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Engineering bacteria expressing active peptides and method of preparing mixed polypeptide

A technology of engineering bacteria and active peptides, applied in the field of bioengineering, can solve the problems of simultaneous expression that have not been reported, and achieve the effect of increasing the ratio

Inactive Publication Date: 2011-08-31
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on the expression of milk-derived hypotensive peptides by engineering bacteria, and there are no reports on the simultaneous expression of two or more antihypertensive peptides by engineering bacteria

Method used

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  • Engineering bacteria expressing active peptides and method of preparing mixed polypeptide
  • Engineering bacteria expressing active peptides and method of preparing mixed polypeptide
  • Engineering bacteria expressing active peptides and method of preparing mixed polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment １

[0019] Example 1: Two kinds of lactating blood pressure reduction peptides CEI-12 and CEI-7 series DNA design and synthesis

[0020] A. Two kinds of milk-derived blood pressure-lowering peptides CEI-12 and CEI-7 series polypeptide sequence design

[0021] The CEI-12 (SEQ ID NO3) and CEI-7 (SEQ ID NO4) involved in the invention involved in the present invention respectively correspond to the residual sequences of cowytan α-casein 23-34 and 177 of cowl β-casein.-183 residue sequence.The N-segment amino acid residues of CEI-12 and CEI-7 are LYS and ARG, respectively, and the enzyme sect sites that are also anotinase.The n segment adds ASP-ASP-ASP-ASP-LYS sequence, and the sequence is shown in SEQ ID NO2.

[0022] B. Two kinds of milk-derived blood pressure peptide CEI-12 and CEI-7 series polypeptide genetic design

[0023] SEQ ID NO2 (SEQ ID NO2) depending on E. coli ( Escherichia color ) BL-21 strain codons prefer sex transformation into nucleotide sequences and add TAATAATAA sequen...

Embodiment 2

[0024] Example 2: The construction of the expression carrier PUC18-CEI-12,7 and the construction of E. coli BL21-MF3A3 engineering bacteria

[0025] A. The nucleotide sequence designed by the Example 1 SEQ ID NO1 is synthesized by Shanghai Xuguan Biotechnology Development Co., Ltd., cloned to PUC18 KPN Ⅰ and Bam Hi, named this restructuring carrier to PUC18-CEI-12,7, and transfer to E. coli ( Escherichia color ) DH5α (purchased from Novagen).

[0026] b. Extraction of E. coli DH5α containing PUC18-CEI-12,7 to extract plasmid KPN Ⅰ and Bam Hi (purchased from Takara Company) dual enzyme cutting and recycling about 200bp.

[0027] C. For the expression carrier PET-30A (purchased from Novagen) KPN Ⅰ and Bam Hi (purchased from Takara Company) dual enzyme cutting, recycling large segments.

[0028]D. The recycling clip of the viscous end of the step B is connected to the large segments recovered in the step C, transferred to the DH5α strain, and the PCR of conventional colonies is used ...

Embodiment 3

[0031] Example 3: The expression and detection of two kinds of lactating blood pressure reduction peptides CEI-12 and CEI-7 series polypeptides

[0032] The LB liquid medium (50 μg / ml) LB liquid medium obtained from E. coli BL21-MF3A3 obtained by example 2 was inoculated at a volume ratio of 1%in volume ratio at a volume ratio of 1%., NaCl 10g / L, pH7.0), 37 ° C, 200R · min -1 When the constant temperature shake bed is cultivated to 1.5, adding the isopropyte-β-D-sulfur sulfurcoside (IPTG) to the final concentration 1mmol·L –1 , Live 12 h, 5000r · min -1 Collect the bacteria for centrifugation, repeatedly frozen the bacteria 3 times, and perform ultrasonic crushing (300W, 2S / 2min, 2min), 8000R · min, 8000R · min -1 Corporation of centrifugal precipitation, dissolved in the 8ml upper column buffer solution, and purified the reorganized protein marked by the activated NI-column (purchased from Novagen) to perform SDS-PAGE electrophoresis, such as figure 2 Show.Electric swings indicat...

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Abstract

The present invention provides escherichia coli engineering bacteria expressing active peptides and a method of preparing mixed polypeptide CEI-12 and CEI-7 with the escherichia coli engineering bacteria, and relates to the bioengineering field. The preservation number of the engineering bacteria is Escherichia Coli BL21-MF3A3 CCTCC No: 2010204. By using smaller His label segment on pET-30a to subject CEI-12 polypeptide and CEI-7 polypeptide to fusion expression and alternative cascade three times, the method increases the ratio of target polypeptide in recombination protein, greatly increases the expression efficiency, and reduces the separating difficulty of small peptides.

Description

Technical field [0001] The invention involves the field of biological engineering, and it is more specifically involved in the construction of a multi -active peptide series DNA, using the engineering bacteria built by its expression carrier and the method of preparing the multi -active peptide. Background technique [0002] Biological activated peptide is the general term of the natural amino acids composed of different compositions and arrangement methods composed of different compositions and arrangements in protein, which is a versatile compound that stems from protein.Active peptides have a variety of human body metabolism and physiological regulatory functions, easy to digest and absorb, and have the functions of promoting immunity, hormone regulation, antibacterial, antiviral, lowering blood pressure, and blood lipids.Projects and functional factor with prospects. [0003] In the treatment of primary hypertension drugs, biological active peptides with blood pressure functi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P21/02C12R1/19
Inventor 李云亮马海乐任晓锋黄六容曲文娟
Owner JIANGSU UNIV
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