CN100482683C

CN100482683C - Active fragment of human glycine-rich protein and use thereof

Info

Publication number: CN100482683C
Application number: CNB2005100769096A
Authority: CN
Inventors: 赵士富
Original assignee: Institute of Radiation Medicine of CAMMS
Current assignee: Institute of Radiation Medicine of CAMMS
Priority date: 2005-06-09
Filing date: 2005-06-09
Publication date: 2009-04-29
Anticipated expiration: 2025-06-09
Also published as: CN1699409A

Abstract

The invention discloses an active fragment of human glycine-rich protein and use, wherein the active fragment is polypeptide having one of the following amino acid residue sequences, (1) SEQ ID No.1 in the sequence table, (2) polypeptides with antibiotic action obtained through subjecting amino acid sequences in sequence table SEQ ID No:1 to one or several subjecting amino acid sequences of substitution and/or deletion and/or addition and/or glycosylation and/or carboxylation and/or acetylation and/or phosphorylation. The active fragment can be used as antibiotic peptides in the preparation of biological medicaments with antibiotic action.

Description

The active fragments of Homo sapiens Glyrichin and application thereof

Technical field

The present invention relates to the active fragments and the application thereof of Homo sapiens Glyrichin.

Background technology

Antibiotic is almost closely related with all biological healthy existence.A large amount of pathogenic infection individualities have been cured in the discovery of antibiotic, have saved countless life, are the medicines of human survival primary need, have brought into play the huge social economic benefit.Yet, nearly two during the last ten years, because the discovery of rare new antibiotic family and because the characteristics and the misapplication of antibiotic itself, anti-medicine, Resistant strain produce fast, the health of serious threat organism, the human infection has drug-fast golden yellow staphylococcus and the existing report of death cases.

Arising at the historic moment of antibacterial peptide will be brought a revolution of biomedicine field.The experimental results shows that antibacterial peptide is one of important composition composition of organism natural immune system, and they have guaranteed organism healthy existence in being full of the environment of the source of infection.So far, a large amount of insects, amphibian animal, higher plant, higher animal and even the mankind identify 700 surplus kind of antibacterial peptide.Discover that the common molecular weight of these antibacterial peptides is less, have good heat stable property, has a broad antifungal spectrum, anti-microbial effect is stronger, to resistant organism effectively and the target bacterium be difficult for or do not produce resistance.Studies show that of the antibacterial peptide mechanism of action, it by with the interaction of cytolemma, directly influence cell membrane stability, and then make cytolemma form ceasma, cause entocyte to leak and killing bacteria.This sterilization mechanism has been given it and has been had the basis of broad-spectrum antibacterial action.The above-mentioned advantage of antibacterial peptide has been established its rosy prospect in biological educational circles widespread use in future.That the part antibacterial peptide also has concurrently is antiviral, protozoacide and antineoplastic action, has further expanded the application prospect of antibacterial peptide.Therefore, closely during the last ten years, the academic paper number of studying about antibacterial peptide linearly rises, and multiple antibacterial peptide has entered preclinical study, and appearing on the market of antimicrobial peptide medicaments is in the near future.

The research of antibacterial peptide is still not fully up to expectations, in the antibacterial peptide of having found, mostly is inhuman source property greatly, influences the process that it develops into bio-pharmaceutical.The part antibacterial peptide has stronger hemolytic action, and having blocked it becomes the possibility of bio-pharmaceutical.Seek and find to have the key that the antibacterial peptide that develops into the good prospect of bio-pharmaceutical has become this research field.

Summary of the invention

The active fragments of Homo sapiens Glyrichin hL52 is the polypeptide with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and/or glycosylation and/or carboxylated and/or acetylize and/or phosphorylation modification and have the polypeptide of anti-microbial effect.

