CN104789513A - A kind of Escherichia coli strain for producing active short peptide - Google Patents

Abstract

The invention discloses an Escherichia coli bacterial strain for preparing an active short peptide. The strain is preserved as Escherichia coli, and is deposited with the China General Microbiological Culture Collection Center (CGMCC). The accession address is No.3 Yard No.1 Beichen West Road, Chaoyang District, Beijing. The accession date is October 24th, 2014. The accession number is CGMCCNo.9842. The Escherichia coli related in the invention is used for preparing an Escherichia coli bacterial preparation. According to the invention, the Escherichia coli bacterial strain for preparing an active short peptide is high in in-vivo activity and low in cost.

Description

A kind of Escherichia coli strain for producing active short peptide

technical field

The invention relates to the field of genetic engineering, in particular to an Escherichia coli strain for preparing active short peptides.

Background technique

Escherichia coli is a Gram-negative round bacillus that lives in the gut of warm-blooded animals. Most strains of E. coli are harmless, but some serotypes can cause severe food poisoning in humans. The harmless Escherichia coli is part of the normal flora of the intestinal tract, and its production of vitamin K2 is beneficial to the host and also prevents the production of other pathogenic pathogenic bacteria in the intestinal tract. Escherichia coli accounts for 0.1% of the entire intestinal flora, and the fecal-oral transmission route is an important way for this pathogen to cause disease. At the same time, Escherichia coli is a widely studied prokaryotic model organism, because it can be used as a host for recombinant DNA, and it is an important bacterium in the field of microbiology and biotechnology. Because Escherichia coli has such an important role, the bacterial mutant strains in the space environment are studied through phenotype, and the three omics research methods of genome, transcriptome and proteome are used to study the adaptability of bacteria to the space environment, and pay attention to its Changes in pathogenicity and drug resistance lay the foundation for preventing the occurrence of infectious diseases in the space station.

At present, there have been a large number of patent reports on "purification of high-purity active peptides by conventional separation methods", although some peptides identified by researchers show strong physiological activities, such as IPP, VPP, IKW, LHP and other hypotensive peptides And YHY, PHH, YKY, YPPAK, HDHPVC and other antioxidant peptides, but due to the low proportion of effective active peptides contained in the parent protein sequence, resulting in low yield and high separation cost in the later stage of enzymatic hydrolysis, except for a very small number of active peptides, Except for products such as IPP and VPP, most highly active functional peptides have not been effectively developed and utilized.

With the development of genetic engineering technology, DNA recombinant technology is used to clone genes expressing active peptides into certain microorganisms or animals, and to directly express the desired peptides through organisms, which can greatly increase their yield and purity. At present, the patent reports on "Using Genetically Engineered Bacteria to Prepare Active Peptides" have been increasing year by year. Although a technical breakthrough has been made in the preparation of highly active short peptides by genetic engineering technology, the gene design, expression efficiency and downstream separation process of active peptide multimers need to be further improved.

In recent years, cell biology and molecular biology studies have shown that in addition to the booster substances and systems in the human body, there are also many endogenous decompression substances and systems to maintain relatively stable blood pressure. If the antihypertensive peptides and antioxidant peptides are used in combination for the prevention and control of cardiovascular diseases, good results will be achieved. Many studies have reported the synchronous preparation of high blood pressure-lowering peptides by enzymatic hydrolysis and the combined effect of anti-oxidation. However, there is no report on the simultaneous preparation of ACE inhibitory peptides and antioxidant peptides by genetically engineered bacteria.

At present, there is a lack of E. coli strains that can produce active short peptides with high in vivo activity and low cost.

Contents of the invention

The purpose of the present invention is to provide an Escherichia coli strain capable of producing active short peptides with high in vivo activity and low cost.

The technical scheme of the present invention is as follows: the present invention provides a kind of Escherichia coli bacterial strain that prepares active short peptide, and this bacterial strain is Escherichia coli; Preservation name is Escherichia coli Escherichia coli; Center (CGMCC), the deposit address is No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing; date of deposit: October 24, 2014; deposit number: CGMCC No.9842.

The Escherichia coli bacterial agent prepared by the Escherichia coli described in the present invention.

