CN105254718A - Preparation method and application of Lactoferricin B - Google Patents

Abstract

The invention relates to a preparation method and an application of Lactoferricin B. According to the method, protein with an amino acid coding sequence NO.1 is selected to serve as target protein, then the amino acid coding sequence NO.1 is converted to a nucleotide coding sequence NO.2, two reversed-phase complementary oligonucleotide chain upstream and downstream primers are designed and artificially synthetized on the nucleotide coding sequence NO.2, restriction enzyme cutting sites of EcoR I and Xho I are introduced at 5' terminals of the upstream and downstream primers respectively, antibacterial peptide gene segments are obtained through PCR (polymerase chain reaction) amplification, recombinant expression vectors are established respectively, a product is converted into Escherichia coli, induction expression is performed, bacteria are subjected to ultrasonic splitting, SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) gel electrophoresis detection is performed, fusion protein GST(glutathione S-transferase)-LfcinB is successfully expressed, subjected to affinity chromatography purification by glutathione agarose and cut by a hydroxylamine shearing buffer solution, and antibacterial peptides are released. Detection of bacteriostasis experiments conducted with an agar diffusion method shows that Lactoferricin B has remarkable bacteriostatic activity for Escherichia coli and staphylococcus aureus, and the problem that the yield is low due to the fact that the molecular weight of Lactoferricin B is small and Lactoferricin B tends to be subjected to protease hydrolysis can be solved.

Description

A kind of preparation method of Bovine lactoferricin and application

Technical field

The invention belongs to biological technical field.Be specifically related to a kind of preparation method and application of Bovine lactoferricin.

Background technology

In recent years from world wide due to antibiotic excessive use, cause bacterial infection and disease negative effect in rising trend increasing, the continuous appearance of endurance strain also more and more causes people worried, therefore finds, finds and produce the agenda that new antibiotic substitute has listed people in.Since New Century, antibacterial peptide (Antibacterlalpeptides) is the many skins of small molecules that the class produced through induction in organism has biologic activity, being present in multiple organism, is an important component part of host immune defenses system.Advantage due to self causes the concern of people day by day, and become the focus of research, wherein Bovine lactoferricin (LactoferricinB), not only participate in the transhipment of iron, and there is the biological functions such as broad-spectrum antimicrobial, anti-oxidant, anticancer, immunity moderation system, be therefore considered to the Ying You Shi Pin ﹑ fodder additives of a kind of novel antibacterials and great exploitation potential for its.

At first, after Japanese researchers finds the degraded of natural Bovinelactoferrin, its anti-microbial activity is stronger than Bovinelactoferrin more than 400 times, finds that active substance is Bovine lactoferricin after separation and Extraction.But from natural origin Separation of Bovine lactoferricin yield poorly, time-consuming length, complex process, cost intensive.After this, commercial Bovine lactoferricin is separation and Extraction Bovinelactoferrin from milk, then through bio-enzyme degradation product, produces Bovine lactoferricin.Because the biological enzyme of Bovinelactoferrin of originally degrading is expensive, therefore the Bovine lactoferricin cost of this method production is still higher.And synthetic Bovine lactoferricin and derivative expense higher, cannot scale operation.Current China is 350,000 dollars from the Bovinelactoferrin of New Zealand's import price per ton.However, along with the development of biotechnology, Bovinelactoferrin is commercially produced, for the manufacture of breast milk infants formula milk or other functional foodstuff in Europe and Japan.Therefore utilize genetic engineering technique Bovinelactoferrin peptide gene to be proceeded in other biology to express, in order to solve, Bovine lactoferricin source is not enough provides new approach.

Gene engineering expression method will be the desirable route producing antibacterial peptide in a large number.Genetically engineered produces expression system mainly pichia spp and the intestinal bacteria of antibacterial peptide, and the codon synthetic gene often selecting expressive host to have a preference for also carries out clonal expression.Prokaryotic expression system is the expression system adopted the earliest, is also the comparatively classical expression system of application at present.Goal gene is mainly connected on carrier by this system, then clones it, is converted into bacterium afterwards, obtains target protein by abduction delivering, purifying.The advantage applies of this expression system can obtain target protein at short notice, and cost is lower.But, a large amount of secretions of antibacterial peptide can produce deleterious effect to host, therefore, at present mainly through recombinant protein technology by same for goal gene glutathione S-transferase, Trx, green fluorescent protein, coat protein, class elastin, ketosteroid isomerase, the amalgamation and expressions such as small molecules ubiquitin sample modified protein, reduce the toxicity of antibacterial peptide to host cell.This research selects glutathione sulfydryl transferase (GlutathioneS-transferase, GST) as fusion tag, attempts utilizing escherichia expression system to prepare Bovine lactoferricin (LfcinB) antibacterial peptide.