SEQ ID № in the sequence table: the 1st, the polypeptide of forming by 19 amino-acid residues, name is called pCM-19, be Homo sapiens Glyrichin (sequence 3) from N-terminal 42-60 amino acids residue.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation and/or glycosylation and/or carboxylated and/or acetylize and/or phosphorylation modification are meant replacement and/or disappearance and/or interpolation and/or glycosylation and/or carboxylated and/or acetylize and/or the phosphorylation modification that is no more than 10 amino-acid residues.

The described proteic active fragments of glycine that is rich in is preferably and has SEQ ID № in the sequence table: the polypeptide of 1 amino acid residue sequence.

The described proteic active fragments of glycine that is rich in can be synthetic by Peptide synthesizer, also can obtain as utilizing microbial expression by biotechnology, and wherein be preferred with the yeast expression system.

The encoding gene of the active fragments of above-mentioned Homo sapiens Glyrichin, and expression vector, clone and the host bacterium of encoding gene that contains the active fragments of above-mentioned Homo sapiens Glyrichin also belongs to protection scope of the present invention.

Experimental results show that the active fragments of above-mentioned Homo sapiens Glyrichin can suppress the growth of gram negative bacterium intestinal bacteria and gram negative bacterium plague bacillus, its anti-microbial effect mechanism is for destroying the integrity of cytolemma, and there is not hemolytic action, can be used as antibacterial peptide, be used to prepare bio-pharmaceutical with anti-microbial effect.Also the active fragments encoding gene of above-mentioned Homo sapiens Glyrichin can be inserted plasmid is used for antibiotic as nucleic acid vaccine.The active fragments of Homo sapiens Glyrichin of the present invention and encoding gene thereof can be widely used in the different field that needs antibacterial therapy and processing.

Description of drawings

Figure 1A induces and the semilog growth curve under the inductive condition not at 0.5mM IPTG for the e. coli bl21 that transformed pET-22b (+) empty carrier

Figure 1B induces and the semilog growth curve under the inductive condition not at 0.5mM IPTG for the e. coli bl21 that transformed pET-22b (+)-UBF and pET-22b (+)-PTH

Fig. 1 C has transformed the positive colony 1 of pET-22b-hL52 and 8 e. coli bl21 to induce and the semilog growth curve under the inductive condition not at 0.5mM IPTG

Fig. 2 A is pCM-19, pMG-19, and pMG-16, pMT-7 is to the influence of intestinal bacteria BL-21 growth

Fig. 2 B is pCM-19, pMG-19, and pMG-16, pMT-7 is to the influence of intestinal bacteria HB101 growth

Fig. 2 C is pCM-19, pMG-19, and pMG-16, pMT-7 is to the influence of bacillus coli DH 5 alpha growth

Fig. 3 is that the local directly point sample of pCM-19 is to the influence of e. coli bl21 bacteria growing

Fig. 4 is the influence of pCM-19 polypeptide to the e. coli bl21 growth

Fig. 5 is that various dose pCM-19 polypeptide and BL-21 bacterial colony generate the dose-effect relationship between the number

Fig. 6 is the influence of pCM-19 polypeptide to the growth of attenuation type plague bacillus

Fig. 7 A adds 30 minutes flow cytometry analysis test result of saline control group

Fig. 7 B is pCM-19 and 5 minutes flow cytometry analysis test result of target bacterium effect

Fig. 7 C is pCM-19 and 10 minutes flow cytometry analysis test result of target bacterium effect

Fig. 7 D is pCM-19 and 20 minutes flow cytometry analysis test result of target bacterium effect

Fig. 7 E is pCM-19 and 30 minutes flow cytometry analysis test result of target bacterium effect

Fig. 8 A-Fig. 8 D is that the e. coli bl21 that obtains amicillin resistance after penbritin and pET22b (+) transform acts on the variation that PI the transfect cell ratio after 5,10,20 and 30 minutes respectively

Fig. 8 E-Fig. 8 H is that the e. coli bl21 that obtains amicillin resistance after pCM-19 polypeptide and pET22b (+) transform acts on the variation that PI the transfect cell ratio after 5,10,20 and 30 minutes respectively

Fig. 9 is that various dose pCM-19 is to the erythrocytic hemolytic action of human peripheral

