CN104448006A

CN104448006A - Hybrid antibacterial peptide CE-PR and application thereof

Info

Publication number: CN104448006A
Application number: CN201410771788.6A
Authority: CN
Inventors: 王宏华
Original assignee: QINGDAO HONGHAO BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: QINGDAO HONGHAO BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2014-12-13
Filing date: 2014-12-13
Publication date: 2015-03-25
Anticipated expiration: 2034-12-13
Also published as: CN104448006B

Abstract

The invention provides a novel hybrid antibacterial peptide CE-PR as well as a preparation method and application of the novel hybrid antibacterial peptide CE-PR. The preparation method comprises the steps of splicing 19 amino acids at the N end of an antibacterial peptide cecropin P1 and 26 amino acids at the N end of an antibacterial peptide PR-39 on the basis of spatial structural analysis on amino acid sequences of swine-derived antibacterial peptides PR-39 and cecropin P1; adding protein Lingker (GPG) in the intermediate; and carrying out mutation (K12N) on an amino acid at the position 1 in the N end of the antibacterial peptide cecropin P1 to obtain the novel hybrid antibacterial peptide CE-PR with a remarkably-improved antibacterial effect. On the basis, a pichia pastoris optimal codon is selected to artificially synthesize a novel hybrid antibacterial peptide CE-PR gene which is cloned into pichia pastoris to express to obtain a novel antibacterial peptide recombinant yeast strain, and the fermentation scale is amplified to the level of a fermentation tank, so that the high-density fermentation and efficient expression of an antibacterial peptide product are realized. After being further purified, a fermentation solution can be prepared into powder, liquid and other antibacterial peptide preparations for preventing and treating livestock and poultry diseases.

Description

A kind of heterozygous antibacterial peptide CE-PR and application thereof

Technical field

The invention belongs to polypeptide triage techniques field, be specifically related to a kind of heterozygous antibacterial peptide and application thereof.

Background technology

Antibacterial peptide (antibacterial peptides) is a kind of polypeptide with strong anti-microbial effect that organism produces, and is the important composition composition of biological congenital immunity.Up to now, discovery more than 600 kind of endogenous antibacterial active peptide in animal, plant and prokaryotic organism has been comprised at many biologies.Pig derived antimicrobial peptide PR-39 (GenBank accession number: NM_214450.1) is made up of 39 amino acid, is that Agerberth B equals first to obtain from the separation of chitling road for 1991.It is rich in arginine (Arg) and proline(Pro) (Pro) two seed amino acid residue, forms a kind of Pro-Arg-Pro structure, may be relevant with bacterial phospholipid membrane interaction.Its N holds 26 amino acid whose synthetic peptides relative to stronger to the restraining effect of multiple Gram-negative bacteria, positive bacteria antibacterial peptide PR-39 itself.Antibacterial peptide cecropin P1 isolates from chitterlings and obtains, it contains 31 amino-acid residues, molecular weight is 3339Da, not containing halfcystine, intramolecular disulfide bond can not be formed, have the C end of alkaline N end and strong-hydrophobicity, wherein amidated C end is very important to its broad-spectrum antibacterial action.Cecropin P1 aminoacid sequence and insect cecropin A have 64% similarity, have the similarity of 75% with cecropin B.Show the theoretical prediction of its secondary structure and CD spectrum and two dimensional NMR data, containing amphiphilic water-based alpha-helix and hydrophobicity alpha-helix in its molecule, centre is glutamic-glycine (Glu-Gly) the curling sequence forming flexible bending.Research shows, its helix-coil-spirane structure has special importance to maintenance high antibacterial activity.

Various shortcoming is there is in natural antibacterial peptide in clinical application, have to pathogenic micro-organism high kill activity while often along with hemolytic action, have inherently to eukaryotic cell toxic (such as natural antibacterial peptide Magainin, alexin and melittin etc.).Along with going deep into of studying antibacterial peptide structure function and sterilization mechanism, people start to attempt the novel heterozygous antibacterial peptide that design disinfection vitality is stronger, antimicrobial spectrum is wider.The method that heterozygous antibacterial peptide design usually adopts is spliced by different sources antibacterial peptide conserved sequence, then modified by biology such as amino acid replacements, and novel antimicrobial peptide that hemolytic low high to anti-microbial activity.At present, design and synthesis many kinds of T1249s both at home and abroad, fungistatic effect is obviously better than native peptides, and greatly reduce toxicity and hemolytic.In a word, the successful design of T1249 and application are to solution current antibacterial peptide Problems existing important in inhibiting and practical value

Summary of the invention

The object of this invention is to provide a kind of heterozygous antibacterial peptide CE-PR, thus make up the deficiencies in the prior art.

