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Recombination expression and application of Chinese prawn antibacterial peptide gene

A technology of Chinese prawns and antimicrobial peptides, applied in the field of molecular biology, can solve problems such as similar activities

Inactive Publication Date: 2003-12-03
SHANDONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Destromux cloned the gene encoding the mature peptide of astaxanthin into the PCRscript SK(+) vector, and transformed it into Saccharomyces cerevisiae for expression and analysis of antibacterial activity, and found that the antibacterial peptide synthesized by genetic engineering was similar to the natural antibacterial Peptides are different in structure but similar in activity

Method used

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  • Recombination expression and application of Chinese prawn antibacterial peptide gene
  • Recombination expression and application of Chinese prawn antibacterial peptide gene

Examples

Experimental program
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Effect test

Embodiment 2

[0031] The GST-CHP fusion protein was cleaved at 23° C. for 8 h at the ratio of 1 μl thrombin: 100 mg GST-CHP fusion protein. Cultivate E.coli DH5α overnight at 37°C in 5ml LB medium, dilute E.coli DH5α at the ratio of 1 μl overnight culture: 1ml LB medium the next day, and add the diluted E.coli DH5α to each well of a 96-well plate 100 μl of the bacterial solution, and then 100 μl of the lysed GST-CHP fusion protein sample was added. LB, GST-CHP fusion protein, PBS, Glutathione Elution Buffer were used as controls. Three parallel experiments were set up for each group, cultured with shaking at 25°C and 30rpm, and the absorbance was measured every 30min at a wavelength of 450nm. Calculate the average value of the parallel experiments of each group, draw the curve of absorbance and time, see the appendix figure 1 . Example 2. Expression of astaxin by using yeast expression system, pPIC vector series. 1) Vector construction (taking pPIC9K as an example)

[0032] Using the s...

Embodiment 3

[0042] As described in Example 2, the difference is that the selection of vector copies is carried out in vitro, and the purification of expression products requires cell destruction. Embodiment 4. Utilize insect cell expression system to express astaxin

Embodiment 4

[0042] As described in Example 2, the difference is that the selection of vector copies is carried out in vitro, and the purification of expression products requires cell destruction. Embodiment 4. Utilize insect cell expression system to express astaxin

[0043] Using chp / pGEM-T easy as a template and using ChpBac F ChpBac R as primers to amplify the astaxin gene. The primer sequence is: ChpBac F 5'GCGC GGA TCC AAG GGT GGT TAC ACA CGC 3'ChpBac R 5'GCGCCTC GAG ACT TAC ATC CCA CAT GCA C3', and Bam H I and Xho I restriction sites are added to both ends of the primer.

[0044] The amplified product was recovered and subcloned into the pFastBac plasmid to obtain the pFastBac / Chp recombinant plasmid, transformed into DH10Bac cells, and under the action of the transposase produced by the helper plasmid (Helper), the m-Chp gene fragment was transposed onto the shuttle vector Bacmid , using blue and white spots to screen recombinant Bacmid. After verification, insect cells were tran...

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Abstract

The recombination, expression and application of Chinese prawn's antibacterial peptide gene are disclosed. Its recombination expression method includes configuring recombinant carrier (pET series, pGEX-4T series, pPIC series, pAO815, and baculovirus expression carrier), transferring the carrier to colibacillus and yeast and transfecting insect cells, screening engineered bacterial strain or cell, and inducing expression. The expressed product can be used to prepare feed additive, antistaling agent, cosmetics and medical products.

Description

(1) Technical field [0001] The invention relates to technologies such as recombinant expression and purification in the technical field of biological genetic engineering, in particular to technologies such as gene expression of prawn antibacterial peptide (astaxin), purification of expression products, activity determination and the like, and belongs to the technical field of molecular biology. (2) Background technology [0002] The culture of prawns in our country occupies an important position in the marine aquaculture industry and has huge economic benefits. Since the 1980s, my country's shrimp farming industry has developed rapidly, with the highest annual output (1988) reaching 200,000 tons, ranking first in the world. With the rapid development of the shrimp industry, the basic research on its disease prevention and treatment is relatively weak, coupled with the environmental pollution caused by excessively high breeding density and other factors, leading to the outbre...

Claims

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Application Information

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IPC IPC(8): C07K1/16C07K14/435C12N15/12C12N15/63C12P21/02C12Q1/68
Inventor 王金星赵小凡相建海李富花
Owner SHANDONG UNIV
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