Summary of the invention

The object of the invention is, a kind of preparation method and application of Bovine lactoferricin are provided, it is target protein that the method chooses amino acid coding NO.1, then by aminoacid sequence NO.1, change into nucleotide coding sequence NO.2, and design and the oligonucleotide chain upstream and downstream primer of synthetic two reverse complement at thuja acid encoding sequence NO.2, restriction enzyme site EcoRI and XhoI is introduced respectively at 5 ' end of upper and lower two primers, pcr amplification obtains antibacterial peptide gene fragment, build recombinant expression vector respectively, abduction delivering after transformation of E. coli.Ultrasonic degradation thalline, sodium lauryl sulphate-polyacrylamide gel electrophoresis detects, fusion rotein GST-LfcinB successful expression.Fusion rotein, after glutathione agarose affinitive layer purification, shears damping fluid cleavage of fusion proteins through azanol, release antibacterial peptide.Detect it by agar diffusion method bacteriostatic experiment, to intestinal bacteria and gold-coloured staphylococci, all there is obvious bacteriostatic activity.Overcome Bovine lactoferricin molecular weight and easily by protease hydrolysis, often cause and yield poorly, be difficult to industrialization.The method of the invention, substantially increases the output of antibacterial peptide.

The preparation method of a kind of Bovine lactoferricin of the present invention, follows these steps to carry out:

The synthesis of a, antibacterial peptide LfcinB gene:

According to the gene order LfcinB of Bovine lactoferricin, choosing amino acid coding NO.1:Ac-FKCRRWQWRMKKLGAP-NH is target protein, then by aminoacid sequence NO.1, change into nucleotide coding sequence NO.2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ', again by nucleotide coding sequence NO.2, design and the oligonucleotide chain upstream and downstream primer of synthetic two reverse complement, restriction enzyme site EcoRI and XhoI is introduced respectively at 5 ' end of upper and lower two primers, obtain upstream primer: 5 '- gATCCTtCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC tAAC,downstream primer: 5 '- tCGAGTTAgAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA tTGAAG, then hydroxylamine cleavage protein loci l-asparagine-glycine is introduced in the amino acid coding NO.1 target protein upstream chosen,

Pcr amplification object antibacterial peptide gene:

PCR reaction system: ultrapure water 40 μ L, 10 × PCR are containing Mg ₂ ⁺damping fluid 5 μ L, 2.5mmol/LdNTPs1 μ L, upstream primer 1.5 μ L, downstream primer 1.5 μ L, Taq enzyme 1 μ L, amount to 50 μ L;

Response procedures is: temperature 94 DEG C keeps after 5 minutes, temperature 94 DEG C reaction 30 seconds, temperature 55 DEG C reaction 30 seconds, temperature 72 DEG C reaction 30 seconds, and after carrying out 30 circulations altogether, temperature 72 DEG C keeps 10 minutes;

Pcr amplification product is after PCR Purification Kit, and temperature-20 DEG C saves backup;

The structure of b, antibacterial peptide LfcinB gene recombined vector:

PCR primer in step a and expression vector pGEX-4T-1 EcoRI and XhoI are carried out double digestion reaction, and reaction system is vector pGEX-4T-142 μ L, EcoRI1.5 μ L, XhoI1.5 μ L, 10 × FastDigest damping fluid 5 μ L, Total50 μ L mixes respectively, temperature 37 DEG C of water-bath 20min;

Connect product conversion in BL21 intestinal bacteria, LB solid plate containing antibiotics ampicillin selects transformant, be inoculated in the 4mLLB liquid nutrient medium containing 50 μ g/mL penbritins respectively, temperature 37 DEG C, 220rpm shaking culture 8-10h, after extracting plasmid, PCR identifies, and called after pGEX-4T-1LfcinB;

The abduction delivering of c, recombinant antibacterial peptide LfcinB gene:

Recombinant bacterium pGEX-4T-1LfcinB is after the induction of 1mmol/L isopropylthiogalactoside through final concentration, detect through sodium lauryl sulphate-polyacrylamide gel electrophoresis, compared with negative control, recombinant bacterium has the target protein band of expression obviously concentrated, testing goal albumen, obtains Bovine lactoferricin.

The Bovine lactoferricin that described method obtains suppresses the application in intestinal bacteria and gold-coloured staphylococci in preparation.

