CN103204938A

CN103204938A - Hybrid antibacterial peptide LFB_Mel and preparation method thereof

Info

Publication number: CN103204938A
Application number: CN201210149898XA
Authority: CN
Inventors: 李本涛; 李帅伟
Original assignee: GUANGZHOU GLAM BIOTECHNOLOGY CO Ltd
Current assignee: GUANGZHOU GLAM BIOTECHNOLOGY CO Ltd
Priority date: 2012-08-15
Filing date: 2012-08-15
Publication date: 2013-07-17

Abstract

The invention relates to a hybrid antibacterial peptide LFB_Mel and a preparation method thereof. The DNA sequence of the antibacterial peptide and a gene Lfcin B_Mel coding the protein is represented by 1 in the sequence list. The amino acid sequence of the protein coded by the gene is represented by 2 in the sequence list. According to the invention, the hybrid antibacterial peptide gene Lfcin B_Mel is synthesized with a PCR primer segmented complementary synthesis method, the DNA sequence is transferred into Escherichia coli, such that hybrid antibacterial peptide genetically engineered bacteria are formed; and the engineered bacteria are subjected to large-scale induced expression and purification, such that hybrid antibacterial peptide is obtained. The method provided by the invention has the advantages that: the hybrid antibacterial peptide has substantial inhibition effects against Gram+ bacteria, Gram- bacteria, fungi, and the like. The hybrid antibacterial peptide shows good application prospects as livestock and poultry feed additives used in livestock and poultry breeding.

Description

A kind of heterozygous antibacterial peptide LFB_Mel and preparation method thereof

Technical field

The present invention relates to the genetically engineered field, specifically a kind of heterozygous antibacterial peptide gene transformation intestinal bacteria and its preparation method and application.

Background technology

Antibiotic abuse widely-used and antibiotic causes germ that antibiotic is produced resistance, and therefore, searching and development of new antibiotic are hot subjects.Cationic antibacterial peptide is present in the multiple organism, has anti-lattice gram-positive microorganism and wide spectrum effects such as negative bacterium, fungi and protozoon in host's natural immunity.This class antimicrobial polypeptide contains more basic aminoacids (having positive charge under physiological condition) and more hydrophobic amino acid, because unique antibiotic mechanism, bacterium is difficult for it is produced resistance, gets a good chance of as the antibiotic that clinical use value is arranged.At present existing nearly 10 cationic antimicrobial peptide analogs enter clinical or preclinical experimental stage.The antibiotic mechanism of cationic antibacterial peptide is the cationic moiety of peptide and the negatively charged ion generation electrostatic interaction of bacterial cell membrane (immobilized artificial membrane), and the hydrophobic position that is formed by hydrophobic amino acid and the fat chain of bacterial cell membrane produce hydrophobic interaction, thus the bacteriostatic action of playing.Most of cationic antibacterial peptide also has the haemolysis side effect except having anti-microbial activity, the research focus in this field or problem to be solved mainly contain at present: the research of antibiotic mechanism; The design of the antibacterial peptide of high anti-microbial activity, low hemolytic activity is synthetic, and the cationic antibacterial peptide of discovery or the anti-microbial activity of its synthetic analogues are lower than the anti-microbial activity of traditional antibiotic, so large usage quantity; Synthetic chain length is the antibacterial peptide of weak point as far as possible, can reduce the antigenicity of peptide and the synthetic cost of reduction peptide.Mellitin is the main active ingredient of apis mellifera Linnaeus (Apismellifera) venom, is made up of 20 amino acid, and primary structure is:

GIGAVLKVLTTGLPALISWIKRKRQQ-NH2, mellitin can form amphipathic α-Luo Xuanjiegou, and 4 C ends (KRKR) that concentrate on peptide chain in 5 basic aminoacidss are typical cationic antibacterial peptides.Mellitin has very strong restraining effect to gram-positive microorganism and negative bacterium, also has very strong hemolytic activity, has limited its clinical application.Mellitin has very high affinity interaction to calmodulin, can be in conjunction with the model of polypeptide combining site according to the calmodulin that we propose, prediction also proves by experiment, the combining site of mellitin and calmodulin is the 12-26 fragment, and find that this fragment still keeps certain anti-microbial activity, but hemolytic activity disappears.

