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Method for using silkworm cultured cell to express antibacterial peptide Cecropin B

A technology for culturing cells and antibacterial peptides of silkworm, which is applied in the fields of DNA recombination technology, protein purification and activity analysis, can solve the problems of less research and achieve high biological activity and is beneficial to extraction.

Inactive Publication Date: 2010-09-29
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Boman, H.G. and Faye, I and others first started the research on Cecropin B gene in 1985, but there are few domestic researches on this aspect

Method used

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  • Method for using silkworm cultured cell to express antibacterial peptide Cecropin B
  • Method for using silkworm cultured cell to express antibacterial peptide Cecropin B
  • Method for using silkworm cultured cell to express antibacterial peptide Cecropin B

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Embodiment Construction

[0017] 1. Materials: DNA extraction kit was purchased from Qiagene. DNA processing and PCR amplification kits were purchased from Takara Biomedicals (Kyoto, Japan). TA cloning kit, baculovirus transfer vector pBlueBacHisa, lipofectin and Ni-NTA resin are all products of Invitrogen Company of the United States. DNA sequencing kits were purchased from PE Applied Biosystems. Fetal calf serum FCS and silkworm culture medium TC-100 are all products of GibcoBRL.

[0018] 2. Workflow:

[0019] 2.1 The recombinant antimicrobial peptide Cecropin B expressed by silkworm cultured cells of the present invention includes the following parts:

[0020] (1) Cloning of the Cecropin B gene of the silkworm:

[0021] Using the genomic DNA extracted from the fat body cells of the silkworm chrysalis as a template, the antimicrobial peptide Cecropin B gene was amplified by PCR technology. The specific method of this PCR amplification is as follows:

[0022] Among them, the primers used for PCR...

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Abstract

The invention discloses a method for using silkworm cultured cell to express antibacterial peptide Cecropin B. The method comprises the following steps: (1) using the total RNA extracted from the fat body cells of wild silkworm chrysalis as template, adopting RT-PCR amplification to obtain wild silkworm antibacterial peptide Cecropin B gene; using 1% agarose gel electrophoresis to perform PCR product analysis to the antibacterial peptide Cecropin B gene, using a PCR purification kit of Qiagene for purification, then cloning the purified PCR product to TA vector pCR2.1 to obtain pCR2.1-Cecropin B, utilizing the dideoxynucleotide chain termination to confirm the correctness of cloned gene order; using restriction endonuclease BamHI and HindIII to digest pCR2.1-Cecropin B and obtain Cecropin B genetic fragments, then cloning rhabdovirus transfer vector pBlueBacHisa in the genetic fragments to obtain reconstituted transfer vector; performing cotransfection of the reconstituted transfer vector and silkworm wild-type nuclear polyhedrosis virus BmNPV DNA in silkworm cultured cell, performing homologous recombination to generate recombinant virus; and (3) inoculating the recombinant virus containing wild silkworm antibacterial peptide Cecropin B gene in the silkworm cultured cell, infecting at 27 DEG C for three days to express the infection, and centrifuging to collect silkworm cultured cell.

Description

technical field [0001] The invention relates to DNA recombination technology, protein purification and activity analysis, in particular to a method for expressing recombinant cecropin antibacterial peptide Cecropin B in silkworm cultured cells. Background technique [0002] Cecropin B is a kind of protein with broad spectrum and strong antibacterial activity produced by silkworm cells. The gene encoding the antibacterial peptide is 105 nucleotides, encoding 35 amino acids, and the molecular weight of the protein is about 4kDa. Boman, H.G. and Faye, I and others first started the research on the Cecropin B gene in 1985, but there are few domestic researches on this aspect. Contents of the invention [0003] The object of the present invention is to provide a method for expressing the antimicrobial peptide Cecropin B in silkworm cultured cells, using genetic engineering expression technology to construct a recombinant virus containing the antibacterial peptide Cecropin B gen...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/866C12N7/01C07K14/435
Inventor 陈琳郭爱芹胡小龙吴小锋
Owner ZHEJIANG UNIV
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