Embodiment

The contriver utilizes the inhibition difference to subtract the method for hybridization, and the mouse bone marrow difference expression gene of LTC (long term culture) being cultivated front and back has carried out a large amount of screenings, has obtained the EST clone of 131 differential expressions.Bioinformatic analysis confirms, these clones have represented gene and 7 brand-new genes of 26 kinds of known or part known functions, wherein 5 have complete open reading frame, article 3, register at GenBank, the mL52 gene in mouse source is exactly one of them, the full length cDNA sequence 521bp of this gene, its GenBank ^TMNumber of registration is AY028425.Associated viscera is referring to " experimental hematology magazine " 2002 the 10th phase 177-182 pages or leaves.

After obtaining mouse source mL52, the contriver is according to a pair of primer of mouse source gene design (5 ' primer Pa:5-CGATGCCGGTGGCCGTGGGTCCCT-3; 3 ' primer Pb:5-TTAGCATCGTATGCCCATTCCA-3), by pcr amplification people tire liver cDNA library, obtained Homo sapiens Glyrichin (hL52) homologous gene sequence, the complete ORF sequence is 240bp (sequence 2) altogether, and the aminoacid sequence of inferring according to the ORF sequence (sequence 3).The contriver utilizes the information biology means that sequence has been carried out detailed analysis: the nucleotide sequence comparative result demonstration hL52 assignment of genes gene mapping is made of 79 the amino acid whose small molecular proteins of encoding in human chromosome 20q11.21 zone three exons; Amino acid sequence analysis is the result show, it has tangible hydrophobic region, and pointing out this albumen may be a kind of secreted protein or membrane-associated protein.

After obtaining to have the hL52 gene of the complete ORF of coding, the contriver is building up to this gene (sequence 2) among the prokaryotic expression carrier pET-22b (+), obtains containing recombinant expression vector pET-22b (+)-hL52 of the human source gene of sequence 2.PET-22b (+)-hL52 is transformed in the e. coli bl21, relatively IPTG exists and does not deposit the variation of bacterial growth state in both cases again.The hL52 gene is not expressed when not adding the IPTG inductor, and only in nutrient solution, add IPTG induce after great expression; Further, under inductive condition,, then can not influence bacterial growth equally if hL52 does not have anti-microbial activity; If but hL52 has anti-microbial effect, then can obviously influence bacterial growth; Under inductive condition, along with incubation time prolongs, IPTG constantly consumes, and the expression of hL52 stops gradually, and the growth of bacterium should recover gradually at this moment in theory.Measured OD every 45 minutes ₆₀₀Once, measure 20 times altogether.With time is transverse axis, OD ₆₀₀Logarithmic value is that the longitudinal axis is done growth curve.The e. coli bl21 that has transformed pET-22b (+) empty carrier is induced and is not drawn semilog growth curve such as Figure 1A under the inductive condition at 0.5mMIPTG, transformed and do not had the independent basis of anti-microbial activity because of pET-22b (+)-UBF (GenBank number:UBF-f1 F294842, China's applied physiology magazine, 20:66,2004) and the e. coli bl21 of pET-22b (+)-PTH (GenBank NM000315) 0.5mM IPTG induce and not the semilog growth curve under the inductive condition see Figure 1B, the e. coli bl21 that has transformed pET-22b (+) to be measured-hL52

positive colony

1 and 8 0.5mM IPTG induce and not the semilog growth curve under the inductive condition see Fig. 1 C.The bacterial strain that the result shows single empty carrier pET-22b of commentaries on classics (+) is at the growth curve that adds and do not add bacterium under two kinds of situations of IPTG coincide substantially (Figure 1A); Change under two known situations that do not have anti-microbial activity gene UBF and a PTH single, add and do not add the growth unaffected equally (Figure 1B) of bacterium under two kinds of situations of IPTG; But, after changing the plasmid that contains the hL52 gene over to, add and do not add two kinds of situations of IPTG under the growth curve of bacterium obviously different, the growth of bacterium obviously is suppressed (Fig. 1 C) under the situation of adding IPTG; It can also be seen that from figure along with incubation time prolongs the IPTG approach exhaustion, the growth of bacterium is again with recovery, and other test cdnas all there is not this phenomenon.The above results proves absolutely that this antimicrobial effect is the effect of hL52 albumen itself.