Escherichia coli bacterial agent of the present invention, its active component is at least one in following (a) (b) (c):

(a) the fermentation culture of the escherichia coli described in claim 1;

(b) the sonication supernatant of the Escherichia coli cell obtained in claim 1;

(c) ultrasonic lysis precipitation of the Escherichia coli cell obtained in claim 1.

The present invention prepares the method for described escherichia coli bacterial agent, comprises the steps:

(1) Put a single colony of the strain in 20ml of LB medium containing 50μg/ml of Kana at a temperature of 37°C and a shaking rate of 200rpm for 12-14h,

(2) Transfer the culture solution into the TB culture solution containing 50 μg/ml of Kana by 2% inoculum size, the temperature is 37° C., and the stirring speed is 200 rpm,

(3) Cultivate E. coli strains in shake flasks until the OD value of the cells reaches 0.6-0.8, add IPTG to a final concentration of 0.02-0.2mM, and induce the culture time at 20-37°C for 5-20h, then sample SDS- PAGE analysis, prepared Escherichia coli bacterial agent.

Further, in step (1), the LB medium is composed of 1% Tryptone, 0.5% Yeast extract, 1% NaCl, and pH 7.0 components by mass percentage.

Further, in step (2), the TB culture solution is composed of 1.2% Tryptone, 2.4% Yeast extract, 0.4% glycerol, 0.2% KH ₂ PO ₄ and 1.6% K ₂ HPO ₄ by mass percentage.

The application of the escherichia coli strain described in the present invention in the preparation of medicine for treating hypertension.

Beneficial effect: the bacterial strain of the present invention has the characteristics of high activity in vivo and low cost. Escherichia coli is a biological agent, and there is no problem of chemical resistance caused by the use of chemical agents. The active small peptide in the obtained recombinant polypeptide can be effectively released by pepsin, trypsin and chymotrypsin, and the prepared active peptide mixture has high antihypertensive activity and antioxidant activity in vivo. The final product of the small peptide mixture of the present invention can synergistically reduce blood pressure while improving the function of vascular endothelial cells, preventing and maintaining cardiovascular health from multiple perspectives; the strain of the present invention has broad application prospects in the preparation of drugs for treating hypertension.

Description of drawings

Fig. 1 is the schematic diagram of recombinant plasmid constructed by the present invention;

Fig. 2 is the schematic diagram of recombinant plasmid PCR identification of the present invention;

Fig. 3 is the schematic diagram of double enzyme digestion identification of the recombinant plasmid of the present invention;

Figure 4 is a schematic diagram of SDS-PAGE identification of recombinant protein inclusion body expression;

Figure 5 is a schematic diagram of SDS-PAGE identification of recombinant protein purification;

Figure 6 is a schematic diagram of the purification of recombinant polypeptide A3 identified by SDS-PAGE;

Fig. 7 is a schematic diagram of SDS-PAGE identification of soluble expression of recombinant protein.

Detailed ways

The present invention will be further described in detail with reference to the drawings and specific examples below, but it should not be construed as limiting the protection scope of the present invention.

Example 1

The invention provides a bacterial strain, an Escherichia coli strain for preparing active short peptides, the bacterial strain is Escherichia coli; the preservation name is Escherichia coli; it is preserved in the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee, The deposit address is No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing; date of deposit: October 24, 2014; deposit number: CGMCC No.9842.

The Escherichia coli bacterial agent prepared by the Escherichia coli described in the present invention.

Escherichia coli bacterial agent of the present invention, its active component is at least one in following (a) (b) (c):

(a) the fermentation culture of the escherichia coli described in claim 1;

(b) the sonication supernatant of the Escherichia coli cell obtained in claim 1;

(c) ultrasonic lysis precipitation of the Escherichia coli cell obtained in claim 1.

The present invention prepares the method for described escherichia coli bacterial agent, comprises the steps:

(1) Bacterial strain single bacterium colony is contained in the LB medium of the Kana of 50 μ g/ml in 20ml, and temperature is 37 ℃, and agitation rate is 200rpm shaker culture 12h, and described LB medium is 1% Tryptone, 0.5% Yeast extract, 1% NaCl, pH 7.0. The LB medium is composed of 1% Tryptone, 0.5% Yeast extract, 1% NaCl, and pH 7.0 by mass percentage.