Bovinelactoferrin peptide (Bovine Lactoferricin is abbreviated as Lfcin B) is that molecular weight is about 3.1kd through 25 amino-acid residues of stomach en-from (17～41) hydrolysis of Bovinelactoferrin (bLF) N-end.Lfcin is the anti-microbial activity center of bLF, to bLF anti-microbial activity play an important role (Kanget al, 1996).And Lfcin B is active the highest among all Lfcin, and fungistatic effect is 400 times of Bovinelactoferrins, can bring into play more important role in gi tract.Two reverse βZhe Die structures that in the aqueous solution, are hydrophilic fat, have heat-resisting, be difficult for being degraded at digestive tube, the feature of no antigen, can regulate immunization, have broad-spectrum antibacterial action, do not disturb characteristics such as intestinal beneficial bacterium.It is to many G ⁺With G_ anti-microbial effect is arranged all, as intestinal bacteria, Salmonella enteritidis, Pseudomonas aeruginosa, streptococcus aureus, clostridium perfringens etc., but do not act on bifidus bacillus etc. to the human body beneficial bacterium, simultaneously can also irritation cell growth, participate in immunomodulatory.Because its special function as the development research of fodder additives with food preservatives, has been subjected to the attention of domestic and international research institution and manufacturers, becomes the research focus.

According to antibacterial peptide Bovinelactoferrin peptide (Lfcin B) and the Core Feature sequence that is rich in the mellitin (Melittin) of glycine, the two couples together with Lfcin B (1-25) and Magainin (12-26), design a kind of novel heterozygous antibacterial peptide LFB-Mel, this antibacterial peptide molecule N end forms two reverse βZhe Die structures of hydrophilic fat, the amphiphatic αLuo Xuanjiegou of intermediate formation, continuous four basic aminoacidss of C end make the antibacterial peptide molecule have positive charge, can be combined with electronegative cytolemma, heterozygous antibacterial peptide has been abandoned the hemolytic activity of mellitin, and its anti-microbial activity and stability all are better than single antibacterial peptide molecule.

Agar hole diffusion process records the streptococcus aureus of heterozygous antibacterial peptide LFB-Mel, intestinal bacteria K ₁₂D ₃₁, staphylococcus, four kinds of bacteriums of Salmonella typhimurium minimal inhibitory concentration (Minimal inhibition concentration MIC) is respectively 1,2,2 and 3 μ gmL-1, does not show hemolytic activity in the Mlc scope.Obtain a kind of novel antibacterial peptide with higher anti-microbial activity and safety non-toxic, provide new approaches for researching and developing desirable novel antibacterial peptide molecule.

Summary of the invention

The object of the present invention is to provide a kind of new heterozygous antibacterial peptide LFB-Mel and the gene order of this albumen of coding.

Another object of the present invention is to provide the production method of this antimicrobial peptide products.

Further purpose of the present invention is to provide the anti-microbial activity of this antibacterial peptide to detect.

Antibacterial peptide is because self good characteristic demonstrates in food storing, feed safety and medicinal industry application prospect widely.Antibacterial peptide does not generally contain rare amino acid and exogenous chemical components, is a kind of healthy and safe product.In animal digestive tract, have good stability, have simultaneously that immunogenicity is little, a good water solubility, broad-spectrum sterilization even can fungicidal, protozoon and do not produce advantages such as endurance strain, can tolerate the degraded of proteolytic enzyme and peptase in the gi tract.

In addition, antibacterial peptide also has good thermostability, particularly under acidic conditions, heating or even high temperature high pressure process are to the not influence of its anti-microbial activity, therefore not only can tolerate the violent condition of High Temperature High Pressure in the feed course of processing, and can in feed preservation process, continue its function of performance, keep feed quality, prolong the shelf-life.Antibacterial peptide is good traditional microbiotic substitute as the green feed additive of a new generation, can solve the residual and chemical sproof problem of pathogenic bacteria of humans and animals medicine effectively.

