CN102127549B - Chicken antimicrobial peptides Cathelicidins and preparation method and applications thereof - Google Patents

Chicken antimicrobial peptides Cathelicidins and preparation method and applications thereof Download PDF

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CN102127549B
CN102127549B CN 201010600240 CN201010600240A CN102127549B CN 102127549 B CN102127549 B CN 102127549B CN 201010600240 CN201010600240 CN 201010600240 CN 201010600240 A CN201010600240 A CN 201010600240A CN 102127549 B CN102127549 B CN 102127549B
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antimicrobial peptides
chicken
thalline
antibacterial peptide
pet30
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CN102127549A (en
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吴静
宋敏训
李玉峰
马秀丽
姜亦飞
黄兵
林树乾
于可响
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Poultry Research Institute Shandong Academy of Agricultural Sciences
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Abstract

The invention discloses children antimicrobial peptides cathelicidins, a preparation method and the applications thereof. The nucleotide sequence and the amino acid sequence of children antimicrobial peptides Cathelicidin1, Cathelicidin2 and Cathelicidin3 are shown as a sequence table. The preparation method of the antimicrobial peptides adopts a solid-phase chemical synthesis method or a genetic engineering method. The genetic engineering method comprises the following steps of: respectively cloning the encoding genes of mature children antimicrobial peptides Cathelicidin1-3 to expression vectors; and then, guiding into host cells to express. By applying the genetic engineering technology in the invention, the children antimicrobial peptides Cathelicidins are expressed in the prokaryotic host cells with high efficiency. Proved by experiments, the antimicrobial peptides have obvious inhibition on various gram-positive bacteria and negative bacteria.

Description

One group of chicken Cathelicidins antibacterial peptide and its preparation method and application
Technical field
The present invention relates to biological technical field, relate in particular to one group of chicken Cathelicidins antibacterial peptide and its preparation method and application.
Background technology
In recent years, multiple communicable disease is perplexing the aquaculture of China, brought into play vital role aspect pre-bacteriological protection infection, the control livestock and poultry since the microbiotic self-discovery, huge pushing effect has been played in the fast development that promotes livestock industry to produce, but long-term a large amount of antibiotics that uses is so that pathogenic bacteria progressively produces resistance, for diseases prevention and treatment work has increased difficulty.Simultaneously, the drug residue that microbiotic causes so that the safety issue of animal food become increasingly conspicuous, R-plasmid intersects to disseminate easily passes to the mankind, bring threat for prevention and the treatment of human health and disease, for this reason, international community proposes to reduce microbiotic growth stimulant (AGP) usage quantity or cancel some antibiotic use.Since in January, 2006, European Union completely forbids food animal and uses the antibiotics feed additive for promoting growth, and in establishment prevention in March antibiotic resistant expansion research network, the developed countries such as Japan and Korea S have also set up corresponding regulations subsequently, and the International Olympic Committee also requires Beijing to meet the extremely low safe meat product of antibiotic content that European Union requires for 2008 Olympic Games provides.Seek Substitutes For Antibiotic, development is prevention and control livestock and poultry, promotion growth of animal effectively, have no side effect again, noresidue, be difficult for to produce chemical sproof green veterinary drug and fodder additives, become the key subjects that face in the fields such as Animal nutrition, food-processing, new veterinary drug research and development.
Antibacterial peptide (antimicrobial peptide) is under inductive condition, and the class that the animal immune system of defense produces is resisted the defensive peptide class active substance of exogenous pathogenic agent pathogenic effects, is an important component part of host immune system of defense.Antibacterial peptide has efficiently, the anti-microbial activity of wide spectrum, to Gram-negative bacteria (G -), gram-positive microorganism (G +), fungi, protobiont, tunicate virus and tumour cell all show stronger activity (Jenssen H, et al.Peptide AntimicrobialAgents.Clin Microbiol Rev, 2006,19 (3): 491-511).And most antibacterial peptides are harmless to the Mammals normal cell, have and be different from the antibiotic unique antibacterial mechanisms of tradition, and to body have no side effect, noresidue, this will provide new way to this thorny global difficult problem of resistance that traditional microbiotic such as penicillin strengthen day by day for solving bacterium, and application prospect is very wide.Separate from comprising in the multiple organisms such as animal, plant, insect, prokaryotic organism so far, identified over thousands of kind of antibacterial active peptide, have tens kinds of antibacterial peptides to enter clinical trial or preclinical study stage.
In poultry body, find at present the three major types antibacterial peptide, namely expressed antibacterial peptide 2 (LEAP-2) in Defensins (Gallinacin), Cathelicidin class and the liver.
From present correlative study result, chicken β-defensin all has bacteriostatic action (Harwig S S external to pasteurella multocida and intestinal bacteria, Candida albicans, Salmonella enteritidis, campylobacter jejuni etc., et al.Gallinacins:cysteine-richantimicrobial peptides of chicken leukocytes.FEBS Lett, 1994,342 (3): 281-285; Evans E W, et al.Antimicrobial activity of chicken and turkey heterophil peptides CHP1, CHP2, THP1, and THP3.Vet Microbiol, 1995,47 (3-4): 295-303).
Cathelicidins class antibacterial peptide is to be found at first the changeable antibacterial peptide of a mammiferous class formation, because of its Cathelin peptide section that contains a high conservative between signal peptide and mature peptide family of having a style of one's own.Cathelicidins has the anti-Gram-negative bacteria of strong wide spectrum and a gram-positive bacteria activity external, comprises the antibiotics resistance bacterium, and what have still has fungicidal activity under 50% serum and physiological salt concentration.The same with alexin, the Cathelicidins antibacterial peptide also is the important component part of host defense system, but their usually more salt tolerant and solvents.Except main kill bacteria, fungi and virus activity, Cathelicidins also has chemotaxis to immunocyte, suppresses tissue injury, promotes trauma repair, in conjunction with various biological function (Zaiou M such as intracellular toxin, induction of vascular generations, et al.Cathelicidins, essential gene-encoded mammalian antibiotics.J Mol Med, 2002,80 (9): 549-61; Ramanathan B, et al.Cathelicidins:microbicidal activity, mechanisms ofaction, and roles in innate immunity.Microbes Infect, 2002,4 (3): 361-372; Carretero M, et al, Invitro and in vivo wound healing-promoting activities of human cathelicidin LL-37.J InvestDermatol, 2008,128 (1): 223-236.).Owing to have so widely biologic activity and function, the Cathelicidinds antibacterial peptide has become the study hotspot in immunology, molecular biology and the biomedicine field, and its medicinal DEVELOPMENT PROSPECT is very wide.
Yet the content of natural antibacterial peptide is extremely low in the poultry body, and molecular weight is little, and the separation and purification difficulty can't be satisfied the demand.Chemosynthesis and gene engineering method just become the Main Means that obtains antibacterial peptide, but chemosynthesis antibacterial peptide cost is too high, also is difficult to be applied to clinical; Therefore, just become an effective approach by engineered method great expression antibacterial peptide in specific host.But because antibacterial peptide itself has certain lethal effect to the prokaryotic hosts bacterium, be not suitable in prokaryotic system, directly expressing, usually adopt the modes such as amalgamation and expression or eukaryotic expression.Intestinal bacteria are clear because of its genetic background, be easy to cultivate, the cycle shortly, with low cost enjoys favor, become one of exogenous gene expression host who is most widely used at present, multiple antimicrobial polypeptide is by fusion form successful expression in escherichia expression system, and product can produce anti-microbial activity after treatment.
Summary of the invention
The object of the present invention is to provide one group of chicken antimicrobial peptides Cathelicidin1 with very strong antimicrobial acivity, 2,3, the present invention utilizes genetic engineering technique to clone Cathelicidins antibacterial peptide gene (CathL-1 from the myeloid tissue of white Leghorn,-2,-3) cDNA fragment, and its mature polypeptide coding sequence is cloned in the fusion expression vectors such as pET-30a (+), recombinant expressed fusion rotein and it is carried out preliminary evaluation in E.coliRosetta (DE3) expression strain, expression product exists with soluble form, do not need the proteolytic enzyme cutting, can prepare one group of chicken recombinant antibacterial peptide Cathelicidin1 with very strong antimicrobial acivity through the affinity chromatography method, 2,3.
The invention also discloses the dna sequence dna of the aminoacid sequence of this group chicken Cathelicidins antibacterial peptide and the described chicken Cathelicidins antibacterial peptide of encoding.
The present invention provides gene engineering preparation method and the solid state chemistry synthesis method of this group chicken Cathelicidins antibacterial peptide simultaneously.
The present invention also further discloses the purposes of this group chicken Cathelicidins recombinant antibacterial peptide in preparation treatment chicken bacterium infection medicine.