(2) The culture solution is transferred into the TB culture solution containing 50 μg/ml of Kana by 2% inoculum size, the temperature is 37° C., and the stirring speed is 200 rpm. The TB culture solution is composed of 1.2% Tryptone, Component composition of 2.4% Yeast extract, 0.4% glycerol, 0.2% KH ₂ PO ₄ and 1.6% K ₂ HPO ₄ .

(3) Cultivate E. coli strains in shake flasks until the OD value of the cells reaches 0.6, add IPTG to a final concentration of 0.2 mM, and induce the culture time at 37°C for 5 hours, then take samples for SDS-PAGE analysis to obtain E. coli bacteria agent.

The application of the escherichia coli strain described in the present invention in the preparation of medicine for treating hypertension.

As shown in Figures 1 to 7, Figure 1 is a schematic diagram of the recombinant plasmid constructed by the present invention; Figure 2 is a schematic diagram of the PCR identification of the recombinant plasmid of the present invention, swimming lane M is the DL10000 DNA standard sample, and swimming lanes 1, 2, and 3 are all the same PCR product (Taking recombinant plasmid pET28a-ESA3 as template, primer 5'-TTCCTGGAACCGGA-3' and 5'-CCAAGCCACACGA AAC-3' as upstream and downstream primers); Fig. 3 is a schematic diagram of double enzyme digestion identification of recombinant plasmid of the present invention, M is DNA Marker, lanes 1-5 are Nco I/Bam HI, Bam HI/Hind III, Hind III/Xho I, Bam HI/Xho I, Nco I/Xho I double digestion products, lane 6 is the recombinant plasmid; 4 is a schematic diagram of SDS-PAGE identification of recombinant protein inclusion body expression, lane M is the protein standard sample, and lanes 1, 2 and 3 are the sonicated stock solution, ultrasonic supernatant and ultrasonic precipitation of the induced recombinant bacteria, respectively; Figure 5 is the SDS-PAGE identification of recombinant Schematic diagram of protein purification, M protein Marker, fusion protein after 0 dialysis, lanes 1, 2, and 3 are fusion proteins purified with final concentrations of 1.0, 1.5, and 2.0 mol/L sodium chloride respectively; Figure 6 is SDS-PAGE identification of recombinant Schematic diagram of polypeptide purification, lane M is the protein standard sample, lane 1 is the product of the fusion protein Elps-SUMO-A3 hydrolyzed by SUMO protease, and lanes 2 and 3 are the final concentration of 1.0mol/L chlorine added to the above enzymatic hydrolyzate Centrifugal supernatant and precipitation after sodium chloride salting out; Figure 7 is a schematic diagram of SDS-PAGE identification of soluble expression of recombinant protein; lane M is the protein standard sample, lanes 1, 2 and 3 are the sonicated stock solution, supernatant and supernatant of recombinant bacteria after induction, respectively. Ultrasonic precipitation;

A recombinant active peptide of the present invention, the recombinant active peptide is composed of active peptide monomers superimposed or directed in series, and the active peptide monomers include blood pressure-lowering peptide monomers and antioxidant peptide monomers, the amino acid sequence of which is Such as sequence listing SEQ ID NO: 1.

A nucleotide sequence encoding the recombinant active peptide of the present invention is shown in the sequence listing SEQ ID NO: 2.

The antioxidant peptide monomers include DTHK, YPIL, FLEPDY, YLEPFR, YLEPDY, YDEPEW, HYRPFW, Y EPDY and IWAPFY; the blood pressure-lowering peptide monomers include MRW, WIR, IRA, AMK, MKR, RGY, VAW, DGL, IPP, IKP, IKPFR, IKPVA, AKF, IW, VAF, VSV, IQY, and IVY.

The N-terminal of the active peptide monomer partial peptide is a pepsin cleavage site; the C-terminal of the active peptide monomer partial peptide is a pepsin or trypsin or chymotrypsin cleavage site; the Part of the active peptide monomers in the recombinant blood pressure-lowering peptide and the anti-oxidation peptide are connected in series by linking fragments with pepsin, trypsin or chymotrypsin cleavage sites.