Description of drawings

Fig. 1 is the schema of the synthetic heterozygous antibacterial peptide gene LFB-Mel of the complementary synthesis method of PCR segmentation primer.

Fig. 2 is the complementary synthetic synthetic heterozygous antibacterial peptide gene map of electrophoresis detection PCR.

Fig. 3 is the physical map of plasmid vector pET-32a.

Fig. 4 is that the special primer PCR of pET32a-Lfcin B Mel recombinant plasmid vector identifies that among the figure: M is DNAMarker DL2000; 1 and the 2 transformant amplified productions for evaluation.

Fig. 5 is that the universal primer PCR of recombinant conversion identifies that M is DNA Marker DL2000; 1 and 2 transformants for evaluation.

Fig. 6 is the abduction delivering that SDS-PAGE detects heterozygous antibacterial peptide, and 1 is Protein Marker, and 2 are the empty carrier contrast,

3 is the product of goal gene abduction delivering.

Fig. 7 is the Ni of fusion rotein pre-LFB Mel ²⁺Chelating Sepharose affinity chromatography stepwise elution figure,

1 is Ni ²⁺Affine layer holds passes the peak, and 2 is the sample of 100mM imidazole wash-out, and 3 is the sample of 250mM imidazole wash-out.

Fig. 8 is the SP strong cation post affinity chromatography separation and purification graphic representation of heterozygous antibacterial peptide, SP cationic exchange coloum gradient

Three protein peaks that the NaC wash-out obtains.

Fig. 9 is that Tris/Tricine SDS-PAGE detects the heterozygous antibacterial peptide that separation and purification obtains, and its molecular weight is 4.9KDa,

1 is protein molecular weight Marker, and 2 for passing the peak, and 3 is first protein peak, and 4 is second protein peak, and 5 is

Three protein peaks.

Figure 10 is the antibacterial experiment result of the streptococcus aureus of heterozygous antibacterial peptide LFB-Mel, among the figure: 1 positive contrast

Amp, 500ug/ml; 2 negative contrasts, the buffered soln of dissolving antibacterial peptide; 3 is the assorted of 1mg/ml

Close antibacterial peptide; 4 is the heterozygous antibacterial peptide of 500ug/ml.

Embodiment

Structure and the abduction delivering of the recombinant expression vector of embodiment 1 heterozygous antibacterial peptide gene LFB-Mel

1. antibacterial peptide gene LFB-Mel's is synthetic:

LFB-Mel is optimized design to antibacterial peptide gene, synthesizes Primer1, Primer2 and three sections primers of Primer3 respectively, and employing design complementary primer carries out the method that the complementary PCR of segmentation is synthetic and increase, directly by PCR synthetic gene LFB-Mel.Synthetic antibacterial peptide gene LFB-Mel schema as shown in Figure 1.Primer after synthetic is diluted to 10 μ mol/L solution ,-20 ℃ of preservations with ddH2O.Primer sequence following (dash area is the NcoI restriction enzyme site, and underscore partly is Xho I restriction enzyme site):

Heterozygous antibacterial peptide LFB-Mel gene

Primer1：

CATG CCATGGTTT AAGTGT AGA AGATGG CAATGGAGA ATG AAGAAGTTGGGT GCTCCA

Primer2：

AAG TTG GGT GCT CCA TCT ATT ACT TGT GTT AGA AGA GCT TTC GGTTTG CCA GCT TTG A

Primer3：CCG CTCGAG TCATTGTTGTCTCTTTCTCTTGATCCAAGAAATCAAAGCTGG

Complementary PCR is synthetic in monogenic segmentation

The 1st stage PCR reacts (Step1):

The PCR reaction system

Illustrate: above-mentioned reaction system mixing is centrifugal, 100 ℃ of water-bath 5min, ice bath 1min adds pfu Taq enzyme (0.5U/ μ L) 2 μ L, and 72 ℃ are extended 10min on the PCR instrument.Carry out the 2nd stage PCR reaction then.