After tentative confirmation hL52 gene had anti-microbial effect, the contriver was according to the aminoacid sequence of hL52 gene, salvage the polypeptide of different lengths, directly prove the proteic anti-microbial effect of hL52 and find the core peptide section from protein level.Article four, the position of peptide section is respectively the 31-46 (pMG-16) of hL52 aminoacid sequence, 42-60 (pCM-19), 53-59 (pMT-7), the individual amino acid of 61-79 (pMG-19).The purity of institute's section of synthesized peptide is 85-90%.Carrying out anti-microbial activity behind the above-mentioned peptide Duan Jingyong sterilization deionized water dissolving detects.The method that adopts is Kirby-Bauer disk diffusion (David T.Kingsbury et al.Microbiology, 2 ^NdEdition, ed by Harwal Publishing, 1990, page 37) method.The target bacteria that is adopted is e. coli bl21 (Fig. 2 A), intestinal bacteria HB101 (Fig. 2 B) and bacillus coli DH 5 alpha (Fig. 2 C) engineering bacteria.Fig. 2 A, label 1-5 is respectively pCM-19 32,64,128,256 and 512 μ g among Fig. 2 B and Fig. 2 C, and label 6 is pMG-19 512 μ g, label 7 is pMG-16 512 μ g, label 8 is pMT-7 512 μ g, and label 9 is penbritin 512 μ g, and label 10 is a solvent control.Can find by result among the figure, above-mentioned four peptide species solution are under the identical condition of concentration, and are similar to the effect of three kinds of target bacterium, and the anti-microbial effect difference of different polypeptide solutions is bigger during with concentration, wherein pCM-19 and pMT-7 have strong anti-microbial effect, and pCM-19 is stronger than pMT-7.

Further, the contriver adopts several different methods that pCM-19 has been carried out a series of analyses.At first, add the LB nutrient solution then and cultivated 3 hours, measure OD at 37 ℃ of shaking tables with pCM-19 and e. coli bl21 elder generation's acting in conjunction for some time when the smaller size smaller ₆₀₀The change of absorption value.The result shows among Fig. 4, and adds the LB nutrient solution merely or adds group of solvents relatively, and pCM-19 has obviously suppressed the growth of target bacteria, and is slightly poorer than effect with penbritin.In addition, have on the agar plate of e. coli bl21 growing, directly pCM-19 is dropped in the part, observe of the influence of this antibacterial peptide bacterial growth.Label 1 is penbritin 30 μ g/15 μ l among Fig. 3, and label 2-5 is respectively pCM-19 peptide solution 300 μ g/15 μ l, 200 μ g/15 μ l, and 100 μ g/15 μ l and 50 μ g/15 μ l, label 6 is a solvent control.Experimental result shows, with group of solvents relatively, pCM-19 has the effect of the engineering bacteria that directly kills local growth, when low partial concn, act on fully inadequately, the point sample circle is interior unintelligible.

In order to measure the amount-result relation between pCM-19 and the target bacteria, the pCM-19 solution that in the test tube of identical BL-21 bacteria concentration, adds different concns respectively, 37 ℃ act on 1 hour, get equal volume bacterium liquid then through suitably dilution, coat 1% agar plate surface, cultivated 20 hours at 37 ℃ again, count formed colony number.Fig. 5 result shows, when pCM-19 concentration is low, between itself and the target bacteria amount-result relation preferably arranged, and as pCM-19 in the system during greatly to 20 μ g, formed colony number reduces suddenly.This experimental result confirms that pCM-19 has definite anti-microbial effect, but only in low dose of scope amount-result relation is arranged, may be relevant with its mechanism of action.