The method for synchronously preparing the recombinant active peptide of the present invention comprises the following steps:

(1) Design and optimization of recombinant antihypertensive peptides and antioxidant peptides and their coding genes;

The gene shown in the artificially synthesized sequence table of the present invention SEQ ID NO: 2; the active peptide monomers in the present invention are screened from the constructed active peptide database, and some active peptides are artificially processed according to the design of the composite functional peptide precursor. remodel. The general characteristics of the screened active peptides are: resistance to digestion by gastrointestinal enzymes; easy complete release of gastrointestinal enzymes; and high biological activity in vivo. After preliminary screening, the selected ACE inhibitory peptide monomers are: MRW, WIR, IRA, AMK, MKR, RGY, VAW, DGL, IPP, IKP, IKPFR, IKPVA, AKF, IW, VAF, VSV, IQY, IVY, etc.; The selected antioxidant peptide monomers are: DTHK, YPIL, FLEPDY, YLEPFR, YLEPDY, YDEPEW, HYRPFW, Y EPDY, IWAPFY, etc.

On the premise that active peptide monomers are theoretically easy to release effectively from gastrointestinal digestive enzymes, direct head-to-tail connection is adopted, or a suitable linking short peptide (2-3 amino acid residues, with biological activity) is used, or some of the same amino acid residues will be included The different active peptides are superimposed, and the screened target active peptides are directional and serially assembled into a composite functional peptide precursor recombinant active peptide, and the protein structure prediction software and gene optimization software are used to guide the design of the recombinant active peptide and its gene, aiming to make Theoretically, the recombinant active peptide gene can be expressed stably and efficiently in engineered bacterial cells.

Based on the above strategies, the amino acid sequences of the recombinant antihypertensive peptides and antioxidant peptides designed by the present invention are as follows:

The nucleotide sequences encoding the recombinant hypotensive peptide and antioxidant peptide are as follows:

(2) Construction of recombinant expression vectors and recombinant bacteria;

As shown in Figure 1, the construction of the recombinant plasmid pET28a-ESA3. Using molecular cloning technology, the recombinant active peptide gene designed and optimized in step (1) was cloned into the Hind III and Xho I sites of pET28a, and the Nco I and BamHI sites upstream of the recombinant active peptide, as well as Bam HI and Hind The ELPs gene of the elastin-like purification tag and the SUMO fusion tag gene were cloned at the III site, and the recombinant expression vector pET28a-ESA3 was constructed and transformed into E.coli BL21(DE3) to obtain engineering bacteria.

As shown in Figure 2, using the recombinant plasmid pET28a-ESA3 as a template, primers 5'-TTCCTGGAACCGGA-3' and 5'-CCAAGCCACACGAAAC-3' as upstream and downstream primers for PCR, a PCR product of about 340bp was obtained, which was consistent with the expected target fragment The recombinant active peptides are similar in size.

Recombinant plasmids were identified by restriction endonucleases Nco I/Bam HI, Bam HI/Hind III, Hind III/Xho I, Bam HI/Xho I, Nco I/Xho I, respectively.

The Nco I/Bam HI double enzyme digestion reaction system is as follows:

Bam HI/Hind III, Hind III/Xho I, Bam HI/Xho I, Nco I/Xho I double enzyme digestion system is the same as above. Mix well and keep warm at 37°C for 2 hours. After the reaction, add 6 μL of electrophoresis loading buffer to terminate the reaction. After 1.5% agarose gel electrophoresis, the enzyme digestion results are observed with a gel imaging system. The results are shown in Figure 3. Lanes 1-5 show the size and ELPs of fragments obtained by double digestion of Nco I/Bam HI, Bam HI/Hind III, Hind III/Xho I, Bam HI/Xho I, and Nco I/Xho I The size of gene fragment (573bp), SUMO gene fragment (294bp), A3 gene fragment (345bp), SUMO-A3 gene fragment (639bp) and ELPs-SUMO-A3 gene fragment (1218bp) was consistent. Further sequencing confirmed that the cloned gene sequence and reading frame were correct, indicating that the recombinant plasmid was constructed successfully. An expression vector capable of efficiently expressing and purifying recombinant hypotensive peptide and antioxidative peptide is constructed; the expression vector is pET28a, and the host bacterium is Escherichia coli E.coli BL21(DE3). Using DNA recombination technology, insert the gene shown in the sequence table SEQ ID NO: 2 into the Hind III and Xho I sites of pET28a, and respectively at the Nco I and Bam HI sites and the Bam HI and Hind III sites upstream of the gene Point cloning into elastin-like purification tag ELPs gene and SUMO fusion tag gene to obtain recombinant expression vector pET28a-ESA3.