The 2nd stage PCR reacts (Step2):

The PCR reaction system:

Illustrate: with the reaction system mixing of the 2nd stage PCR, the PCR reaction parameter is: 94 ℃ * 5min → (94 ℃ * 1min → 55 ℃ * 30s → 72 ℃ * 1min) * 30cycles → 72 ℃ * 10min → 4 ℃ * ∞.

The 3rd stage PCR reacts (Step3):

The PCR reaction system:

With the reaction system mixing among the stp3, pcr amplification is carried out to the synthetic complete fragment of single-gene in centrifugal back.The PCR reaction conditions is: 94 ℃ * 5min → (94 ℃ * 1min → 55 ℃ * 30s → 72 ℃ * 1min) * and 30cycles → 72 ℃ * 10min → 4 ℃ * ∞, amplified production detects with 2% agarose gel electrophoresis, UVP gel imaging system record result.

The building-up process of primer as shown in Figure 1, the synthetic rear electrophoresis of heterozygous antibacterial peptide gene PCR detects as shown in Figure 2.

2. the structure of antibacterial peptide gene LFB-Mel recombinant vectors

Antibacterial peptide gene LFB-Mel and carrier pET-32a behind Nco I and Xho I double digestion, are connected antibacterial peptide gene LFB-Mel with the T4DNA ligase enzyme with carrier pET-32a, make up the pET-32a-LFB-Mel recombinant plasmid, transformed into escherichia coli DH5 α.The physical map of plasmid pET-32a as shown in Figure 3.Select transformant at the LB solid plate that contains penbritin, extract the transformant plasmid DNA and carry out the amplification of T7/T7T primer PCR, as shown in Figure 4.Further measure antibacterial peptide gene LFB-Mel and implementation sequence is in full accord and the reading frame is correct through ABI 7300DNA automatic sequencer.

3. recombinant expression vector transforms the expressive host bacterium

With identifying that correct recombinant plasmid transformed arrives expressive host bacterium E.coli Rosetta (DE3), use the antibiotic-screening positive colony, PCR as shown in Figure 5, selects the abduction delivering that recombinant expressed bacterium carries out target protein after identifying.

4. the abduction delivering of target protein

(1) will on the LB solid plate, (contain AMP100 μ g/mL, Cam34 μ g/mL) through the bacterium liquid that order-checking is accredited as the bacterium liquid that contains correct recombinant plasmid pET32a-Lfcin B Mel and contains empty carrier pET-32a line, 37 ℃ of incubated overnight;

(2) the single bacterium colony of picking is inoculated in the LB liquid nutrient medium of 3mL (containing AMP100 μ g/mL, Cam34 μ g/mL), and 37 ℃, 220rpm jolting overnight incubation;

(3) by 2% volumetric ratio the bacterium liquid of overnight incubation is transferred (contain AMP100 μ g/mL, Cam34 μ g/mL) in the LB of 10mL liquid culture, 37 ℃ of 250rpm joltings are cultivated about 3.5hr, make its OD ₆₀₀=0.5～0.8;

(4) add IPTG, making its final concentration is 0.5mmol/L, and the sampling of 1～5h after inducing 1mL is in the centrifuge tube of 1.5mL respectively then, and 4 ℃, behind the centrifugal 5min of 6000rpm ,-20 ℃ of preservations are standby.

SDS-PAGE electrophoresis detection T1249 abduction delivering result as shown in Figure 6.

Abduction delivering and the separation and purification of embodiment 2 heterozygous antibacterial peptides

1. the great expression of fusion rotein:

Picking inserts in the 50mLLB liquid nutrient medium (containing AMP100 μ g/mL) 37 ℃ of 220rpm jolting overnight incubation through being accredited as the positive E.coli Rosetta (DE3) that contains recombinant plasmid respectively; Volumetric ratio by 2% is transferred the bacterium liquid of overnight incubation and (contain AMP100 μ g/mL) in the LB of 1000mL liquid culture, and 37 ℃ of 250rpm joltings are cultivated about 3.5h, make its OD ₆₀₀=0.5～0.8; Add IPTG, making its final concentration is 0.5mmol/L, induce 5h after, 4 ℃, the centrifugal 10min of 8000rpm collects thalline ,-20 ℃ of preservations are standby.