First the present invention provides a kind of heterozygous antibacterial peptide CE-PR, and the aminoacid sequence of its proteins encoded is SEQ ID NO:1;

Heterozygous antibacterial peptide CE-PR of the present invention can be used as the prevention and therapy preparation of additive for farm animal feed or livestock and poultry.

The present invention also provides the preparation method of the recombinant yeast pichia pastoris of a kind of heterozygous antibacterial peptide CE-PR, and its preparation process is as follows:

1) according to the aminoacid sequence of heterozygous antibacterial peptide CE-PR, select pichia spp preferred codons, synthetic antibacterial peptide gene order, be connected in recombinant yeast expression vector, be built into recombinant expression;

2) the recombinant expression electricity built is transformed host yeast, build the recombination Engineering Yeast bacterium expressing heterozygous antibacterial peptide CE-PR; Heterozygous antibacterial peptide CE-PR is given expression to this recombination engineering bacteria high density fermentation;

3) to after recombinant expressed heterozygous antibacterial peptide CE-PR further concentrated, purifying, mensuration is tired.Dilution packing, is antimicrobial peptide preparation.

The present invention is carrying out on the basis of space Structure Analysis to the aminoacid sequence of pig derived antimicrobial peptide PR-39 and cecropin P1, the N of 19 amino acid and antibacterial peptide PR-39 is held by the N of antibacterial peptide cecropin P1 to hold 26 amino acid to splice, centre adds albumen Lingker (GPG), and (K12N) is suddenlyd change to 1 place's amino acid in the N end of antibacterial peptide cecropin P1, obtain a kind of novel heterozygous antibacterial peptide CE-PR, its antibacterial efficacy is obtained and significantly improves.

Embodiment

The present invention is further described below in conjunction with embodiment; but what those skilled in the art should understand that is; can modify to the details of technical scheme of the present invention and form when not departing from technical scheme of the present invention or replace, these amendments and replacement all fall in scope.

The acquisition of embodiment 1 heterozygous antibacterial peptide CE-PR and gene thereof

1, the aminoacid sequence of biosoftware to pig derived antimicrobial peptide cecropin P1 (GenBank accession number: AB186032) and antibacterial peptide PR-39 (GenBank accession number: NM_214450.1) is utilized to carry out space Structure Analysis, the N end choosing antibacterial peptide cecropin P1 has based on 19 amino acid of α-helixstructure, and its 12nd hydrophobic amino acid is replaced to hydrophilic amino acid Methionin (K12N), then 26 amino acid are held to splice N stronger to itself and antibacterial peptide PR-39 bacteriostatic activity, centre adds albumen Lingker (GPG), obtain a kind of novel heterozygous antibacterial peptide CE-PR, its aminoacid sequence is SEQ ID NO:1.

2, according to the aminoacid sequence SEQ ID NO:1 of the novel heterozygous antibacterial peptide CE-PR obtained, redesign according to pichia spp Characteristic of Codon Usage, obtain the nucleotide sequence of encoding novel heterozygous antibacterial peptide CE-PR.And hold introducing Kex2 restriction enzyme site at its N.XhoI and XbaI enzyme cutting site are introduced in two ends, so that be cloned in yeast expression vector.

The structure of embodiment 2 genetically engineered heterozygous antibacterial peptide CE-PR expression vector and the acquisition of engineering bacteria

1, the carrier and Yeast expression carrier that contain antibacterial peptide gene are all used XhoI and XbaI double digestion, digestion products reclaims and connects, and carries out PCR qualification, order-checking.

2, positive plasmid adds in pichia spp competent cell suspension after the linearizing of SacI single endonuclease digestion.The YPDS that electricity is spread evenly across after transforming containing 100 μ g/mL Zeocin selects, on flat board, to hatch 3-5 days for 30 DEG C.Treat that the positive transformant growth on YPDS flat board is larger, each transformant successively dibbling is selected dull and stereotyped to the YPDS containing Zeocin 200 μ g/mL, 500 μ g/mL, 1000 μ g/mL, with the bacterium colony of normal growth on high density Zeocin flat board for possibility high copy recombinant bacterial strain.