(3) construct genetically engineered bacterium with described expression vector transformation host bacterium; And transform E.coli BL21 (DE3), obtain engineered bacterium; Recombinant protein Elps-SUMO-A3 induces expression in E.coli BL21 (DE3)

Pick a single colony of E.coli BL21 (DE3) containing the recombinant plasmid pET28a-ESA3 and place it in 20 ml of LB medium (1% Tryptone, 0.5% Yeast extract, 1% NaCl, pH 7.0) containing 50 μg/ml of Kana, at 37°C , cultivate overnight on a shaker at 200rpm, then transfer the overnight culture solution into the TB culture solution containing 50 μg/ml Kana (1.2% Tryptone, 2.4% Yeast extract, 0.4% glycerol, 0.2% KH ₂ PO _4. In 1.6% K ₂ HPO ₄ ), 37°C, 200rpm, shake the flask until the cell OD _600nm value reaches 0.6-0.8, add IPTG to the final concentration of 0.2mM, 37°C, after induction for 5 hours, sample SDS- According to PAGE analysis, as shown in Figure 4, after the induced culture of the engineered bacteria for 5 hours, a protein band with a molecular weight of about 45kDa appeared relative to the sample before induction, which indicated that the recombinant protein Elps-SUMO-A3 was successfully expressed in E.coli BL21 (DE3). The fusion expression was obtained, and the gray-scale scanning analysis showed that the expression of the recombinant protein accounted for more than 65% of the total protein in the cell, of which more than 90% was expressed in the form of inclusion bodies, and the purity of the target protein in the inclusion bodies was more than 80%.

(4) Utilize the engineering bacteria to express the recombinant active peptide; Elps-SUMO-A3 inclusion body preparation and renaturation

Divide the cultured bacterial solution, centrifuge at 4000rpm for 5min to obtain bacterial pellet, add 20ml of pH 8.0 PBS buffer to the pellet to resuspend, resuspend the bacterial solution at 300W, work for 2s, interval of 8s, and sonicate in ice bath Broken for 50min. The crushed solution was centrifuged at 4°C and 8000 rpm for 10 min, and the obtained inclusion body precipitate was resuspended with an equal volume of 6 mol/l urea, and kept in an ice bath for 10 h. Centrifuge at 4°C, 8000rpm for 10min, and keep the supernatant. The supernatant after reconstitution of the inclusion bodies was respectively dialyzed in dialysate 1, dialysate 2, and dialysate 3 in an ice bath for 6 hours (the dialysate contained 10mmol/l Tris-HCl pH 8.0, 1mmol/l EDTA, 150mmol/l NaCl , 10% glycerol). Dialysate 1 has a urea concentration of 4 mol/l, dialysate 2 has a urea concentration of 2 mol/l, and dialysate 3 has a urea concentration of 1 mol/l. After the dialysis, the sample was centrifuged at 4°C and 8000rpm for 10 min, and the supernatant was retained for renaturation to obtain the soluble fusion protein Elps-SUMO-A3.

(5) Separation and purification of recombinant protein Elps-SUMO-A3 and recombinant polypeptide A3