In order to realize purpose of the present invention, the invention provides following technical scheme: one group of Cathelicidins antibacterial peptide provided by the invention, it is the antibacterial peptide of white Leghorn genes encoding, belong to poultry Cathelicidins antibacterial peptide family, the a series of sequences of they this are named as Cathelicidin 1,2,3, the aminoacid sequence of its precursor protein (from aminoterminal to carboxyl terminal) is as follows: Cathelicidin1 (SEQ ID NO.5):
Met Leu Ser Cys Trp Val Leu Leu Leu Ala Leu Leu Gly Gly Ala Cys Ala Leu Pro Ala Pro Leu GlyTyr Ser Gln Ala Leu Ala Gln Ala Val Asp Ser Tyr Asn Gln Arg Pro Glu Val Gln Asn Ala Phe ArgLeu Leu Ser Ala Asp Pro Glu Pro Gly Pro Asn Val Gln Leu Ser Ser Leu His Asn Leu Asn Phe ThrIle Met Glu Thr Arg Cys Gln Ala Arg Ser Gly Ala Gln Leu Asp Ser Cys Glu Phe Lys Glu Asp GlyLeu Val Lys Asp Cys Ala Ala Pro Val Val Leu Gln Gly Gly Arg Ala Val Leu Asp Val Thr Cys ValAsp Ser Met Ala Asp Pro Val Arg Val Lys Arg Val Trp Pro Leu Val Ile Arg Thr Val Ile Ala Gly TyrAsn Leu Tyr Arg Ala Ile Lys Lys Lys
Cathelicidin 2(SEQ ID NO.10)：
Met Leu Ser Cys Trp Val Leu Leu Leu Ala Leu Leu Gly Gly Val Cys Ala Leu Pro Ala Pro Leu SerTyr Pro Gln Ala Leu Ile Gln Ala Val Asp Ser Tyr Asn Gln Arg Pro Glu Val Gln Asn Ala Phe ArgLeu Leu Ser Ala Asp Pro Glu Pro Gly Pro Gly Val Asp Leu Ser Thr Leu Arg Ala Leu Asn Phe ThrIle Met Glu Thr Glu Cys Thr Pro Ser Ala Arg Leu Pro Val Asp Asp Cys Asp Phe Lys Glu Asn GlyVal Ile Arg Asp Cys Ser Gly Pro Val Ser Val Leu Gln Asp Thr Pro Glu Ile Asn Leu Arg Cys Arg AspAla Ser Ser Asp Pro Val Leu Val Gln Arg Gly Arg Phe Gly Arg Phe Leu Arg Lys Ile Arg Arg PheArg Pro Lys Val Thr Ile Thr Ile Gln Gly SerAlaArg Phe Gly
Cathelicidin 3(SEQ ID NO.15)：
Met Leu Ser Cys Trp Val Leu Leu Leu Ala Leu Leu Gly Gly Ala Cys Ala Leu Pro Ala Pro Leu GlyTyr Ser Gln Ala Leu Ala Gln Ala Val Asp Ser Tyr Asn Gln Arg Pro Glu Val Gln Asn Ala Phe ArgLeu Leu Ser Ala Asp Pro Glu Pro Gly Pro Asn Val Gln Leu Ser Ser Leu His Asn Leu Asn Phe ThrIle Met Glu Thr Arg Cys Gln Ala Arg Ser Gly Ala Gln Leu Asp Ser Cys Glu Phe Lys Glu Asp GlyLeu Val Lys Asp Cys Ala Ala Pro Val Val Leu Gln Gly Gly Arg Ala Val Leu Asp Val Thr Cys ValAsp Ser Met Ala Asp Pro Val Arg Val Lys Arg Phe Trp Pro Leu Val Pro Val Ala Ile Asn Thr ValAla Ala Gly Ile Asn Leu Tyr Lys Ala Ile Arg Arg Lys
Chicken antimicrobial peptides Cathelicidin1, Cathelicidin2 and Cathelicidin3, its nucleotide sequence are respectively as described in SEQ ID NO.1, SEQ ID NO.6 and the SEQ ID NO.11.
One group of chicken Cathelicidins antibacterial peptide of the present invention, its encoding gene cathelicidin 1,2,3, come from: in Adult White Leghorn myeloid tissue, extract total RNA, behind synthetic cDNA the first chain of reverse transcription, take cDNA as template, carry out PCR with 3 couples of gene-specific primer CathL1 P1/P2, CathL2 P1/P2 and CathL3 P1/P2 respectively again, amplified production is cathelicidin1,2,3cDNA complete sequence.The PCR product separates and recovery purpose fragment (seeing Fig. 1) through 2% agarose gel electrophoresis, the purpose fragment is connected with cloning vector pMD18-T and Transformed E .coli DH5 α competent cell, positive strain checks order with M13 F/R primer, obtain containing cathelicidin1,2, the complete nucleotide sequence of 3 open reading frame (ORF), its ORF sequence total length is respectively 447bp, 465bp, 456bp, and sequence to 3 ' end sees sequence table (SEQ ID NO.1, SEQ ID NO.6 and SEQ ID NO.11) for details from 5 ' end for it.
One group of chicken Cathelicidins antibacterial peptide provided by the invention, its preparation method can be the solid state chemistry synthesis method, also the encoding gene of antibacterial peptide can be cloned on the expression vector, then imports and expresses rear the acquisition in the specific host cell.Wherein expression vector can be a kind of in plasmid or the virus, and host cell can be prokaryotic cell prokaryocyte, comprises intestinal bacteria, subtilis etc., and host cell can be eukaryotic cell also, comprise yeast cell, insect cell and mammalian cell etc.The antibacterial peptide of preparation can be identified by mass spectroscopy.
The invention provides a kind of chicken antimicrobial peptides Cathelicidin1,2,3 efficient gene engineering preparation methods, mainly realize by following steps:
(1) encoding gene of chicken antimicrobial peptides Cathelicidin1, chicken antimicrobial peptides Cathelicidin2 and chicken antimicrobial peptides Cathelicidin3 mature peptide is cloned into respectively among the intestinal bacteria fusion expression vector pET30a (+), obtains fusion expression vector pET30-CathL1S, pET30-CathL2S, pET30-CathL3S;
(2) pET30-CathL1S, pET30-CathL2S, pET30-CathL3S plasmid are transformed respectively intestinal bacteria Rosetta (DE3) bacterial strain, obtain the recombination bacillus coli engineering strain;
Under (3) 37 ℃, in the LB substratum, inductor IPTG concentration is under the condition of 1.0mmol/L, and above-mentioned recombination bacillus coli genetic engineering bacterium was carried out abduction delivering 3 hours;
(4) the above-mentioned expression thalline of centrifugal collection, ultrasonic disruption, isolated cell soluble constituent and inclusion body;
(5) Ni 2+-NTA Sepharose 4B affinity column purified fusion protein.
Its concrete steps are:
(1) adopts RT-PCR method amplification coding chicken antimicrobial peptides Cathelicidin1 from white Leghorn myeloid tissue, 2, the nucleotide sequence of 3 precursor proteins (cDNA sequence), described gene is cloned into respectively in the pMD-18T carrier, transform e.colistraindh5α, and carry out sequencing;
(2) PCR method amplification coding chicken antimicrobial peptides Cathelicidin1,2, the nucleotide sequence of 3 mature peptides, the PCR product is connected through pET-30a (+) (or pET-32a (+)) fusion expression vector that EcoR I is connected with Xho I double digestion with same enzyme is cut, transform the bacillus coli DH 5 alpha competent cell, extract in a small amount plasmid, use the method screening positive clone of PCR and plasmid enzyme restriction, by the exactness of dna sequencing checking Insert Fragment reading frame, thereby construct prokaryotic expression carrier.With fusion expression vector difference called after pET30-CathL1S, pET30-CathL2S, the pET30-CathL3S that makes up;
Recombinant expression plasmid pET30-CathL1S, the pET30-CathL2S that (3) will check order correct, pET30-CathL3S transform respectively intestinal bacteria Rosetta (DE3) bacterial strain, and the picking positive strain is seeded to 37 ℃ of cultivation 8～12h in the LB substratum.Next day, the centrifuging and taking thalline was resuspended to fresh LB substratum, treated nutrient solution OD 600nmValue reaches at 0.5～0.6 o'clock and add IPTG, and to make its final concentration be 1.0mmol/L, begins to induce; 37 ℃ were continued to cultivate 4h, every 1 hour sampling 1mL.Thalline behind the centrifugal collection inducing culture washs thalline 2 times with the PBS damping fluid, adds lysis buffer (50mmol/L Tris-HCl again, pH 8.0,50mmol/L NaCl, 1mmol/L EDTA, 5% glycerine, add before use proteinase inhibitor PMSF to final concentration be 1mmol/L), ultrasonication thalline in the ice bath, each broken 5s, interval 2s, power 600W, broken 10min.Ultrasonic rear 12000r/min, 4 ℃ of lower centrifugal 10min, separate soluble constituent with and inclusion body, carry out SDS-PAGE electrophoresis and Western Blot and detect.Concentrated gum concentration is 5%, and resolving gel concentration is 15%.With pET-30a (+) plasmid by same method transform, abduction delivering, get 3h abduction delivering product (supernatant) in contrast.Western Blot operation is carried out with reference to the method in " molecular cloning experiment guide ".Behind the electrophoresis with the band electrotransfer in the SDS-PAGE gel to nitrocellulose filter, take mouse-anti His monoclonal antibody as primary antibodie, sheep anti-mouse igg-HRP is two anti-, DAB is that chromogenic substrate carries out Western-blot and analyzes, and identifies expression product.
(4) affinitive layer purification of expressing fusion protein condition optimizing and expression product
By adopting height to ooze the methods such as concentration that inductor IPTG was induced, regulated to substratum (being the LB/SB substratum), low temperature (25 ℃ or 15～20 ℃) for a long time, suppress inclusion body and form, improve soluble proteins proportion in the expression product.Evidence is inducing culture 8 hours under the condition of 1.0mmol/L at 25 ℃, IPTG concentration, and the shared ratio of solubility target protein is beneficial to the downstream purification operation than obviously improving under 37 ℃ of conditions.
Carry out the abduction delivering of enlarged culturing and target protein by the condition after the above-mentioned optimization, get the thalline after inducing, add an amount of binding buffer liquid, ultrasonic disruption thalline in the ice bath, isolation of occlusion bodies and soluble constituent are splined on immobilization metal part affinity column chromatography post (Ni with the cracking supernatant liquor 2+-NTA Sepharose 4B post), sample is collected the fusion rotein elution peak and is carried out the SDS-PAGE detection through containing the buffer solution for gradient elution of 0.1～0.5mol/L imidazoles.The result shows that target protein presents single band in the elutriant in the target location, shows through affinity chromatography to obtain the higher fusion rotein of purity, for the acquisition of maturation products creates conditions.
(5) adopt standard agar hole diffusion process (AWDA) to detect the In Vitro Bacteriostatic to indicator strain of recombination chicken antibacterial peptide Cathelicidin 1,2,3.
One group of new chicken antimicrobial peptides Cathelicidin1 of the present invention, 2,3, also can adopt easily the solid state chemistry synthetic method to be prepared.Derive the aminoacid sequence (Seq-1) of its precursor protein from chicken Cathelicidin1 antibacterial peptide nucleotide sequence, according to the understanding of at present the ripe peptase of chicken Cathelicidins class antibacterial peptide being cut Processing position (elastoser cleavage site), be that elastoser generally is that cutting processing is carried out in first α-amino-isovaleric acid (Val) site from the 4th exons coding aminoacid sequence of this gene in the chicken body, discharge the N-end of the mature peptide of biologic activity; In conjunction with the analysis to the Cathelin domain peptides section of high conservative between signal peptide and mature peptide, can infer that in its precursor protein Arg 123-Lys 148 is the mature peptide sequence (Seq-4) of chicken Cathelicidin1 antibacterial peptide.
