of Gene Engineering Expression in Vitro

Method used

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  • A kind of American white moth sumo‑abp‑dhc‑Cecropin A fusion protein and application thereof
  • A kind of American white moth sumo‑abp‑dhc‑Cecropin A fusion protein and application thereof
  • A kind of American white moth sumo‑abp‑dhc‑Cecropin A fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cloning of Cecropin A cDNA

[0026] 1. Extract the total RNA of white moth.

[0027] Use the RNA extraction reagent (TIANGEN) to extract the total RNA of the fat body of the white moth pupa according to its operation manual, and identify its purity by formaldehyde-denatured agarose gel electrophoresis, which is greater than 95%, and its concentration is 720ng / µL, to meet the needs of use.

[0028] 2. Use Transgen cDNA reverse transcription kit to transcribe into first-strand cDNA.

[0029] 1) Primer design: According to the sequence alignment of Cecropin A in NCBI, Bombyx mori and Drosophila, degenerate primers were designed:

[0030] Cec-A1: 5'-ATGAATTTCNCAANAATNNNTNTNCTTCGT-3',

[0031] Cec-A2: 5'-NTATTTNCNAANGNTTTTTNCNGTACCCAG-3'.

[0032] 2) Reverse transcription: Add 3 μL of total RNA extract in sequence to a 1.5 mL Eppendorf tube treated with DEPC, Oligod (T) 18 (0.5 μg / μL) 1 μL, 2×TS Reaction Mix 10 μL, DEPC water 5 μL, TransScript RT / RI EnzymeMix...

Embodiment 2

[0034]Example 2 American white moth ABP- dHC -Cecropin A Escherichia coli in vitro expression

[0035] Construct the ABP-Cecropin A sequence of American white moth into the pET-SUMO plasmid region, such as figure 1 As shown, obtain pET-SUMO-ABP- dHC -Cecropin A, the specific construction method is as follows:

[0036] 1) According to the ABP obtained in Example 1- dHC - Cecropin A sequence design primers A1, A2,

[0037] A1 sequence: 5'-CAGGTGGAAGATCTTTAAGAAAATCG-3',

[0038] A2 sequence: 5'-CCCAAGCTTTTATTTTCTTAATGCTTTTGC-3';

[0039] where the 5' end of A1 has Stu1 Restriction site, the 5' end of A2 has HindⅢ Restriction sites.

[0040] Carry out PCR with American white moth DNA as a template, tap rubber to recover the PCR product, and recover the product for Stu1 and HindⅢ Enzyme digestion, SUMO vectors only use HindⅢ Enzyme digestion, T4 DNA Ligase ligation of the digested vector and PCR product, transformation of the ligation product E. coli In top10, t...

Embodiment 3

[0043] Example 3 American white moth ABP- dHC -Identification of antifungal activity of Cecropin A

[0044] 1) Anti-Candida albicans Canidia albicans activity identification

[0045] Determination of the antimicrobial peptide ABP- dHC -Cecropin A against Candida albicans Canidia Antibacterial activity of albicans. inoculation Canidia Albicans were cultured in liquid potato medium at 25°C and 200rpm for 2 hours, then added 1% of the bacteria to the melted potato agarose medium (35-50°C), mixed evenly and spread on sterile disposable plates at 10mL / dish . After solidification, punch a hole under sterile conditions, add the sample to be tested and place it in a 25°C incubator for 48 hours to observe the antibacterial activity. The results are shown in Image 6 A, has obvious antifungal activity.

[0046] 2) Resistance to Neurospora crassa Neurospora crassa activity identification

[0047] Determination of the antimicrobial peptide ABP- dHC -Cecropin A agains...

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Abstract

The invention discloses fall webworm SUMO-ABP-dHC-Cecropin A fusion protein and an application thereof. An expression method of the fall webworm SUMO-ABP-dHC-Cecropin A fusion protein comprises the following steps: establishing a pETSUMO-ABP-dHC-Cecropin A expression carrier, expressing in escherichia coli, performing ultrasonic crushing, collecting supernate Ni<+>, performing affinity purification, and performing gradient elution, thereby obtaining the fall webworm SUMO-ABP-dHC-Cecropin A fusion protein. The fall webworm SUMO-ABP-dHC-Cecropin A fusion protein is obtained by using a genetic recombinant method and can be efficiently expressed in escherichia coli, after HPLC analysis, target polypeptide can achieve the purity greater than 95%, and through mass spectrometry, the molecular weight fits for prediction polypeptide molecular weight. The detection shows that the polypeptide has remarkable antifungal activity, and the prepared polypeptide is high in activity and low in cost.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an American white moth SUMO-ABP-dHC-CecropinA fusion protein and application thereof. Background technique [0002] With the extensive application of antibiotics, the emergence of drug resistance in pathogenic bacteria and the screening bottleneck of new antibiotics restrict the application of antibiotics to a certain extent. Recent studies have found that antimicrobial peptides (AMPs) have become ideal drugs that are expected to replace traditional antibiotics due to their unique mechanism of action and molecular characteristics. [0003] To apply antimicrobial peptides to production practice, there are still many problems to be solved, the most important of which is how to obtain large quantities of antimicrobial peptides. At this stage, antimicrobial peptides are mainly obtained through the following three ways: (1) natural extraction; (2) chemical synt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70A61P31/10
Inventor 诸葛强张嘉鑫武小龙桑明王晓立刘铃宋雪晴
Owner NANJING FORESTRY UNIV
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