The preparation method of a kind of Bovine lactoferricin of the present invention, the Bovine lactoferricin that the method obtains is through glutathione gelose gel-4B (GlutathioneSepharose4B) affinity chromatography gel-purified fusion rotein result:

Mass propgation induces recombinant expressed bacterium pGEX-4T-1LfcinB, the broken thalline of high pressure after collecting, centrifugally get supernatant liquor and deposit sample respectively afterwards, trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) detects, result display supernatant liquor and precipitation have expression, main expression is at supernatant liquor, supernatant liquor is after 0.45 μm of membrane filtration removal of impurities, through glutathione gelose gel-4B (GlutathioneSepharose4B) gel affinitive layer purification, one and predicted molecular weight band of the same size is detected with 20%Tricine-SDS-PAGE gel electrophoresis.

The preparation method of a kind of Bovine lactoferricin of the present invention, aminoacid sequence No1:Ac-FKCRRWQWRMKKLGAP-NH2 in the method; The gene order LfcinB of Bovine lactoferricin, nucleotide coding sequence NO.2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 '.Its preparation method, builds the expression vector of the amino acid whose nucleotide sequence of gene order LfcinB of Bovine lactoferricin, and is transformed in host cell by expression vector, forms the reconstitution cell can expressing LfcinB antibacterial peptide; Cultivation of recombinant cells, makes it can express LfcinB antibacterial peptide; After isolated cell cracking, remove label with proteolytic cleavage, product has anti-microbial activity.

Accompanying drawing explanation

Fig. 1 is the pcr amplification result figure of LfcinB antibacterial peptide gene of the present invention;

Fig. 2 is the PCR qualification figure of LfcinB antibacterial peptide gene recombinant plasmid of the present invention;

Fig. 3 is the double digestion qualification figure of LfcinB antibacterial peptide gene recombinant plasmid of the present invention;

Fig. 4 is that the present invention recombinates LfcinB antibacterial peptide gene abduction delivering figure;

Fig. 5 is affinity chromatography gel-purified LfcinB antibacterial peptide fusion protein figure of the present invention;

Fig. 6 is that LfcinB antibacterial peptide fusion protein of the present invention is to the inhibition figure of gold-coloured staphylococci;

Fig. 7 is that LfcinB antibacterial peptide fusion protein of the present invention is to colibacillary inhibition figure.

Embodiment:

Embodiment

The synthesis of a, antibacterial peptide LfcinB gene:

According to the gene order LfcinB of Bovine lactoferricin, choosing amino acid coding NO.1:Ac-FKCRRWQWRMKKLGAP-NH is target protein, then by aminoacid sequence NO.1, change into nucleotide coding sequence NO.2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ', again by nucleotide coding sequence NO.2, design and the oligonucleotide chain upstream and downstream primer of synthetic two reverse complement, restriction enzyme site EcoRI and XhoI is introduced respectively at 5 ' end of upper and lower two primers, obtain upstream primer: 5 '- gATCCTtCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC tAAC,downstream primer: 5 '- tCGAGTTAgAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA tTGAAG, then hydroxylamine cleavage protein loci l-asparagine-glycine is introduced in the amino acid coding NO.1 target protein upstream chosen,

Pcr amplification object antibacterial peptide gene:

PCR reaction system: ultrapure water 40 μ L, 10 × PCR are containing Mg ₂ ⁺damping fluid 5 μ L, 2.5mmol/LdNTPs1 μ L, upstream primer 1.5 μ L, downstream primer 1.5 μ L, Taq enzyme 1 μ L, amount to 50 μ L;

Response procedures is: temperature 94 DEG C keeps after 5 minutes, temperature 94 DEG C reaction 30 seconds, temperature 55 DEG C reaction 30 seconds, temperature 72 DEG C reaction 30 seconds, and after carrying out 30 circulations altogether, temperature 72 DEG C keeps 10 minutes;

Pcr amplification product is after PCR Purification Kit, and temperature-20 DEG C saves backup;

The structure of b, antibacterial peptide LfcinB gene recombined vector:

PCR primer in step a and expression vector pGEX-4T-1 EcoRI and XhoI are carried out double digestion reaction, and reaction system is vector pGEX-4T-142 μ L, EcoRI1.5 μ L, XhoI1.5 μ L, 10 × FastDigest damping fluid 5 μ L, Total50 μ L mixes respectively, temperature 37 DEG C of water-bath 20min;

Connect product conversion in BL21 intestinal bacteria, LB solid plate containing antibiotics ampicillin selects transformant, be inoculated in the 4mLLB liquid nutrient medium containing 50 μ g/mL penbritins respectively, temperature 37 DEG C, 220rpm shaking culture 8-10h, after extracting plasmid, PCR identifies, and called after pGEX-4T-1LfcinB;

The abduction delivering of c, recombinant antibacterial peptide LfcinB gene:

Recombinant bacterium pGEX-4T-1LfcinB is after 1mmol/L isopropylthiogalactoside (IPTG) induction through final concentration, detect through sodium lauryl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis, compared with negative control, recombinant bacterium has the target protein band of expression obviously concentrated, testing goal albumen, obtains Bovine lactoferricin.