Derive the aminoacid sequence of ripe antibacterial peptide according to the nucleotide sequence of coding chicken Cathelicidin1 antibacterial peptide, according to the standard solid phase peptide synthesis program with synthetic its mature peptide sequence of full-automatic polypeptide synthetic instrument (its N holds to the aminoacid sequence of C end: ArgVal Lys Arg Val Trp Pro Leu Val Ile Arg Thr Val Ile Ala Gly Tyr Asn Leu Tyr Arg Ala Ile Lys LysLys, see Seq-4 in the sequence table).(RP-HPLC, Column:X Bridge4.6 * 250mm) desalination, purifying adopt acetonitrile/water/trifluoroacetic acid system wash-out, the sample purity of synthetic peptide＞95% (95.4%) to synthetic peptide through anti-phase-high-pressure liquid chromatography.Adopting electrospray ionization mass spectrum (ESI-MS) to measure its molecular weight is 3142.80, basically identical with theoretical molecular (3141.86).
The present invention detects by bacteriostatic activity and finds, the recombinant C athelicidins antibacterial peptide that utilizes Rosetta (DE3)/pET30a expression system to express does not need enzyme the processing such as to cut, Gram-positive, negative bacterium all there are good bacteriostatic activity, the reference cultures such as streptococcus aureus, genus bacillus, micrococcus luteus, intestinal bacteria, white dysentery Salmonellas C79-13, pasteurellosis bacillus, riemerella anatipestifer, Pseudomonas aeruginosa and clinical isolates strain are all had stronger restraining effect.Compare discovery with conventional drug sensitive test result, the reference cultures such as micrococcus luteus, intestinal bacteria are to the equal resistance of employed most antibiotics class medicine in the test, and recombinant C athelicidins antibacterial peptide has obvious inhibition to these bacterial strains.Show that recombinant C athelicidins antibacterial peptide may surpass traditional microbiotic to a certain extent to the restraining effect of some bacterial isolates.
The present invention shows the chick test of streptococcus aureus acute infection bacteriostatic activity by antibacterial peptide Cathelicidin1, antibacterial peptide gives 5.0mg/kg dosage, just can reach 100% sterilization inhibiting rate, fail to reach obvious sterilization inhibiting rate and give 5.0mg/kg dosage as the penbritin that press down to kill streptococcus aureus, show that antibacterial peptide of the present invention has very significant sterilization effect to the streptococcus aureus acute infection, therefore antibacterial peptide of the present invention can be applicable to preparation treatment gram-positive microorganism, the medicine of the disease that Gram-negative bacteria or fungi infestation cause especially is applied to preparation treatment streptococcus aureus, genus bacillus, intestinal bacteria, the medicine of Salmonellas etc.Antibacterial peptide of the present invention simultaneously can also be for the preparation of animal feedstuff additive and sanitas.
Beneficial effect of the present invention is:
(1) the present invention selects intestinal bacteria/fusion expression vector as the prokaryotic expression system of foreign gene, select the fusion expression vectors such as pET30a, make chicken Cathelicidins antibacterial peptide obtain to efficiently express, and it is directly lethal to avoid expression product that host cell is caused.
(2) method expression efficiency provided by the invention is high, and the expression product separation and purification is simple, and production cost is low, easily amplifies, and good stability is applicable to large-scale industrial production, has wide application prospect.
(3) using gene engineering technique of the present invention efficiently expresses chicken Cathelicidins antibacterial peptide in prokaryotic host cell, through this group antibacterial peptide of experiment confirm multiple gram-positive microorganism, negative bacterium is all had obvious restraining effect.This group antibacterial peptide can be applicable to prepare antibacterials, or for the preparation of animal feedstuff additive and sanitas.
(4) compare the beneficial features such as that this group antibacterial peptide also has is simple in structure, synthetic convenient, antibiotic pedigree is wide with the alkaline antibacterial peptide in other source.
Description of drawings
Fig. 1 is the white Leghorn Cathelicidin1 of pcr amplification, 2% agarose gel electrophoresis figure of 2,3 antibacterial peptide gene products.In Fig. 1,1 is DL15000 DNA marker, and wherein the base number of each nucleic acid fragment (number unit is bp) is respectively 15000,10000,7500,5000,2500,1000,250; 2,3,4 are respectively Cathelicidin-1, and-2, the pcr amplification product of-3cDNA sequence; 5 is DL2000 DNA Marker among the figure, and wherein the base number of each nucleic acid fragment (number unit is bp) is respectively 2000,1000,750,500,250,100.
Fig. 2 is for containing chicken Cathelicidin-1, and the positive plasmid pMD-CathL enzyme of-2 ,-3 gene cDNA sequences is cut rear 2% agarose gel electrophoresis figure.1 is λ-EcoT14 I digest DNA marker among Fig. 2, and wherein the base number of each nucleic acid fragment (number unit is bp) is respectively 19329,7743,6223,4254,3472,2690,1882,1489,925; 2 is band behind the plasmid pMD-CathL1/EcoR I+Hind III double digestion, 3 is band behind the plasmid pMD-CathL1/EcoR I+Not I double digestion, 4 is band behind the plasmid pMD-CathL2/EcoR I+HindIII double digestion, 5 is band behind the plasmid pMD-CathL2/EcoR I+SmaI double digestion, 6 is band behind the plasmid pMD-CathL3/EcoR I+HindIII double digestion, 7 is band behind the plasmid pMD-CathL3/EcoR I+Not I double digestion, 8 is DL2000 DNA marker, wherein the base number of each nucleic acid fragment (number unit is bp) is respectively 2000,1000,750,500,250,100.
Fig. 3 is 1.5% agarose gel electrophoresis figure after recombinant expression plasmid pET30-CathLS enzyme is cut.1 is λ-EcoT14I digest DNA marker among Fig. 3, and wherein the base number of each nucleic acid fragment (number unit is bp) is respectively 19329,7743,6223,4254,3472,2690,1882,1489,925; 2 is band behind the plasmid pET-30a Sma I+Xba I double digestion; 3 is band behind the plasmid pET30-CathL1S Sma I+Xba I double digestion; 4 is band behind the plasmid pET30-CathL2S Sma I+Xba I double digestion; 5 is band behind the plasmid pET30-CathL3S Sma I+Xba I double digestion.
Fig. 4 is chicken antimicrobial peptides Cathelicidin1, the structure synoptic diagram of 2,3 mature peptide segment composition expression vector pET30-CathLS.In Fig. 4, the fusion expression vector pET30-CathLS size that the 5.5Kb representative makes up is 5.5Kb; P1, P2 represent respectively Cathelicidin1, the upstream and downstream primer that 2,3cDNA sequence clone is used; MCS represents multiple clone site; LacZ represents the encoding gene of beta-galactosidase enzymes; LacI represents the encoding gene of lac repressor; The CathL representative connects the Cathelicidin1 that inserts cloning vector, 2,3cDNA sequence; The CathLS representative connects the Cathelicidin1 that inserts expression vector, 2,3 mature polypeptide coding sequences; Amp represents the ampicillin resistance gene site; Kan represents the kalamycin resistance gene site; PUCorigin, f1 origin, pBR322 origin all represents replication origin.
Fig. 5 is that SDS-PAGE and Western-Blot analyze chicken Cathelicidin1, the expression product figure of 2,3 mature peptide fusion roteins.Wherein, the left side is the SDS-PAGE electrophorogram, and the right side is Western blot figure.Swimming lane 1,1 ' is that empty carrier conversion bacterial strain Rosetta (DE3)/pET30a IPTG induces 3h thalline soluble proteins after ultrasonication; Swimming lane 2,2 ' is to dye in advance lower molecular weight standard protein Marker, and wherein the molecular weight of each protein fragments is respectively 117,85,48,34,26,19kDa, and KDa represents the unit of molecular weight of albumen, i.e. kilodalton; Swimming lane 3,3 ' is that Rosetta (DE3)/pET30-CathL1S bacterial strain IPTG induces 3h thalline soluble proteins after ultrasonication; Swimming lane 4,4 ' is that Rosetta (DE3)/pET30-CathL1S bacterial strain IPTG induces 3h thalline inclusion body protein after ultrasonication; Swimming lane 5,5 ' is that Rosetta (DE3)/pET30-CathL2S bacterial strain IPTG induces 3h thalline soluble proteins after ultrasonication; Swimming lane 6,6 ' is that Rosetta (DE3)/pET30-CathL2S bacterial strain IPTG induces 3h thalline inclusion body protein after ultrasonication; Swimming lane 7,7 ' is that Rosetta (DE3)/pET30-CathL3S bacterial strain IPTG induces 3h thalline soluble proteins after ultrasonication; Swimming lane 8,8 ' is that Rosetta (DE3)/pET30-CathL3S bacterial strain IPTG induces 3h thalline inclusion body protein after ultrasonication.
Fig. 6. the synthetic chicken antimicrobial peptides Cathelicidin1 of solid state chemistry anti-phase-high-pressure liquid phase (RP-PHLC) color atlas.
Fig. 7. mass spectrum (ESI-MS) figure of the synthetic chicken antimicrobial peptides Cathelicidin1 of solid state chemistry.
Embodiment
Below in conjunction with the drawings and specific embodiments, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually " molecular cloning lab guide " (the Molecular cloning:Alaboratory manual that writes according to ordinary method such as Sambrook etc., 2nded.New York:Cold Spring Harbor Laboratory Press, 1999) condition described in is carried out, or carries out according to the specification sheets that manufacturer provides.
Employed toolenzyme and other reagent in the specific embodiment: RNAiso Plus total RNA extraction reagent, ReverseTranscriptase M-MLV (Rnase H -), ribonuclease inhibitor-RNAsin, Taq archaeal dna polymerase, restriction enzyme, pMD18-T Vector, T4 dna ligase be TAKARA company product except specializing; Mouse-anti His monoclonal antibody is all available from Tiangen company; UNIQ-10 pillar plasmid a small amount of extraction agent box and UNIQ-10 pillar DNA glue reclaim test kit and are the living chemical product in Shanghai; IPTG is BBI company product; HRP mark sheep anti-mouse igg is SouthernBiotech company product; Pre-dsred protein molecular weight standard is Fermentas (MBI) company product; Metal chelate chromatography resin Ni 2+-NTASepharose 4B is available from Beijing ancient cooking vessel state company; Microbial culture is Qingdao GaoKeYuan Hai Bo biotech company product with solid mediums such as TMP, TSA, the preparation of by specification supplying method; Conventional drug susceptability test paper sheet is sky, Hangzhou and microorganism reagent company limited product, and other reagent are import or domestic analytical pure.