3, by the positive recombinant bacterium list colony inoculation that screens in containing in the YPD nutrient solution of 100 μ g/mL Zeocin, 28 DEG C of joltings cultivate 18 hours.Getting this bacterium liquid transfers in 5ml BMGY substratum by 4% volume ratio, and 28 DEG C of shakes are cultivated about 18-24h, OD600 value and are about 5-6.Culture is directly transferred in 25ml BMMY substratum, and 28 DEG C are continued shake and cultivate.In order to maintain abduction delivering, adding 100% methyl alcohol every 24h and making final concentration reach 1%.After 48h, 4 DEG C of centrifugal 10min of 5000r/min, collect supernatant, carry out Antibacterial Activity.The restructuring yeast strains that can produce bacteriostatic activity is the positive strain that can produce novel heterozygous antibacterial peptide CE-PR.

Embodiment 3 is fermented, the preparation of the novel heterozygous antibacterial peptide CE-PR of purifying

1, zymotechnique

1) triangular flask is inoculated in screening after the positive recombinant obtained activates by 1%-10% inoculum size, 28-30 DEG C, 200r/min shaking table accesses 10L fermentor tank (actual load substratum 6L) with 5%-20% inoculum size after cultivating 16-24h, temperature 28-30 DEG C, rotating speed 500-1500r/min, Medium's PH Value 5.0-6.0, air flow 0.1-1.0VVM (amount of oxygen that 1L fermented liquid 1min passes into), ferment in dissolved oxygen >20% situation, after cultivation 18-24h, stream adds 50% glycerine 4h, when dissolved oxygen rises to suddenly 100%, stream adds methyl alcohol to fermentation ends, whole fermentation lasts 48-72h.

2) former tank steam 100 DEG C of sterilizing 10-20min after fermentation ends, blowing, the centrifugal 10min of 5000r/min, collects fermentation supernatant and is antibacterial peptide work in-process.

3) novel antibacterial peptide formulations

Liquid preparation is obtained after the pulvis that antibacterial peptide work in-process are produced through micro-filtration, ultrafiltration, spraying dry, freeze-drying etc. and, purifying refining with biochemical method.

Said gene engineering antibacterial peptide can make the prevention and therapy preparation of additive for farm animal feed or livestock and poultry.

The minimal inhibitory concentration of the novel heterozygous antibacterial peptide CE-PR of embodiment 4 measures (MIC)

1, test strain

Streptococcus aureus (Cowan I), pig Staphylococcus aureus enterotoxin 75-1, e. coli k12 D31, swine escherichia coli O78, swine escherichia coli O157-H71 and swine paratyphoid Salmonellas C782

2, bacterial strain process: bacterial classification is recovered, rules in solid LB media, 37 DEG C of incubated overnight in incubator.The bacterium of incubated overnight is chosen single bacterium colony in the triangular flask containing 25ml liquid MHB substratum, triangular flask is put in shaking table 37 DEG C and cultivates 12-18h.Bacterium liquid after cultivating is surveyed its absorbance under the condition of OD600, regulates bacterial concentration with MHB substratum, make it be between 0.6-0.8.Bacterium liquid being diluted to concentration is afterwards 5 × 10 ⁵cFU/mL, gets 90 μ l successively and joins in each hole of 96 orifice plates.

3, antibacterial peptide quantitatively takes turns doing doubling dilution with MHB substratum afterwards.The antibacterial peptide diluted respectively being got 10 μ l joins in each hole of 96 orifice plates successively, and now the reaction system in each hole is 100 μ l.After 96 orifice plate lid lids, after 37 DEG C of shaking culture 24h, observe and use microplate reader measure at 600nm place and record experimental result.Antibacterial peptide sample, after doubling dilution, becomes a series of concentration, defines minimal inhibitory concentration (MIC) with the Cmin of bacteria growing inhibiting.Utilize bacterium liquid and MHB substratum to be used for respectively doing feminine gender and positive control, representing bacteriostasis rate is separately 0 and 100%.Set up pig derived antimicrobial peptide PR-39 and cecropin P1 standard substance test group respectively in contrast, for observing the bacteriostatic activity change before and after antibacterial peptide transformation simultaneously.

4, recombined new heterozygous antibacterial peptide CE-PR is measured to the minimum inhibitory concentration of various bacteria with micro-dilution method, result shows that novel heterozygous antibacterial peptide CE-PR is compared with pig derived antimicrobial peptide PR-39, cecropin P1 standard substance, it increases the bacteriostasis of antibacterial peptide to Gram-negative bacteria and positive bacteria greatly, has more market using value.

Table 1: antibacterial peptide is to the minimum inhibitory concentration of various bacteria

The minimum hemolytic concentration of embodiment 5 novel heterozygous antibacterial peptide CE-PR measures (MHC)

The hemolytic activity of antibacterial peptide to erythrocyte weighs it to the main method of eukaryotic cell toxicity.Namely this test objective is whether the novel heterozygous antibacterial peptide CE-PR of checking has cytotoxicity.

