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Product expressed by housefly embryonic cell construction antibacterial peptide attacin eukaryotic expression system and construction method

A technology of embryonic cells and expression products, applied in the field of biogenetic engineering, can solve problems such as undiscovered cell lines

Inactive Publication Date: 2016-01-27
ZUNYI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, some new cell lines have been established from Lepidoptera insects, but no cell lines with excellent performance in protein expression have been found

Method used

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  • Product expressed by housefly embryonic cell construction antibacterial peptide attacin eukaryotic expression system and construction method
  • Product expressed by housefly embryonic cell construction antibacterial peptide attacin eukaryotic expression system and construction method
  • Product expressed by housefly embryonic cell construction antibacterial peptide attacin eukaryotic expression system and construction method

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Experimental program
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Effect test

Embodiment Construction

[0093] Preparation steps and methods:

[0094] 1.1) Cells and Plasmids

[0095] The housefly embryonic cell line (MDEC-07114) and the recombinant baculovirus Bacmid-pFastBacTM-attacin were established and preserved by the parasite teaching and research department;

[0096] 1.2) Main experimental reagents

[0097] Jinpu Nuoan Insect Cell Culture Medium (GPM-115) (Jinpu Nuoan Protein Engineering Technology Co., Ltd.); Cellfectin Transfection Reagent (Invitrogen, USA); One-step Insect Cell Active Protein Extraction Kit (Shanghai Shenggong), Ni-NTASefinoseTM ResinKit (Shanghai Sangong), UNIQ-200 Column Plasmid Medium Extraction Kit (Shanghai Sangong); Fetal Bovine Serum (FBS) (product of Hangzhou Sijiqing Institute of Bioengineering); Protein Marker (purchased from Dalian) TaKaRa Company); BCA protein quantification kit, Coomassie brilliant blue R-250, goat anti-mouse HRP-labeled secondary antibody, goat anti-rabbit HRP-labeled secondary antibody (Biyuntian Institute of Biotec...

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Abstract

The present invention relates to a product expressed by a housefly embryonic cell construction antibacterial peptide attacin eukaryotic expression system and a construction method, housefly antibacterial peptide attacin is used as a target gene, constructed housefly embryonic cell line MDEC-07114 is transfected by recombinant baculovirus Bacmid-pFastBacTM-attacin, and an antibacterial peptide attacin housefly embryonic cell eukaryotic expression system is constructed. The housefly antimicrobial peptide attacin is obtained by complete gene synthesis, the target gene attacin is cloned into PUC57plasmid, after double enzyme digestion, recombinant plasmid is connected with pFastBacTMHTA donor plasmid for recombination of baculovirus Bacmid DNA; the recombinant baculovirus Bacmid DNA is transfected into housefly embryonic cell MDEC-07114, culture supernatant and cell lysates crude protein are extracted, and purified by a nickel column, SDS-PAGE and Western-Blot are used for detection and identification of protein expression, and the antimicrobial activity of the expressed product can be detected.

Description

Technical field [0001] The present invention relates to biogenetic engineering technology and provides candidate insect cell lines for baculovirus expression vector systems, and particularly relates to products and construction methods expressed by housefly embryonic cells constructing a eukaryotic expression system for the antimicrobial peptide attacin. Background technique [0002] As the most common insect, houseflies live in dirty environments for a long time without being infected. They are one of the best materials for studying insect antimicrobial peptides. At present, the natural antibacterial peptide attacin isolated from house flies has broad-spectrum antibacterial effects and strong antibacterial activity. Since insect antimicrobial peptides are difficult to extract and purify, chemical synthesis is expensive, and difficult to standardize, their inability to be produced on a large scale is the biggest obstacle restricting their practical application. Therefore,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C07K14/435A61P31/04
Inventor 刘晖贺莉芳王灵军王鑫
Owner ZUNYI MEDICAL UNIVERSITY
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