Embodiment 1:
Chicken Cathelicidin1, the Clone and sequence determination and analysis of the complete encoding gene of 2,3 antibacterial peptides specifically comprises:
1. the design of primer
According to the jungle fowl Cathelicidins class antibacterial peptide gene sequence (DQ092350 that delivers among the GenBank, DQ092351, DQ092352, DQ092353) and the principle of design of PCR primer, and computer software DNASTAR carries out assistant analysis, designed three pairs of pcr gene Auele Specific Primers chicken Cathelicidin1 that is respectively applied to increase, 2, the cDNA sequence of 3 encoding genes, primer is respectively CathL1 P1/P2 in the table, CathL2 P1/P2, CathL3 P1/P2, wherein P1 is forward primer, and P2 is reverse primer.Primer is synthetic by Shanghai Bo Shang company.
Primer sequence used among present embodiment and the embodiment 2 is as shown in table 1.At CathL1S F/R, CathL2S F/R, CathL3S F/R primer centering, the base at underscore place represents respectively EcoR I, the Xho I restriction endonuclease sites of introducing in the upstream and downstream primer.
Table 1 chicken Cathelicidins class antibacterial peptide gene amplification the primer
Figure BDA0000039921290000101
Annotate: underscore place base is respectively EcoR I, the Xho I restriction endonuclease sites of introducing in the upstream and downstream primer.
2. chicken Cathelicidin1, the clone of 2,3 antibacterial peptide gene cDNA sequences
(1) the total RNA of chicken myeloid tissue extracts (the used utensil of following experiment and reagent are all through processing, without RNase): leaching process operates by the reagent specification sheets.1 monthly age of aseptic collection white Leghorn thigh bone fresh bone marrow (1g) contains its adding in the glass homogenizer of 1mLRNAiso Plus cell pyrolysis liquid (TAKARA company product), places ice bath homogenate, until homogenate is without the particle transparence.Homogenate is transferred to the 2ml centrifuge tube under room temperature, acts on 10min; The chloroform that adds RNAiso Plus 1/5 volume (200 μ L) is with hand thermal agitation 15 seconds (the chloroform boiling point is low, volatile, and care should be used to centrifuge tube lid flicks suddenly during vibration).Behind solution fully emulsified (without noted phase separation phenomena), under room temperature, left standstill 5 minutes again; In 4 ℃, centrifugal 15min under 12000 * g condition; The careful centrifuge tube that takes out from whizzer, absorption supernatant liquor are transferred in another new 2ml centrifuge tube and (must guard against sucking-off white middle layer); In supernatant, add isopyknic Virahol, behind the abundant mixing of the centrifuge tube that turns upside down, under-20 ℃, leave standstill 20min; 4 ℃, centrifugal 15min under 12000 * g; Careful supernatant discarded adds 75% ethanol 1ml (being sure not to touch precipitation) lentamente along centrifugal tube wall, the washing centrifuge tube tube wall that turns upside down gently, and 12000 * g, 4 ℃ carefully discard ethanol after centrifugal 5 minutes; Drying at room temperature precipitation 2～5 minutes adds an amount of RNase-free water dissolution precipitation, can obtain the total RNA of chicken medullary cell, and dissolving is rear in-80 ℃ of preservations fully until the RNA precipitation.
(2) synthetic (the mRNA reverse transcription) of cDNA the first chain
At first in the 0.2mL Eppendorf tube, carry out the synthetic cDNA of reverse transcription reaction according to following reaction system.In 20 μ L reaction systems, add respectively following component: 5 * RT Buffer, 4.0 μ L, dNTP (10mM each) 2.0 μ L, 25mM MgCI 20.8 μ L, RNA enzyme inhibitors Rnasin 0.5 μ L (～20U), primer P2 (CathL1 P2, CathL2 P2 or CathL3 P2,20 μ M) 1.0 μ L, cell total rna 10.7 μ L (～1 μ g), above-mentioned reaction mixture is slightly done centrifugal on desk centrifuge, then carried out reverse transcription reaction by following reaction conditions on the PCR instrument: place rapidly chilling 2min on ice behind 70 ℃ of insulation 5min; Of short duration centrifugal after, in reaction system, add Reverse Transcriptase M-MLV (Rnase H -) 1.0 μ L, in the PCR instrument, finish following program behind the mixing: in 42 ℃ of reaction 60min, finish reaction behind 95 ℃ of sex change 2min, with the cDNA solution that obtains be stored in-20 ℃ for subsequent use or be directly used in next step pcr amplification.
(3) pcr amplification of chicken Cathelicidins gene and colony screening
Take cDNA the first chain product of obtaining as template, it is synthetic to carry out cDNA the second chain.Carry out according to following reaction system: 10 * PCR Buffer, 5.0 μ L, 25mmol/L MgCl 21.4 μ L, primer P1 (CathL1 P1, CathL2 P1 or CathL3 P1,20 μ M) 1.0 μ L, RT product 10 μ L, Taq archaeal dna polymerase 0.6 μ L adds ddH 2O to cumulative volume be 50 μ L.
At the PCR instrument following response procedures is set: 95 ℃ of denaturation 3min, 1 circulation; 94 ℃ of 30S, 54 ℃ of 30S, 72 ℃ of 30S extend 10min with 72 ℃ after 30 circulations and finish reaction, 4 ℃ of preservations.Get 5～10 μ L PCR products and carry out 2% sepharose-TAE electrophoresis, the testing goal band, the result is as shown in Figure 1.
The PCR product is separated through 2% agarose gel electrophoresis, after rubber tapping recovery and purifying (reclaiming the test kit explanation by gel carries out) the purpose fragment, be connected with the pMD18-T cloning vector; The heat shock method transforms CaCl 2The bacillus coli DH 5 alpha competent cell that legal system is standby, bacterium liquid was uniformly coated on the LB nutrient agar flat board that contains 100ug/ml penbritin (Amp) 37 ℃ of overnight incubation after centrifuging and taking transformed.Next day picking list bacterium colony, after the shaking culture, alkaline process extracts plasmid (pressing in a small amount extraction agent box specification sheets operation of plasmid) in a small amount; By the method screening positive clone (as shown in Figure 2) that utilizes primer CathL1 P1/P2, CathL2 P1/P2, CathL3 P1/P2 to carry out respectively PCR detection and the evaluation of plasmid restriction enzyme digestion, and with positive plasmid called after pMD-CathL1, pMD-CathL2 and pMD-CathL3.Send the positive strain of identifying the order-checking of to Shanghai Bo Shang company, the dna sequence dna of mensuration carries out homology search with the Blast program, and carries out the sequence alignment analysis.
4. the sequencing of chicken cathelicidins gene and interpretation of result:
Get chicken Cathelicidins cDNA clone and use the order-checking of M13 F/R primer, obtain Cathelicidins1, the complete nucleotide sequence of 2,3 precursor protein encoding genes.The sequencing result of gene is shown in sequence table.
Sequencing is the result show, clone's Cathelicidin-1 ,-2 ,-3 gene open reading frame (ORF) sizes are respectively 447bp, 465bp, 456bp, coding comprises the precursor protein of 148,154,151 amino-acid residues respectively, and wherein 17 of N-terminal amino-acid residues are signal peptide sequence, 105 amino-acid residues of middle portion consist of the Cathelin structural domain, 26,32,29 amino-acid residues of C end form respectively Cathelicidin-1, the mature peptide of-2 ,-3 antibacterial peptides.
The sequence alignment analysis finds that ORF is in full accord for clone's white Leghorn Cathelicidin-1 gene order and reference sequences (Fowlicidin-1, GenBank accession number DQ092351).With Gallus Domesticus Cathelicidin-1 (GenBank accession number FJ938357) ORF sequence similarity be 99.6%, only have 432 base differences, the former is A, the latter is G.The amino acid sequence similarity of deriving is 100%.
Clone's white Leghorn Cathelicidin-2 gene order and reference sequences (Fowlicidin-2, GenBank accession number DQ092352) the ORF sequence similarity is 98.7%, there are differences at 36,44,48,57,63,453 respectively, the amino acid sequence similarity of deriving is 99.4% (15 the former is A, and the latter is V).White Leghorn Cathelicidin-2 gene order and Gallus Domesticus Cathelicidin-2ORF (GenBank accession number FJ938358) nucleotide sequence similarity are 98.9%, there are differences at 44,48,57,63,453, the amino acid sequence similarity of deriving is 99.4% (15 the former is A, and the latter is V).
Clone's white Leghorn Cathelicidin-3 gene order and reference sequences (Fowlicidin-3, GenBank accession number DQ092353) the ORF sequence similarity is 99.6%, respectively have 1 base difference at 168,420, the former is respectively A, G, and the latter is respectively G, A.Amino acid sequence similarity is 100%.SPF chicken Cathelicidin-3ORF and Gallus Domesticus Cathelicidin-3ORF (GenBank accession number FJ938359) nucleotide sequence similarity is 99.8%, has 1 base difference at 168, and the former is A, and the latter is G.Amino acid sequence similarity is 100%.
Sequential analysis shows that the chicken Cathelicidins antibacterial peptide gene in different varieties source lists at nucleotides sequence and has small difference, and aminoacid sequence is of slight difference, thereby shows that the Cathelicidins antibacterial peptide gene is high conservative in evolution.
Embodiment 2:
The gene engineering preparation method of recombination chicken Cathelicidins antibacterial peptide fusion protein may further comprise the steps:
1. chicken Cathelicidins1,2,3 mature polypeptide coding sequences preparation and structure (1) the chicken Cathelicidins1 of fusion expression vector, 2,3 mature polypeptide coding sequences amplification: according to the chicken Cathelicidins 1 that has cloned, the aminoacid sequence of 2,3 antibacterial peptide gene sequences and derivation designs 3 pairs with the Auele Specific Primer CathL1S F/R of restriction enzyme site, CathL2S F/R, CathL3S F/R, downstream primer is with terminator codon TGA, and primer sequence sees Table 1.Respectively containing chicken Cathelicidin-1, the positive plasmid pMD-CathL1 of-2 ,-3 gene cDNA sequences, pMD-CathL2, pMD-CathL3 are template, with PCR method amplification chicken Cathelicidins 1,2,3 mature polypeptide coding sequences (～100bp).