2.Ni ²⁺Affinity chromatography preliminary purification fusion rotein

Adopt the Chelating of Pharmacia company

Fast Flow filler is with Ni ²⁺Be part.Ni ²⁺Chelating Sepharose affinity column size is that (2.6cm * 10cm), the entire operation process is kept the constant flow rate of maximum 6ml/min.

The Ni that seals up for safekeeping with the washed with de-ionized water ethanol of 5 times of column volumes ²⁺Chelating Sepharose affinity column is removed ethanol; 0.2M NiSO with 2 times of column volumes ₄Cross post, make the post material in conjunction with last Ni ²⁺After placing for some time, with the washed with de-ionized water of 10 times of column volumes, to remove free NiSO ₄With the 50mmol/L PB (pH 6.0) of 5 times of column volumes, 300mmol/L NaCl or 50mmol/LTris, 300mmol/L NaCl, pH 7.0 solution equilibrias; With the direct upper prop of the sample after centrifugal or will cross protein solution upper prop after Buffer is changed in the SephadexG-25 desalination; Wash post to ultraviolet with balanced solution and reach baseline; Collect the percolation peak and carry out the SDS-PAGE detection; Carry out the condition that linear gradient elution is determined stepwise elution with the imidazoles gradient, collect each protein elution peak, SDS-PAGE detects recombinant protein in the distribution situation of each elution peak.The preliminary purification result as shown in Figure 7.

3. the recombinant enterokinase enzyme of fusion rotein is cut and separation and purification:

At Ni ²⁺The fusion rotein of the preliminary purification that the imidazoles wash-out comes out in the Chelating Sepharose Fast Flow affinity chromatography, mistake

(using the enzyme cutting buffering liquid balance), exchange buffering liquid is enzyme cutting buffering liquid, the Enterokinase proteolytic enzyme that in enzyme cutting buffering liquid, slowly adds different volumes, soft mixing, enzyme is cut the different time under different temperature, cuts situation at the different time points enzyme that carries out Tris/Tricine SDS-PAGE electrophoresis detection fusion rotein of taking a sample respectively.

Before using, preserves SP Sepharose Fast Flow cationic exchange coloum the ethanol of pillar earlier with the washed with de-ionized water of 3～5 times of column volumes, use the 2M NaCl (pH10) of 3 times of column volumes to go up column regeneration then, use sample-loading buffer (50mmol/L PB, pH6.0) the balance cation exchange column of 5～10 times of column volumes then.Enzyme is cut the product upper prop, wash uncombined albumen to baseline with sample-loading buffer, with 50mmol/L PB (pH6.0), the 500mmol/LNaCl buffer solution elution, collect elution peak, elution curve as shown in Figure 8, Tris/Tricine SDS-PAGE detected result as shown in Figure 9, the molecular weight of the heterozygous antibacterial peptide that separation and purification obtains is 4.9KDa.

The vacuum lyophilization of recombinant protein, the freeze-drying system is: 50mmol/L NH ₄HCO ₃, protein solution is freezed fully under-80 ℃, vacuum lyophilization 4896 hours.

The anti-microbial activity of embodiment 3 heterozygous antibacterial peptides detects

1. the resistance activity of target protein is measured

Nutrient agar is heat fused in water-bath, is cooled to about 50 ℃, draws giving birth to of 60 μ L with aseptic method and surveys bacterium (OD ₆₀₀=0.3), add in the 20mL nutrient agar, rapid mixing, pouring into diameter is that thickness is about 1.5mm in the aseptic plane ware of 9cm, horizontal positioned is waited to solidify.Beat the circular hole that diameter is 2.7mm at agar, in the hole, add each 10 μ L of the fusion protein sample of purifying (Trx-BmA1, Trx-BmA2, Trx-BmB2) respectively, do negative control with sterilized water and each 10 μ L of 0.05M Spirit of Mindererus.Behind the application of sample plate is put into 4 ℃ of refrigerators, treat that sample fully is diffused into agar after, plate is inverted in 37 ℃ of overnight incubation, next day observations as shown in figure 10.Supplying the examination bacterium in this test is streptococcus aureus (Staphylococcus aureus).