Glutathione gelose gel-4B (GlutathioneSepharose4B) affinity chromatography gel-purified fusion rotein result:

Mass propgation induces recombinant expressed bacterium pGEX-4T-1LfcinB, the broken thalline of high pressure after collecting, centrifugally get supernatant liquor and deposit sample respectively afterwards, trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) gel electrophoresis detects, result display supernatant liquor and precipitation have expression, main expression is at supernatant liquor, supernatant liquor is after 0.45 μm of membrane filtration removal of impurities, through glutathione gelose gel-4B (GlutathioneSepharose4B) gel affinitive layer purification, one and predicted molecular weight band of the same size is detected with 20% trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) gel electrophoresis.

The antibacterial effect of antibacterial peptide:

Gel after purifying is scaled off from glue, be dissolved in the acetate buffer solution of 0.01%, get supernatant liquor, agar diffusion method is adopted to detect bacteriostatic activity, result shows: the Bovine lactoferricin obtained by the method for the invention has obvious bacteriostatic activity to intestinal bacteria and gold-coloured staphylococci, Fig. 6, Fig. 7.

Title: a kind of preparation method of Bovine lactoferricin and application

Applicant: Chinese Academy Of Sciences Xinjiang physics & chemistry Technology Research Institute

The gene order LfcinB of Bovine lactoferricin, amino acid coding NO.1:Ac-FKCRRWQWRMKKLGAP-NH2.

The gene order LfcinB of Bovine lactoferricin, nucleotide coding sequence NO.2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 '.

Claims (2)

1. a preparation method for Bovine lactoferricin, is characterized in that following these steps to carry out:

The synthesis of a, antibacterial peptide LfcinB gene:

According to the gene order LfcinB of Bovine lactoferricin, choosing amino acid coding NO.1:Ac-FKCRRWQWRMKKLGAP-NH is target protein, then by aminoacid sequence NO.1, change into nucleotide coding sequence NO.2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ', again by nucleotide coding sequence NO.2, design and the oligonucleotide chain upstream and downstream primer of synthetic two reverse complement, restriction enzyme site EcoRI and XhoI is introduced respectively at 5 ' end of upper and lower two primers, obtain upstream primer: 5 '- gATCCTtCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC tAAC,downstream primer: 5 '- tCGAGTTAgAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA tTGAAG, after introducing restriction enzyme site, hydroxylamine cleavage protein loci l-asparagine-glycine is introduced in amino acid coding NO.1 target protein upstream,

Pcr amplification object antibacterial peptide gene:

PCR reaction system: ultrapure water 40 μ L, 10 × PCR are containing Mg ₂ ⁺damping fluid 5 μ L, 2.5mmol/LdNTPs1 μ L, upstream primer 1.5 μ L, downstream primer 1.5 μ L, Taq enzyme 1 μ L, amount to 50 μ L;

Response procedures is: temperature 94 DEG C keeps after 5 minutes, temperature 94 DEG C reaction 30 seconds, temperature 55 DEG C reaction 30 seconds, temperature 72 DEG C reaction 30 seconds, and after carrying out 30 circulations altogether, temperature 72 DEG C keeps 10 minutes;

Pcr amplification product is after PCR Purification Kit, and temperature-20 DEG C saves backup;

The structure of b, antibacterial peptide LfcinB gene recombined vector:

PCR primer in step a and expression vector pGEX-4T-1 EcoRI and XhoI are carried out double digestion reaction, and reaction system is vector pGEX-4T-142 μ L, EcoRI1.5 μ L, XhoI1.5 μ L, 10 × FastDigest damping fluid 5 μ L, Total50 μ L mixes respectively, temperature 37 DEG C of water-bath 20min;

Connect product conversion in BL21 intestinal bacteria, LB solid plate containing antibiotics ampicillin selects transformant, be inoculated in the 4mLLB liquid nutrient medium containing 50 μ g/mL penbritins respectively, temperature 37 DEG C, 220rpm shaking culture 8-10h, after extracting plasmid, PCR identifies, and called after pGEX-4T-1LfcinB;

The abduction delivering of c, recombinant antibacterial peptide LfcinB gene:

Recombinant bacterium pGEX-4T-1LfcinB is after the induction of 1mmol/L isopropylthiogalactoside through final concentration, detect through sodium lauryl sulphate-polyacrylamide gel electrophoresis, compared with negative control, recombinant bacterium has the target protein band of expression obviously concentrated, testing goal albumen, obtains Bovine lactoferricin.

2. the Bovine lactoferricin that method obtains as claimed in claim 1 is preparing the application suppressed in intestinal bacteria and gold-coloured staphylococci.