Reaction system is: 10 * PCR Buffer (contains Mg 2+) 5.0 μ L, dNTP (2.5mmol/L) 4.0 μ L, primers F (CathL1F, CathL2F or CathL3F, 20 μ M) 1.0 μ L, primer R (CathL1R, CathL2R or CathL3R, 20 μ M) 1.0 μ L, pMD-CathL (pMD-CathL1, pMD-CathL2 or pMD-CathL3) plasmid DNA 0.5 μ L, Taq archaeal dna polymerase 0.5 μ L adds ddH 2O 38 μ L, cumulative volume are 50 μ L.On the PCR instrument, react according to follow procedure behind the mixing: 95 ℃ of denaturation 3min, 1 circulation; 94 ℃ of 30S, 55 ℃ of 30S, 72 ℃ of 30S extend 10min with 72 ℃ after 30 circulations and finish reaction, 4 ℃ of preservations.All PCR reaction solutions are carried out 2% sepharose-TAE electrophoresis, and reclaim test kit with the gel of giving birth to worker company and reclaim specific fragment.The PCR product is dissolved in an amount of EB elutriant after cutting the glue recovery.(2) preparation of pre-connection dna fragmentation: carried out double digestion with EcoR I and Xho I with reclaiming product in the upper step.The endonuclease reaction system is specially: 10 * endonuclease reaction damping fluid H, 10 μ L, and PCR reclaims product 35 μ L, restriction enzyme EcoR I 3.0 μ L, Xho I 3.0 μ L, distilled water 49 μ L, total reaction volume is 100 μ L.Place 37 ℃ of water-baths to react 3h reaction solution.Endonuclease reaction liquid is transferred in the new 1.5ml centrifuge tube, and adding TE damping fluid (pH 8.0 for 10mmol/L Tris-HCl, 1mmol/L EDTA) to cumulative volume is 500 μ L; Add isopyknic phenol/chloroform (volume ratio is 25: 24) albumen extract, mixing fully vibrates; In 4 ℃, centrifugal 10min under 12000 * g condition; The careful upper strata inorganic phase of drawing is transferred in the new centrifuge tube; Add the dehydrated alcohol of 2.5 times of supernatant liquor volumes, under-20 ℃, leave standstill 30min; 4 ℃, centrifugal 15min under 12000 * g; Careful supernatant discarded adds 75% ethanol 1ml washing centrifuge tube tube wall lentamente along centrifugal tube wall, 12000 * g, and 4 ℃ carefully discard ethanol after centrifugal 5 minutes; Drying at room temperature precipitation 5～10 minutes adds an amount of TE damping fluid dissolution precipitation, namely obtains containing the pre-connection mature peptide encode fragment of the sticky end that mutually matches with pET-30a (+) linear carrier of handling well.(3) preparation of linearizing expression vector: DH5 α/pET-30a (+) prokaryotic expression carrier deposited of going bail for transforms bacterial strain, after the shaking culture, extracts in a small amount plasmid.Get pET-30a (+) plasmid and carry out double digestion with EcoR I and Xho I.The endonuclease reaction system is: 10 * endonuclease reaction damping fluid H, 8 μ L, and pET-30a (+) plasmid 20 μ L, restriction enzyme EcoR I 3.0 μ L, Xho I 3.0 μ L, distilled water 46 μ L, total reaction volume is 80 μ L.In 37 ℃ of water-baths the 3h endonuclease reaction fully after, after reaction solution carried out 1% (W/V) sepharose-TAE electrophoresis, reclaim test kit with gel and reclaim specific fragment, the carrier of handling well has the sticky end of EcoR I and Xho I, be stored in-20 ℃ stand-by.
(4) Cathelicidins1,2, the structure of 3 mature peptide segment composition expression vectors, transform with identify will be after above-mentioned a series of processing with pET-30a (+) prokaryotic expression carrier of identical sticky end and contain Cathelicidins1,2, the dna fragmentation of 3 mature polypeptide coding sequences carries out external connection, and reaction system is as follows:
10×T4 DNA Ligase Buffer 2μL
Cathelicidins1,2,3 mature peptide encode fragments, 14 μ L
Linearizing expression vector 3 μ L
T4 DNA Ligase 1μL
Total reaction volume 20 μ L
16 ℃ of reactions are spent the night; All be converted into ligation liquid in the JM109 competent cell next day, is coated on overnight incubation on the LB agar plate that contains kantlex (100 μ g/ml).Next day, picking list bacterium colony after the shaking culture, in a small amount extracted plasmid, utilized CathL1S F/R, CathL2S F/R, and CathL3S F/R primer pair take plasmid as template, detects and plasmid enzyme restriction method evaluation positive colony bacterial strain according to aforementioned PCR.Positive strain sent to by Shanghai Bo Shang company carry out nucleotide sequencing, sequencing result shows that coding Cathelicidins mature protein gene correctly is inserted into the purpose site of prokaryotic expression carrier, and the open reading frame of each section nucleotide sequence (ORF) is continuous, namely obtain recombinant expression plasmid, with its difference called after pET30-CathL1S, pET30-CathL2S, pET30-CathL3S.The restriction analysis of recombinant expression plasmid pET30-CathLS the results are shown in Figure 3, and the building process of fusion expression vector sees Fig. 4 for details.
2. chicken Cathelicidins1, abduction delivering and the expression product of 2,3 mature peptide fragments in intestinal bacteria identified
Rosetta (DE3) is that Novagen company is used for the F-strain that pET series prokaryotic expression carrier is expressed foreign protein, the Rosetta Host Strains is to derive and the λ DE3 lysogenic bacteria that comes from BL21, can strengthen eukaryotic gene with the intestinal bacteria rare codon in prokaryotic system expression level.The genotype of this bacterial strain is: F -OmpT hsdS B(r B -m B -) gal dcm lacY1 (DE3) pRARE (argU, argW, ileX, glyT, leuW, proL) (Cm R).The pET30-CathLS recombinant expression plasmid is transformed Rosetta (DE3) F-strain, can obtain recombinant expressed Cathelicidins1, the engineering bacteria Rosetta (DE3) of 2,3 mature peptide fusion roteins/pET30-CathLS.
(1) structure of the engineering bacteria Rosetta (DE3) of expression recombination fusion protein/pET30-CathLS
1) gets aforementionedly through identifying correct fusion expression plasmid pET30-CathL1S, pET30-CathL2S, pET30-CathL3S and pET-30a (+) empty plasmid 0.5 μ L, transform respectively CaCl with the heat shock method 2Rosetta (DE3) the host competent cell that legal system is standby is got the rear bacterium liquid of 200 μ L conversion and is evenly coated on the LB agar plate that contains 100 μ g/mL kantlex (kana) 37 ℃ of overnight incubation.
2) next day, the picking positive colony is seeded in the 4ml LB substratum (containing 100 μ g/mL kantlex) and carries out liquid culture, behind 37 ℃ of shaking culture 8h, bacterium liquid takes a morsel, utilize respectively CathL1S F/R, CathL2S F/R, CathL3S F/R primer detects the bacterial strain (PCR method, condition are as previously mentioned) of determining that conversion is successful to carrying out PCR.The engineering bacteria called after Rosetta (DE3) that success is transformed/pET30-CathLS (be respectively Rosetta (DE3)/pET30-CathL1S, Rosetta (DE3)/pET30-CathL2S and Rosetta (DE3)/pET30-CathL3S), bacterial strain in containing the LB substratum of 15% glycerine in-80 ℃ of preservations.
(2) Cathelicidins1, the small-scale abduction delivering of 2,3 mature peptide fusion roteins
1) gets respectively single bacterium colony of the engineering bacteria Rosetta (DE3) that builds/pET30-CathL1S, Rosetta (DE3)/pET30-CathL2S, Rosetta (DE3)/pET30-CathL3S, be seeded to 37 ℃ of shaking culture 8～12h in the 4ml LB liquid nutrient medium (containing 100 μ g/mL kantlex);
2) bacteria suspension is spent the night in 4 ℃ of preservations, next day, the centrifuging and taking thalline was resuspended in the fresh LB substratum that 50mL contains 100 μ g/mL kantlex;
3) about 37 ℃ of shaking culture 3h to nutrient solution OD 600nmValue reaches 0.5～0.6, takes out first the 1mL culture as inducing front contrast, and adding isopropyl-β-D-thiogalactoside(IPTG) (IPTG) stock solution (500mmol/L) to final concentration in remaining culture is that 1.0mmol/L induces;
4) 37 ℃ were continued to cultivate 4h, got the 1mL nutrient solution every 1 hour; Thalline after centrifugal collection is induced washs thalline 2 times with the PBS damping fluid, in the ratio of the centrifugal rear adding 100uL lysis buffer of 1mL bacterium liquid, the lysis buffer of adding ice precooling (50mmol/LTris-HCl, pH 8.0,50mmol/LNaCl, 1mmol/LEDTA, 5% glycerine, add before use proteinase inhibitor PMSF to final concentration be 1mmol/L) resuspended thalline, ultrasonication thalline in the ice bath, each broken 5s, interval 2s, power 600W, broken 10min.
5) ultrasonic rear cellular lysate liquid is in 12000 * g, 4 ℃ of lower centrifugal 10min, separate soluble constituent with and inclusion body, carry out next step evaluation.
6) Rosetta (DE3) bacterial strain of getting simultaneously the conversion of pET-30a (+) empty plasmid carries out inducing culture and cellular lysate for contrast according to above-mentioned condition.
(3) Cathelicidins1, the evaluation of 2,3 mature peptide expression products
Polyacrylamide gel electrophoresis (SDS-PAGE) and protein immunoblotting analysis (Western Blot detection) by expression product carry out preliminary evaluation to fusion rotein.
SDS-PAGE detects the expression of fusion rotein:
1) preparation of gel: prepare 15% separation gel and 5% concentrated glue with reference to the prescription in " molecular cloning experiment guide " and method;
2) sample preparation: get the protein example of above-mentioned evaluation to be analyzed, add isopyknic 2 * sds gel sample loading buffer, 5min is boiled in 100 ℃ of water-baths;
3) application of sample: will add 1 * Tris-glycine electrophoretic buffer (25mmol/LTris-HCl, pH8.0,250mmol/L glycine, 0.1%SDS, pH8.3) in the electrophoresis chamber, carefully take out the point sample comb, then blow and beat well with syringe.Draw respectively sample to be analyzed with micro sample adding appliance, determine the application of sample amount according to protein concentration and well volume;
4) electrophoresis: begin with low voltage 80V constant voltage electrophoresis, after the bromophenol blue indicator forward position enters separation gel, improve voltage to the 120V electrophoresis, until after tetrabromophenol sulfonphthalein migrates to the separation gel bottom, stop electrophoresis;
Take out gel, scanning analysis after coomassie brilliant blue staining, the decolouring.