2. the mensuration of the heterozygous antibacterial peptide minimal inhibitory concentration (MIC) of recombinating

MIC: test strain is cultured to the bacterium liquid dilution of logarithmic phase for about 5 * 10 ⁶CFU/mL adds in 96 well culture plates, and the every hole of test hole adds bacterium liquid 90uL, adds the antibacterial peptide solution of the different concns of doubling dilution then, the 10uL/ hole.Positive control is the bacterium liquid in 100uL/ hole, and negative control is corresponding 100uL substratum.Slowly shake in 37 ℃ then and cultivate about 16h, survey OD with microplate reader ₆₃₀The minimum concentration of bacteria growing inhibiting is MIC, the results are shown in Table 1.

The MIC of table 1 recombinant antibacterial peptide Lfcin B

3. hemolytic activity experiment

(the 35mmol/L phosphate buffered saline buffer contains 150mmol/LNaCl, is suspended in the PBS damping fluid after pH=7.4) washing 4 times, gets erythrocyte suspension (volume fraction 2.5%) with the PBS damping fluid with human red cell.Heterozygous antibacterial peptide is dissolved in the PBS damping fluid, be made into storing solution, get the polypeptide storing solution of different volumes in 0.5mL 2.5% erythrocyte suspension, add the PBS damping fluid to final volume 1mL, shake up, behind 37 ℃ of insulation 60min, with the centrifugal 10min of 4000r/min, get supernatant colorimetric under 414nm, being suspended in the PBS damping fluid with erythrocyte is blank, and being suspended among the TritonX 100 with erythrocyte is 100% haemolysis.After testing, the heterozygous antibacterial peptide of preparation does not have obvious hemolytic activity.

Claims

1. heterozygous antibacterial peptide LFB_Mel is characterized in that a kind of heterozygous antibacterial peptide gene Lfcin B_Mel Transformed E .coli Rosetta (DE3) and the engineering bacteria that makes.

2. heterozygous antibacterial peptide LFB_Mel according to claim 1 is characterized in that described carrier is prokaryotic expression carriers such as pET-32a.

3. heterozygous antibacterial peptide LFB_Mel according to claim 1 is characterized in that described gene is heterozygous antibacterial peptide gene Lfcin B_Mel, and its nucleotide sequence is as follows: TTT AAGTGTAGA AGA TGGCAATGG

AGAATGAAGAAGTTGGGTGCTCCATCTATTACTTGTGTTAGAAGAGCTTTCGGTTTGCCA GCT TTG ATT TCT TGG ATC AAG AGA AAG AGACAACAA。

4. according to the described gene coded protein LFB_Mel of claim 2, it is characterized in that its aminoacid sequence is: FKCRRWQWRMKKLGAPSITCVRRAFGLPALISWIKRKRQQ.

5. antibacterial peptide LFB_Mel goods according to claim 1 is characterized in that the preparation method comprises the steps:

(1) with antibacterial peptide gene Lfcin B_Mel through the complementary synthesis method synthetic gene sequence of PCR primer segmentation;

(2) antibacterial peptide gene Lfcin B_Mel is cloned into expression vector pET-32a, obtains recombinant expression vector pET32a-Lfcin B_Mel;

(3) recombinant expression vector pET32a-Lfcin B_Mel is transformed expressive host bacterium E.coli Rosetta (DE3);

(4) engineering bacteria is cultivated in a large number, adding IPTG is 0.5mM to final concentration, 28 ℃ of abduction deliverings 10 hours, separation and purification fusion rotein;

(5) with fusion rotein after the enteropeptidase enzyme is cut, separation and purification gets heterozygous antibacterial peptide LFB_Mel, detects its anti-microbial activity and MIC value.

6. antibacterial peptide LFB_Mel goods according to claim 5 is characterized in that anti-microbial activity and the antimicrobial spectrum of the antibacterial peptide narrated.