Use tag purification and tag removal technology to obtain recombinant active peptides; after dialysis, the refolded recombinant protein solution still contains a small amount of impurity proteins, which need to be purified. Since the recombinant protein prepared above contains the Elps tag, it can be purified by adding salt and centrifuging. Take the protein solution obtained by dialysis, add NaCl with a final concentration of 1.0, 1.5 and 2.0 mol/L, mix evenly, then bathe in water at 30°C for 10 minutes, centrifuge at room temperature for 10 minutes, collect the precipitate, and add 1 mL of 4°C pre-cooled pH 8.0 to the precipitate Resuspend in PBS buffer, ice-bath for 1 h, and centrifuge to get the supernatant for electrophoresis analysis. The results are shown in Figure 5, where the concentrations in the figure are 1.0, 1.5, 2.0mo/L NaCl precipitation, and the fusion protein redissolved in pH 8.0 PBS electrophoresis. Comparing the target protein bands in the figure, the impurities in the precipitate formed by the target protein Elps-SUMO-A3 under the induction of sodium chloride were reduced. Since the salt concentration in a certain range will lead to the dissolution or precipitation of proteins, different concentrations of sodium chloride have different effects on impurity proteins in the solution. Comparing the results in the figure, 1.5mol/L NaCl is used to purify the solution, and the obtained precipitate has the highest purity. Therefore, 1.5mol/L sodium chloride was used to purify the refolded recombinant protein Elps-SUMO-A3. For the soluble expressed target protein as shown in the sequence table SEQ ID NO: 1, the labeling technology is directly used for purification, and the target protein expressed in the inclusion body is first denatured and refolded, and then the labeling technology is purified, and the salt is added to the centrifugal precipitation. Add an equal volume of pre-cooled 4°C pH 8.0 PBS buffer to resuspend, ice-bath for 6 hours and then centrifuge. The supernatant is the purified protein solution. After two centrifugation cycles, the target protein with a purity of more than 95% can be obtained. Purify the fusion protein by Elps tag purification technology and use SUMO protease cleavage and salt centrifugation to remove the doublet fusion tag Elps-SUMO to obtain recombinant polypeptide A3. The purification schematic diagram is shown in Figure 6.

(6) Enzymatic hydrolysis with pepsin and/or trypsin and/or chymotrypsin to prepare active peptide

Pepsin, trypsin and chymotrypsin were used to enzymolyze the recombinant antihypertensive peptide and antioxidative peptide, ultrafiltration to obtain a small peptide mixture, and then desalted to remove impurities, freeze-dried the sample, and quality control to prepare antihypertensive peptide and antioxidative peptide .

The application of the recombinant antihypertensive peptide and antioxidative peptide of the present invention in the preparation of medicine for treating hypertension.

Example 2

The difference between Example 2 and Example 1 is that the engineered bacteria pET28a-ESA3/E.coli BL21(DE3) was constructed according to step (1) and step (2) in Example 1.

In step (1), a single colony of the strain was placed in 20 ml of LB medium containing 50 μg/ml Kana at a temperature of 37° C. and a stirring rate of 200 rpm for 13 hours on a shaker.

In step (3), the Escherichia coli strain was cultured in a shaking flask until the OD value of the thalline reached 0.7, and IPTG was added to a final concentration of 0.06mM, and the temperature was 20° C. After the induction culture time was 20h, a sample was taken for SDS-PAGE analysis, and the prepared Escherichia coli bacteria.

As shown in Figure 7, after the engineered bacteria were induced and cultured for 20 hours under the condition of low temperature and low inducer concentration, a protein band with a molecular weight of about 45 kDa appeared relative to the sample before induction; after the cells were ultrasonically disrupted, the soluble expression level of the target protein reached 60% above. The supernatant was sonicated, added NaCl with a final concentration of 1.5 mol/l, mixed evenly, placed in a water bath at 30°C for 10 min, and centrifuged at room temperature to retain the precipitate. Add an equal volume of pre-cooled 4°C pH 8.0 PBS buffer to the precipitate to resuspend, and centrifuge after 6 hours in ice bath. The supernatant is the purified protein solution. After two centrifugation cycles, the target protein with a purity of more than 95% can be obtained .

Example 3

The difference between embodiment 3 and embodiment 1 is:

In step (1), a single colony of the strain was placed in 20 ml of LB medium containing 50 μg/ml Kana at a temperature of 37° C. and a stirring rate of 200 rpm for 14 hours on a shaker.

In step (3), the Escherichia coli strain was cultured in a shake flask until the OD value of the thalline reached 0.7, and IPTG was added to a final concentration of 0.1 mM, and the temperature was 30° C. After the induction culture time was 10 h, a sample was taken for SDS-PAGE analysis, and the prepared Escherichia coli bacteria.