5) dyeing: pry open gently layer glass, take out gel, soak gel with the coomassie brilliant blue R_250 staining fluid (0.25g Xylene Brilliant Cyanine G R-25 is dissolved in the solution that contains 45% (V/V) methyl alcohol, 10% (V/V) acetic acid) of at least 5 times of volumes, place on the steady decolorization swinging table room temperature dyeing 2h that slowly vibrates;
6) decolouring: change staining fluid, soak gel with destainer (10% (V/V) acetic acid solution), slowly shake 4～8h, change destainer therebetween 3～4 times, until clean background, till band is clear.
7) SDS-PAGE the analysis showed that, Cathelicidin-1,2,3 mature peptide fragments are the energy successful expression all, expression product exists with soluble constituent and two kinds of forms of inclusion body, but the expression amount of 3 fragment genes difference to some extent, wherein the expression amount of Cathelicidin-3 is high than other 2 kinds of products.The apparent molecular weight of 3 kinds of expression products is about 12kDa.And empty carrier transforms control group without the corresponding protein band, the results are shown in Figure 5.
Western blot analysis (Western-Blot detection):
1) electrotransfer of target protein (wet type transfer):
1. by preceding method the induction expression protein sample is carried out the SDS-PAGE electrophoresis;
2. after the SDS-PAGE electrophoresis finishes, carefully take out gel, be immersed in balance 5～10min in the transferring film damping fluid;
3. filter paper, nitrocellulose filter (NC) are cut into gel onesizely, the NC film is soaked 5min to eliminate bubble in distilled water, again NC film, filter paper, absorbent sponge layer etc. are immersed balance 10min in the transfering buffering liquid.
NC is immersed first 10-20min in the distilled water, immerse again balance 30min in the transfering buffering liquid.
4. transferring film: putting on one's gloves installs electrotransfer device (Beijing Liu Yichang) as follows: black sponge plastics is folded up at the lowest layer, place successively absorbent sponge layer, multi-layer filter paper, gel, NC film, multi-layer filter paper, sponge layer, colourless sponge plastics folder by order from bottom to top again, filter paper, gel, NC film are wanted Accurate align, slowly mobile on every layer of surface with a test tube or glass stick, to remove the bubble between gel and NC film, the filter paper; Close up electric transfer printing and folded up in the transfer groove (black in black, white leaning on is red), filled it up with transfering buffering liquid; The exact connect ion electrode, namely gel is towards negative electrode, and the NC film makes the protein on the gel shift to NC film trace towards anode; 200mA constant current (about voltage 100V) adds electrotransfer 3h under the ice condition.Initial current should less than 250mA, finish electric current and should not surpass 400mA.
2) immune labeled:
1. from the electrotransfer groove, take out the NC film, wash film 2 times with the TBS damping fluid, to remove the gel on the NC film.The NC film packed into one can be added in the plastics bag of heat sealing, adds confining liquid (3% skim-milk is dissolved in TBS), is placed on the shaking table that shakes gently and seals 3h;
2. sealing finishes, and film is transferred in the plastics bag of new added heat sealing, presses 0.1mL/cm 2Amount add confining liquid and an amount of first antibody (mouse-anti His monoclonal antibody is diluted in confining liquid at 1: 1000), sealing, 4 ℃ of gentle joltings are spent the night;
3. cut off plastics bag, wash film 3 times with about 200mL TBST rinsing liquid, each 10min is to remove excessive primary antibodie;
4. film is changed over to another plastics bag, add the sheep anti-mouse igg-HRP two anti-(dilution in 1: 3000,7uL sheep anti-mouse igg-HRP is diluted to 21mL) that is dissolved in two anti-diluents, 1～2h is shaken in sealing gently under the room temperature;
5. take out the NC film, wash film 3～5 times with a large amount of TBST rinsing liquids, each 10min, anti-to remove unconjugated two;
6. wash film once with TBS more at last, remove Tween-20.
3) immunostaining:
The DAB nitrite ion that the NC film is changed over to the proper amount of fresh preparation (faces time spent adding H 2O 2) in, put dark place reaction, (color reaction reaches optimum extent, and is about 2～3min), washs termination reaction with distilled water immediately, and film is dried preservation when treating that the protein band color depth reaches requirement.
The compound method that Western-Blot detects used each damping fluid is as follows:
1. transfering buffering liquid: 39mmol/L glycine, 48mmol/LTris-HCl, 0.037% (W/V) SDS, 20% (V/V) methyl alcohol; Preparation 1L transfering buffering liquid need take by weighing the 2.9g glycine, 5.8g Tris-base, and 0.37g SDS, and add 200mL methyl alcohol, adding water to total amount is 1L.
2. confining liquid: 3% (W/V) skim-milk, 0.02% sodium azide is dissolved in TBS;
3. rinsing liquid I-TBS damping fluid (150mmol/L NaCl, 50mmol/L Tris-Cl, pH7.5);
Rinsing liquid II-TBST:150mmol/LNaCl, 50mmol/LTris-Cl, pH7.5,0.02%Tween-20
4. two anti-diluents (3% skim-milk, 150mmol/L NaCl, 50mmol/L Tris-HCl, pH7.5)
5. nitrite ion DAB (3.3-diaminobenzidine, 3.3-diaminobenzidine) preparation: 7.5mg DAB is dissolved among the 10mL TBSbuffer, faces time spent adding 30%H 2O 210 μ L (facing the time spent now joins) use behind the mixing immediately.
SDS-PAGE and Western-blot the analysis showed that, Cathelicidin-1,2,3 mature peptide fragments are the energy successful expression all, expression product exists with soluble constituent and two kinds of forms of inclusion body, but the expression amount of 3 fragment genes difference to some extent, wherein the expression amount of Cathelicidin-3 is high than other 2 kinds of products, induce comparatively clearly specific band of rear generation, and empty carrier transforms control group without the corresponding protein band.The apparent molecular weight of 3 kinds of fusion protein expression products is about 12kDa (the results are shown in Figure 5).
Embodiment 3:
The affinitive layer purification of Cathelicidins expressing fusion protein condition optimizing and expression product
1. expressing fusion protein condition optimizing
For improving soluble component proportion in expressing total protein, by reducing inducing temperature (being down to 25 ℃ or 15～20 ℃), the prolongation induction time (3h～8h), (IPTG respectively concentration is 0.4mmol/L to regulate inductor concentration, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L) etc. method, suppress the formation of inclusion body, thereby simplify purification process.
Suppress the purpose that inclusion body forms, improves soluble proteins proportion in the expression product by adopting height to ooze the methods such as concentration that inductor IPTG was induced, regulated to substratum (being the LB/SB substratum), low temperature (25 ℃ or 15～20 ℃) for a long time, reaching.Evidence is under the inductive condition of 1.0mmol/L at 25 ℃, IPTG concentration, and the shared ratio of solubility target protein is beneficial to the downstream purification operation than obviously improving (figure slightly) under 37 ℃ of conditions.
2. the affinitive layer purification of fusion protein expression products
His label protein purifying resin (Ni-NTA Resin) is for a kind of purification media of purifying with 6 * His label recombinant protein, this purification media and His label protein have high avidity, are used in 6 * His label recombinant protein that any expression system of purifying is expressed under non-sex change or the Denaturing.6 * His can with Ni 2+Chelating, thus the His label protein is combined on the Ni-NTA purification media, unconjugated albumen is washed down, and the albumen that is combined on the medium passes through eluting of certain density imidazoles or low pH damping fluid gentleness, thereby obtains highly purified target protein.The present invention utilizes Ni 2+-NTA Sepharose4B affinitive layer purification medium is purified into highly purified Cathelicidins fusion rotein under non-Denaturing.Operate as follows:
(1) protein example preparation:
1) get the engineering bacteria Rosetta (DE3) of successful expression Cathelicidins fusion rotein/pET30-CathLS, line separates single bacterium colony on the LB/Kan agar plate.
2) get single colony inoculation 37 ℃ of shaking culture 8～12h to the 4ml LB liquid nutrient medium (containing 100 μ g/mL Kan);
3) centrifuging and taking thalline is resuspended in enlarged culturing in the fresh LB substratum that 200mL contains 100 μ g/mL Kan, and 37 ℃ of shaking culture are to culture OD 600nmValue reaches at 0.5～0.6 o'clock, and adding isopropyl-β-D-thiogalactoside(IPTG) (IPTG) stock solution (500mmol/L) is that 1.0mmol/L induces to final concentration, induces by the rear change condition of above-mentioned optimization;
4) centrifugal collection thalline, the ratio that is suspended from 4mL binding buffer liquid (20mmol/LTris-HCl, pH 8.0,500mmol/L NaCl) according to the bacterial sediment of every 100mL culture adds an amount of binding buffer liquid;
5) thalline is suspended, add N,O-Diacetylmuramidase to final concentration 1mg/mL, mixing is placed 30min on ice, and ultrasonic or broken cell homogenate is hatched 10min with mixture again on 4 ℃ of shaking tables.
6) add Triton X-100, DNase (5U/ μ L) and RNase (10mg/mL), to final concentration be respectively 1%, 5U/mL and 5 μ g/mL, on 4 ℃ of shaking tables, hatch 10min again.
7) 12000r/m (more than 20, the 000xg) is more than 4 ℃ of centrifugal 15min.Remove insoluble cell debris, supernatant (cell pyrolysis liquid) is transferred in another new pipe, place for subsequent use or-20 ℃ of preservations on ice.