1, the 8% swine erythrocyte 100 μ l be resuspended in PBS is added in 96 orifice plates, add the antibacterial peptide 100 μ l of PBS serial dilution again, make the concentration of antibacterial peptide in each hole be respectively 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.12 μ g/mL, 1.56 μ g/mL and 0.78 μ g/mL.Positive control wells adds 100 μ l 0.2%Triton X-100, negative control hole adds 100 μ lPBS, 37 DEG C hatch 1h after, after the centrifugal 5min of 3000rpm, 100 μ l supernatants are drawn to another 96 orifice plate from each hole, 550nm wavelength measures OD value, calculates percent hemolysis=[(experimental port OD value-negative hole OD value)/(positive hole OD value-negative hole OD value)] × 100.Set up pig derived antimicrobial peptide PR-39 and cecropin P1 standard substance in contrast simultaneously.

2, result shows: novel heterozygous antibacterial peptide CE-PR is the same with cecropin P1 standard substance with pig derived antimicrobial peptide PR-39, to swine erythrocyte substantially without hemolytic activity, is a kind of safe antibacterial peptide.

Detected by antibacterial peptide synthetic of the present invention, effect and recombinant expressed recombinant polypeptide are consistent.Above-mentioned result shows that the novel heterozygous antibacterial peptide CE-PR that the present invention obtains has better effect, can be used in business development.

Claims

1. a heterozygous antibacterial peptide CE-PR, is characterized in that, the aminoacid sequence of described heterozygous antibacterial peptide CE-PR is SEQ ID NO:1.

2. the preparation method of heterozygous antibacterial peptide CE-PR according to claim 1, is characterized in that, described method comprises the steps:

1) according to the aminoacid sequence of novel heterozygous antibacterial peptide CE-PR, select pichia spp preferred codons, synthetic antibacterial peptide gene order, and hold introducing Kex2 restriction enzyme site at its N, XhoI and XbaI enzyme cutting site are introduced in two ends, so that be cloned in yeast expression vector;

2) carrier and expression vector that contain antibacterial peptide gene are all used XhoI and XbaI double digestion, digestion products reclaims and connects, and carries out PCR qualification, order-checking;

3) positive plasmid adds in pichia spp competent cell suspension after the linearizing of SacI single endonuclease digestion, and the YPDS that electricity is spread evenly across after transforming containing 100 μ g/mL Zeocin selects, on flat board, to hatch 3-5 days for 30 DEG C; Treat that the positive transformant growth on YPDS flat board is larger, each transformant successively dibbling is selected dull and stereotyped to the YPDS containing Zeocin200 μ g/mL, 500 μ g/mL, 1000 μ g/mL, with the bacterium colony of normal growth on high density Zeocin flat board for possibility high copy recombinant bacterial strain;

4) by the positive recombinant bacterium list colony inoculation that screens in containing in the YPD nutrient solution of 100 μ g/mL Zeocin, 28 DEG C of joltings cultivate 18 hours; Getting this bacterium liquid transfers in 5ml BMGY substratum by 4% volume ratio, and 28 DEG C of shakes are cultivated about 18-24h, OD600 value and are about 5-6; Culture is directly transferred in 25mlBMMY substratum, and 28 DEG C are continued shake and cultivate; In order to maintain abduction delivering, adding 100% methyl alcohol every 24h and making final concentration reach 1%; After 48h, 4 DEG C of centrifugal 10min of 5000r/min, collect supernatant, carry out Antibacterial Activity; The restructuring yeast strains that can produce bacteriostatic activity is the positive strain that can produce novel heterozygous antibacterial peptide CE-PR;

5) zymotechnique of restructuring yeast strains: be inoculated in triangular flask by 1%-10% inoculum size by screening after the positive recombinant obtained activates, 28-30 DEG C, 200r/min shaking table accesses fermentor tank with 5%-20% inoculum size after cultivating 16-24h, at 28-30 DEG C, 500-1500r/min, pH value 5.0-6.0, air flow 0.1-1.0VVM, ferment in dissolved oxygen >20% situation, after cultivation 18-24h, stream adds 50% glycerine 4h, when dissolved oxygen rises to suddenly 100%, stream adds methyl alcohol to fermentation ends, whole fermentation lasts 48-72h;

Former tank steam 100 DEG C of sterilizing 10-20min after fermentation ends, blowing, the centrifugal 10min of 5000r/min, collects fermentation supernatant and is antibacterial peptide work in-process.

3. heterozygous antibacterial peptide CE-PR according to claim 1 is preparing the application in additive for farm animal feed or immunostimulant.