Preparation of recombinant small peptide mixture and identification of its activity. Take an appropriate amount of the prepared Elps-SUMO-A3, add SUMO protease according to the addition amount of 3%, and enzymatically hydrolyze at 30°C for 5h. After the enzymolysis, add sodium chloride with a final concentration of 1.5mol/l to the enzymolysis solution, bathe in water at 30°C for 10 minutes, centrifuge at 8000r/min for 10 minutes, and keep the supernatant, which is the recombinant active peptide. Simulate the natural physiological digestion process of the human body in vitro, adjust the pH of the 2mg/ml recombinant active peptide solution to 2.0 with hydrochloric acid, add 2% pepsin, hydrolyze it at 37°C for 4 hours, stop the reaction in a boiling water bath, adjust the pH to 7.0, and take part of the hydrolyzed solution Determination of peptide activity. Add 2% trypsin (or a mixture of trypsin and chymotrypsin) to the remaining hydrolyzate, hydrolyze at 37°C for 4 hours, and terminate the reaction in a boiling water bath. Pepsin hydrolyzate (H1); trypsin hydrolyzate (H2); compound enzyme hydrolyzate (H3). In vitro ACE inhibitory activity and antioxidant activity detection, the results showed that the gastrointestinal digestive enzyme hydrolyzate H1, H2 and H3 of the multimer recombinant active peptide all showed strong ACE inhibitory activity and antioxidant activity, among which, the hydrolyzate The ACE inhibition IC50 of H2 is below 1.0 μg/ml, and the SHR animal experiment also confirmed that the gastrointestinal enzyme hydrolyzate H1, H2 and H3 of the recombinant active peptide all have a significant antihypertensive effect, among which H3 is administered at a dose of 0.5 mg/kg orally After 4 hours of intragastric administration, the range of blood pressure reduction can reach 45mmHg.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have For various changes and improvements, the protection scope of the present invention is defined by the appended claims, description and their equivalents.

Claims (7)

1. An Escherichia coli strain for preparing active short peptides, characterized in that: the strain is Escherichia coli; the preservation name is Escherichia coli; it is preserved in CGMCC, General Microbiology Center of China Microbiological Culture Collection Management Committee, and the preservation address is Beijing No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, City; date of preservation: October 24, 2014; preservation number: CGMCC No.9842. 2. the escherichia coli bacterial agent prepared by escherichia coli according to claim 1. 3. Escherichia coli bacterial agent according to claim 1, its active ingredient is at least one in following (a) (b) (c): (a) the fermentation culture of the escherichia coli described in claim 1; (b) the sonication supernatant of the Escherichia coli cell obtained in claim 1; (c) ultrasonic lysis precipitation of the Escherichia coli cell obtained in claim 1. 4. prepare the method for the described escherichia coli bacterial agent of claim 3, it is characterized in that comprising the steps: (1) A single colony of the bacterial strain was placed in 20 ml of LB medium containing 50 μg/ml of Kana at a temperature of 37 ° C and a stirring rate of 200 rpm for 12-14 hours; (2) Transfer the culture solution into the TB culture solution containing 50 μg/ml of Kana according to the inoculum size of 2%, the temperature is 37° C., and the stirring rate is 200 rpm; (3) Cultivate E. coli strains in shake flasks until the OD value of the cells reaches 0.6-0.8, add IPTG to a final concentration of 0.02-0.2mM, and induce the culture time at 20-37°C for 5-20h, then sample SDS- PAGE analysis, prepared Escherichia coli bacterial agent. 5. the preparation method of escherichia coli bacterial agent according to claim 4 is characterized in that: in step (1), described LB culture medium is made of 1%Tryptone, 0.5%Yeast extract, 1%NaCl, Component composition at pH 7.0. 6. the preparation method of Escherichia coli bacterial agent according to claim 4 is characterized in that: in step (2), described TB culture fluid is made of 1.2%Tryptone, 2.4%Yeast extract, 0.4%glycerol, Composition _of 0.2% _KH2PO4 and 1.6% _K2HPO4 . 7. according to the application of the escherichia coli bacterial strain described in claim 1 in the preparation treatment hypertension drug.