(2) affinitive layer purification of fusion rotein and wash-out
1) the post bed is prepared: put upside down gently mixing immobilization Ni 2+-NTA resin, in the chromatography column that it is suitable that it is packed into, the resin natural subsidence with the aseptic water washing resin of 3 times of column volumes, is used the binding buffer liquid balance resin of 5 times of column volumes again, places stand-by;
2) the supernatant sample of the upper step being handled well is splined on the good Ni of balance behind 0.45 μ m filtering with microporous membrane 2+In-NTASepharose 4B the affinity column, flow rate control is at 15mL/h～20mL/h, collects penetrating component, be used for the SDS-PAGE analysing protein in conjunction with situation;
3) wash post with the binding buffer liquid of 5 times of column volumes, flow rate control is about 30mL/ hour, to remove other solubility compositions of unconjugated albumen and thalline;
4) wash post with the lavation buffer solution (20mmol/L Tris-HCl, pH 8.0,500mmol/L NaCl, 50mmol/L imidazoles) of 4 times of column volumes, the protein ingredient of further flush away non-specific binding is until flow through liquid OD 280nm＜0.01;
5) under magnetic agitation at the uniform velocity, utilize the gradient mix device preparation to contain the elution buffer (imidazole concentration is by 0.1mol/L to 0.5mol/L linear increment in the binding buffer liquid) of the linear imidazole concentration gradient of 0.1mol/L～0.5mol/L;
6) with the target protein of the imidazoles gradient elution buffer solution elution combination of 5 times of column volumes, collect step by step by every part of 1mL, and monitoring OD 280nm
7) wash post with the elution buffer that contains high density imidazoles (1mol/L) of 3 times of column volumes again, the albumen that will be combined with the post bed fully elutes;
8) eluted protein of getting 20 μ L equal portions carries out the 15%SDS-PAGE detection, determines the distribution situation of target protein in elutriant;
The elutriant that 9) will contain target protein is packed in the dialysis tubing of handling well, in 4 ℃ of dialysis 12～16h, (dialyzate is 50mmol/L Tris-HCl, 50mmol/LNaCl to change the several dialyzate therebetween, pH 7.6), to remove the imidazoles composition in the target protein solution;
10) again dialysis tubing is placed beaker, be embedded in an amount of solid polyethylene glycol (PEG)-20000,4 ℃ are concentrated into proper volume, and the flushing dialysis tubing is removed residual PEG.The sample freeze-drying can be preserved and done and further analyze.
The fusion rotein elution peak of collecting is through SDS-PAGE, coomassie brilliant blue staining, show that target protein presents single band in the elutriant in the target location, show through affinity chromatography to obtain the higher fusion rotein of purity, create conditions for utilizing on a large scale genetic engineering means to prepare recombinant antibacterial peptide.
As seen SPS-PAGE electrophoretic analysis, coomassie brilliant blue staining obtain the single band that molecular weight is 12kD after purified.
(3) Ni 2+The regeneration of-NTA Sepharose 4B resin
Ni 2+-NTA resin is after using some number of times (3-5 time), and joint efficiency descends to some extent, can regenerate with following methods, improves the work-ing life of resin and the joint efficiency of protein.All solution that need before the NTA resin regeneration to drain off from the chromatography column lower end estimate the resin volume of NTA, by following order regeneration reagent are added in the chromatography column, and a regeneration solution stream is done and added next regeneration dissolving again on waiting.Need the following regeneration soln of preparation:
Stripping Solution I：6M GuHCl，0.2M acetic acid
Stripping Solution II：2％SDS
Stripping Solution III：100mM EDTA，pH 8.0
Ni Charging Solution：100mM NiSO 4
Also to prepare 25%, 50%, 75%, 100% (V/V) ethanol and deionized water etc.
Ni 2+The regeneration step of-NTA is as follows:
1) all solution that drain off from the chromatography column lower end are washed with the Stripping Solution I of 2 times of NTA resin volumes.
2) wash with the deionization of 2 times of volumes.
3) the Stripping Solution II with 3 times of volumes washes.
4) wash with 25% ethanol of 1 times of volume.
5) wash with 50% ethanol of 1 times of volume.
6) wash with 75% ethanol of 1 times of volume.
7) wash with 100% ethanol of 5 times of volumes.
8) wash with 75% ethanol of 1 times of volume.
9) wash with 50% ethanol of 1 times of volume.
10) wash with 25% ethanol of 1 times of volume.
11) wash with the deionization of 1 times of volume.
12) the Stripping Solution III with 5 times of volumes washes.
13) wash with the deionization of 3 times of volumes.
14) if use immediately, wash with the Ni Charging Solution of 5 times of volumes, use again the binding buffer liquid balance chromatography column of 10 times of volumes.
15) such as the need standing storage, add 20% ethanol of 1 times of volume, 4 ℃ of preservations need performing step 14 before the use, the new clothes post of laying equal stress on.
Embodiment 4: the solid state chemistry synthetic method of chicken Cathelicidin1 antibacterial peptide:
Cathelicidin1 mature peptide aminoacid sequence according to the deduction of coding chicken cathelicidin antibacterial peptide gene, according to standard solid phase peptide synthesis program synthetic chicken Cathelicidin1 antibacterial peptide: CATH1 (Arg Val Lys Arg Val Trp Pro Leu Val IleArg Thr Val Ile Ala Gly Tyr Asn Leu Tyr Arg Ala Ile Lys Lys Lys), synthetic peptide is through anti-phase-high-pressure liquid chromatography (RP-HPLC, Column:X Bridge 4.6 * 250mm) desalinations, purifying adopts acetonitrile/water/trifluoroacetic acid system wash-out (Pump A:0.065%trifluoroacetic in 100%water; Pump B:0.05%trifluoroacetic in 100%acetonitrile), adopt electrospray ionization mass spectrum (ESI-MS) to measure its molecular weight.Antibacterial peptide is synthetic to be finished by Nanjing Genscript Biotechnology Co., Ltd. (Genscript Co., Ltd.Nanjing, China), sample purity＞95% (being 95.4%), and its HPLC figure is as shown in Figure 6; Its ESI mass spectroscopy figure sees Fig. 7.Its theoretical molecular is 3141.86, and the actual measurement molecular weight is 3142.80, and is basically identical.Synthetic Cathelicidin1 antibacterial peptide is dissolved in the sterilization distilled water, is used for active the detection.
Embodiment 5: the bacteriostatic activity of recombination chicken antibacterial peptide Cathelicidin 1,2,3 detects
Employing standard agar hole diffusion process (Agar Well Diffusion Assay, AWDA) (Parente E, et al.Acomparison of methods for the measurement of bacteriocin activity.J Microbiol Methods, 1995,22 (1): 95-108) detect recombination chicken antibacterial peptide Cathelicidin 1,2,3 In Vitro Bacteriostatic, and compare with traditional microbiotic.
The indicator strain that bacteriostatic test is used: streptococcus aureus (Staphylococcus aureus CMCC 26003; ATCC6538), subtilis (Bacillus subtilis CMCC 63501), bacillus pumilus (Bacillus pumilus CMCC63501), micrococcus luteus (Micrococcus luteus CMCC 28001), colon bacillus (Escherichia coli CMCC44102; ATCC 8099), white dysentery Salmonellas (Salmonella pullorum CVCC533, C79-13), Candida albicans (Candida albicans ATCC 10231), pasteurella multocida (Pasteurella multocida CVCC474, C48-7) etc. be reference culture, all available from China Veterinery Drug Inspection Office; Colon bacillus K 12D 31Bacterial strain (CGSC#5165) is given by DSMZ of Yale University (coli strain storehouse); Clostridieum welchii (Clostridieum welchii), serum I type, II type riemerella anatipestifer (Riemerellosis ahatipestifer), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Proteus mirabilis (Proteus mirabilis), Arizona bacterium (Salmonella arlzonae, Arizona Salmonella), Salmonella paratyphi A (Salmonella paratyphi A) etc. are clinical separation, identify that bacterial strain is by Shandong Province's Poultry diseases and the preservation of immune key lab.
Concrete steps are as follows:
1. recombinant C athelicidins antibacterial peptide In Vitro Bacteriostatic detects
Antibacterial Activity adopts standard agar hole diffusion process: above-mentioned reference culture and clinical isolates strain are inoculated in the fresh LB liquid nutrient medium of 5mL 37 ℃ of overnight incubation; Transfer in the fresh LB of 5mL with 1% inoculum size, 37 ℃ are continued shaking culture 2.5h～3h to logarithmic growth mid-term again; Centrifugal collection thalline is used 10mM phosphate buffered saline buffer (PBS, the pH 7.4) washing of precooling once, and is regulated OD 600To 0.4～0.6; Get respectively each reference culture and the clinical isolates strain suspension (OD that are in logarithmic phase 600≈ 0.5) 200 μ L, behind 45 ℃ TSA solid medium 20mL mixing, to entering in the 90mm plate, after it solidifies, with punch tool (micro-pore diameter the is 4mm) punching of sterilization; Every hole drips respectively 40 μ L testing samples, 30 ℃ of overnight incubation, with the negative contrast of pET30a empty carrier transformant expressing protein (abduction delivering 3h cracking supernatant liquor) with volume, antibacterial circle diameter is measured in the positive contrast of Amp (100 μ g/mL) next day.
2. the drug sensitivity testing in vitro of the reference culture such as streptococcus aureus, intestinal bacteria
Explanation with reference to the drug susceptability test paper box, select 27 kinds of drug susceptability test paper sheets such as Ancef (30 μ g/ sheet), gentamicin (10 μ g/ sheet), Streptomycin sulphate (10 μ g/ sheet), Prostaphlin (1 μ g/ sheet), to 7 strain G such as streptococcus aureus, subtilis, bacillus pumilus, intestinal bacteria, micrococcus luteuses +, G -Reference culture carries out drug sensitive test, as the microbiotic contrast of recombinant antibacterial peptide extracorporeal bacteria inhibitor test.
Bacteriostatic activity detects to be found, the recombinant C athelicidins antibacterial peptide that utilizes Rosetta (DE3)/pET30a expression system to express does not need enzyme the processing such as to cut, Gram-positive, negative bacterium all there is good bacteriostatic activity, the reference cultures such as streptococcus aureus, genus bacillus, micrococcus luteus, intestinal bacteria, white dysentery Salmonellas C79-13, pasteurellosis bacillus, riemerella anatipestifer, Pseudomonas aeruginosa and clinical isolates strain are all had stronger restraining effect, and the antibacterial circle diameter measurement result sees Table 2.
Find with conventional drug sensitive test result (table 3) comparison, the reference culture such as micrococcus luteus, intestinal bacteria is to the equal resistance of employed most antibiotics class medicine in the test, and recombinant C athelicidins antibacterial peptide has obvious inhibition to these bacterial strains.Show that recombinant C athelicidins antibacterial peptide may surpass traditional microbiotic to a certain extent to the restraining effect of some bacterial isolates.
Table 2 recombinant C athelicidins antibacterial peptide is to the bacteriostatic activity of indicator strain
Table 2 Detection of the antibacterial activity of recombinant Cathelicidins against indicator strains
Figure BDA0000039921290000231
Figure BDA0000039921290000241
The drug sensitive test result of the reference cultures such as table 3 streptococcus aureus, intestinal bacteria
Table 3 Drug susceptibility test for standard strains of Staphylococcus aureus，Escherichia coli，etc
Figure BDA0000039921290000242
Figure BDA0000039921290000251
Embodiment 6:
Cathelicidin1 antibacterial peptide and microbiotic are to the comparison of chick infection of staphylococcus aureus model inhibition
Sample in the present embodiment is the chicken Cathelicidin1 antibacterial peptide that adopts the solid phase method chemosynthesis, and concrete preparation method and aminoacid sequence are seen specific embodiment 4.
For determining antibacterial peptide Cathelicidin1 anti-microbial activity in vivo, use male chick in 2 ages in week (body weight 100 ± 2.0 grams), set up artificial challenge streptococcus aureus (Staphylococcus aureus, S.A.) experimental model of ATCC6538 bacterial strain, injection antibacterial peptide Cathelicidin1 and traditional microbiotic are determined its restraining effect.Experimental procedure is as follows:
Get 36 of the close chick of body weight, be divided at random six groups, 6 every group.1～5 group is respectively the ATCC6538 test group, and every chicken contains 1 * 10 at neck subcutaneous injection 0.1mL respectively 8The bacterium liquid of streptococcus aureus ATCC6538,1～3 group is the antibacterial peptide treatment group, attacks poison and injects respectively simultaneously the antibacterial peptide Cath1 of various dose by 2.5mg/kg, 5mg/kg, 10mg/kg body weight; The 4th group is the antibiotic therapy control group, presses 5mg/kg injection penbritin; The 5th group for attacking malicious control group, do not inject any medicine; The 6th group is the blank group, neck subcutaneous injection stroke-physiological saline solution 0.1ml.
48h observes mortality ratio, the daily behavior of chick and begins morbidity and clinical disease course every 12h continuously.Not dead test group chick is slaughtered rear aseptic its liver organization 0.5g that takes in the 48h, and homogenate in the PBS of 9 times of volumes damping fluid (4.5mL) is carried out 10 * doubling dilution with homogenate with aseptic PBS damping fluid, gets respectively 10 -3, 10 -4, 10 -5, 10 -6, 10 -7Each dilution bacterium liquid 100 μ L coating TMP streptococcus aureus is differentiated dull and stereotyped, and each extent of dilution repeats 2 times.Carry out bacterial colony (CFU) counting behind 37 ℃ of cultivation 24h.
The bacterium of cultivating carries out gramstaining, microscopy.Choose the culture dish of colony number (CFU) between 30～300 and count standard as total number of bacterial colony, calculate the bacterial count of every gram content, and represent with its logarithmic value, the results detailed in Table 4.
In-house bacterial count=the colony number of every gram * 2 * 10 2* extension rate.
Simultaneously, calculate weightening finish, average weight gain and standard deviation according to the body weight of every chicken before and after the test; The relative weight gain rate is to calculate according to the ratio of each test group with the average weight gain of normal healthy controls group, and wherein the normal healthy controls group is made as 100%.
Table 4Cathelicidin1 antibacterial peptide is on the impact of S.aureus colony number and weightening finish in the test chick liver organization
Table 4 Influences of Cathelicidin1 on the counts of S.aureus in silver and weight of chickens
Figure BDA0000039921290000261
As shown in table 4, antibacterial peptide Cathelicidin1 fungistatic effect in vivo is obvious: wherein inject middle and high dosage antibacterial peptide test group (5mg/kg, 10mg/kg), after off-test, get hepatic homogenate liquid and coat the TMP flat board, be not separated to staphylococcus, test chicken there is no abnormal response.Each antibacterial peptide test group average weight gain all is higher than the infection control group, and wherein low dosage antibacterial peptide test group (2.5mg/kg) is though the interior fungistatic effect of body is suitable with common microbiotic, and its gaining effect is particularly evident.
By this experimental example as seen, synthetic chicken Cathelicidin family antibacterial peptide has the effect of significant bacteria growing inhibiting breeding in the chicken body, and has no side effect, and can be used as anti-microbial active matter and is applied to prepare the anti-microbial infection preparation.
Figure IDA0000039921380000011
Figure IDA0000039921380000021
Figure IDA0000039921380000031
Figure IDA0000039921380000051
Figure IDA0000039921380000061
Figure IDA0000039921380000081
Figure IDA0000039921380000091
Figure IDA0000039921380000101
Figure IDA0000039921380000111
Figure IDA0000039921380000121
Figure IDA0000039921380000131
Figure IDA0000039921380000141
Figure IDA0000039921380000151
Figure IDA0000039921380000161

Claims (3)

1. the preparation method of chicken antimicrobial peptides Cathelicidin1 is characterized in that, described chicken antimicrobial peptides Cathelicidin1, and its nucleotides sequence is as described in the SEQ ID NO.1;
(1) nucleotide sequence of PCR method amplification coding chicken antimicrobial peptides Cathelicidin1 mature peptide, PCR product warp EcoR I and XhoPET-30a (+) fusion expression vector of cutting with same enzyme behind the I double digestion is connected, transform the bacillus coli DH 5 alpha competent cell, extract plasmid, use the method screening positive clone of PCR and plasmid enzyme restriction, by the exactness of DNA sequence verification Insert Fragment reading frame, thereby construct prokaryotic expression carrier; With the prokaryotic expression carrier called after pET30-CathL1S that makes up;
(2) will check order plasmid pET30-CathL1S correct transforms intestinal bacteria Rosetta (DE3) bacterial strain, obtains the engineering bacteria Rosetta (DE3) of recombinant expressed Cathelicidins1 mature peptide fusion rotein/pET30-CathL1S; Picking engineering bacteria list colony inoculation to the LB substratum 37 ℃ cultivate 8 ~ 12h; Next day, the centrifuging and taking thalline was resuspended to fresh LB substratum, treated nutrient solution OD 600nmValue reaches at 0.5 ~ 0.6 o'clock and add IPTG, and to make its final concentration be 1.0mmol/L, begins to induce; 37 ℃ were continued to cultivate 4h, every 1 hour sampling 1mL; Thalline behind the centrifugal collection inducing culture washs thalline 2 times with the PBS damping fluid, adds lysis buffer, ultrasonication thalline in the ice bath, each broken 5s, interval 2s, power 600W, broken 10min again; Ultrasonic rear 12000r/min, 4 ℃ of lower centrifugal 10min separate soluble constituent and inclusion body, carry out SDS-PAGE electrophoresis and Western Blot and detect; Behind the electrophoresis with the band electrotransfer in the SDS-PAGE gel to nitrocellulose filter, take mouse-anti His monoclonal antibody as primary antibodie, sheep anti-mouse igg-HRP is two anti-, DAB is that chromogenic substrate carries out Western-blot and analyzes, and identifies expression product;
(3) then carry out the abduction delivering of engineering bacteria enlarged culturing and target protein, condition is: 25 ℃, IPTG concentration are inducing culture 8 hours under the condition of 1.0mmol/L; Get the thalline after inducing, add binding buffer liquid, ultrasonic disruption thalline in the ice bath, isolation of occlusion bodies and soluble constituent are splined on Ni with the cracking supernatant liquor 2+-NTA Sepharose 4B affinity column is washed post with the binding buffer liquid of 5 times of column volumes, and flow rate control is at 30mL/ hour, to remove other solubility compositions of unconjugated albumen and thalline; Wash post with the lavation buffer solution of 4 times of column volumes again, the protein ingredient of further flush away non-specific binding is until flow through liquid OD 280nm＜0.01; Sample is again through containing the buffer solution for gradient elution of 0.1 ~ 0.5mol/L imidazoles, with the target protein of the imidazoles gradient elution buffer solution elution combination of 5 times of column volumes, collects step by step by every part of 1mL, and monitoring OD 280nmWash post with the elution buffer of the high density imidazoles that contains 1mol/L of 3 times of column volumes again, the albumen that will be combined with the post bed fully elutes; Collect the fusion rotein elution peak and carry out the SDS-PAGE detection.
2. the preparation method of chicken antimicrobial peptides Cathelicidin1 as claimed in claim 1, it is characterized in that, the primer of the nucleotide sequence of described step (1) amplification coding chicken antimicrobial peptides Cathelicidin1 mature peptide is respectively CathL1S F/R, wherein F is forward primer, R is reverse primer, and the nucleotide sequence of described CathL1S F/R is respectively as described in the SEQ ID NO.22-23.
3. the preparation method of chicken antimicrobial peptides Cathelicidin1 as claimed in claim 1 or 2, it is characterized in that, the acquisition methods of the encoding gene of chicken antimicrobial peptides Cathelicidin1 is: extract total RNA in Adult White Leghorn myeloid tissue, behind synthetic cDNA the first chain of reverse transcription, again take cDNA as template, carry out PCR with gene-specific primer CathL1 P1/P2, amplified production is cathelicidin1; The PCR product separates and reclaims the purpose fragment through 2% agarose gel electrophoresis, the purpose fragment is connected with cloning vector pMD18-T and transforms E. coliDH5 α competent cell, positive strain checks order with M13 F/R primer, obtains containing the complete nucleotide sequence of cathelicidin1 open reading frame; The nucleotide sequence of described CathL1 P1/P2 is respectively as described in the SEQ ID NO.16-17.
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CN103333889A (en) * 2013-07-12 2013-10-02 南京敖众生物技术有限公司 Primer and method for preparing recombinant chicken antibacterial peptide Fowlicidin-3 by using primer
CN104211794B (en) * 2014-08-21 2018-01-23 大连理工大学 A kind of pigeon Cathelicidin-Cl CATH2 peptides and its gene, application
CN104650208B (en) * 2015-01-22 2018-04-13 东北农业大学 Derived peptide of one breeder derived antimicrobial peptide and its preparation method and application
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CN105753959A (en) * 2016-03-30 2016-07-13 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） Yeast expressed chicken antibacterial peptide Cathelicidin 2 and preparation method and application thereof
CN113493791A (en) * 2021-07-26 2021-10-12 广西壮族自治区兽医研究所 Cattle antibacterial peptide gene Cath and preparation method and application thereof
CN116262781B (en) * 2021-12-13 2024-05-14 中国农业大学 Antibacterial peptide descensin derivative, prokaryotic expression method and application thereof
CN115873076B (en) * 2022-11-24 2024-07-16 青岛农业大学 Antibacterial peptide, and expression and application thereof in bacillus subtilis

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Lynn,D J et al.GenBank：AY534900.1.《GenBank：AY534900.1》.2004, *
史春林 等.Fowlicidins及其功能研究.《动物医学进展》.2008,第29卷(第3期),54-59. *
姜亦飞.禽流感病毒抗体检测间接竞争ELISA方法的建立和初步应用.《中国优秀硕士学位论文全文数据库 农业科技辑》.2008,全文. *

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