Above-mentioned experimental result confirms that pCM-19 has definite anti-microbial effect, but just effective to engineering bacteria.In order to confirm whether it is effective equally to pathogenic bacterium, 100 μ g/200ul and 200 μ g/200ul pCM-19 peptide solutions are joined respectively contain 1 X 10 ⁴In the attenuation plague bacillus liquid of CFU/200ul, 37 ℃ act on 1 hour, then all mixed solutions are coated on 1% agar plate surface and continue cultivation 3 days.The result of Fig. 6 shows that the attenuation plague bacillus after pCM-19 handles has almost completely lost energy for growth.Illustrate that pCM-19 has lethal effect equally to the attenuation plague bacillus.

The analysis of the mechanism of action is useful replenishing to antibiotic effect assessment.Antibacterial peptide is normally realized its anti-microbial effect by changing membrane structure.Therefore, in pCM-19 and the effect of BL-21 bacterium, add the PI fluorescence dye, at different time sampling flush away free PI dyestuff and carry out flow cytometry analysis, observe fluorescence dye and the transfect cell number then with pCM-19 and the Changing Pattern of target bacteria interaction time.The results are shown in Figure 7A-E.The result shows among the figure, with solubilizing agent control group (30 minutes measurement results) relatively, pCM-19 effect promptly can be observed PI in 5 minutes and the transfect cell ratio and increase, to acting on 20 minutes and reach the peak, 30 minutes the transfect cell ratio reduce on the contrary.This experimental result has pointed out pCM-19 that the integrity of target bacteria cytolemma is had destruction.Not hard to imagine according to this experimental result, the different bacterium of pCM-19 cell membrane similar all has same effect.For this reason, it is target bacteria that the contriver selects pET22b (+)/BL21 that obtains amicillin resistance for use, with the penbritin of same dose as parallel control, analyzed pCM-19 and penbritin respectively with the interactional process of pET22b (+)/BL21 in, the Changing Pattern that the ratio that PI transfect cell prolonged with action time.Fig. 8 E-Fig. 8 H result shows, in pCM-19 effect group, PI the transfect cell ratio with prolonging action time and significantly increasing, and acts on 20 minutes and reaches the peak, and downtrending was arranged in 30 minutes.On the contrary, penbritin effect group (Fig. 8 A-Fig. 8 D) is in the process of effect in 30 minutes, and PI almost no change of transfect cell ratio.This experimental result confirms that penbritin is obviously different with the anti-microbial effect mechanism of pCM-19, and pCM-19 has obviously influenced the integrity of target bacteria film.

The multiple antibacterial peptide of having found has hemolytic action, and can this effect have determined antibacterial peptide develop into the destiny of bio-pharmaceutical.The contriver is with the positive contrast of 0.1%TritonX-100, compare with the negative contrast of solvent and with penbritin, respectively with 10 μ g/100 μ l, 20 μ g/100 μ l, 50 μ g/100 μ l, 100 μ g/100 μ l, 200 μ g/100 μ l, the pCM-19 of 300 μ g/100 μ l joins in the anti-freezing human red cell suspension of 100 μ l, and 37 ℃ act on 1 hour, and the centrifuging and taking supernatant is measured the absorbance value of 570nm with Cary 50 Bio UV-visible Spectrophotometer instrument.Fig. 9 result shows that the pCM-19 of various dose does not almost have hemolytic action to HRBC.Further, with solvent in contrast, give the pCM-19 solution of normal Balb/c mouse mainline 5mg/0.2ml/kg and 10mg/0.2ml/kg respectively, injected back 20 minutes and 2 hours, tail vein is analyzed with SysmexF-820 hemocyte automatic analyser.Table 1 and table 2 result show, behind the injection various dose pCM-19 after 20 minutes and 2 hours, and the no abnormal variation of mouse active state, peripheral red blood cells counting and content of hemoglobin and control group are than no marked difference.Experimental result shows that all pCM-19 does not have the effect of lysed erythrocyte in the above-mentioned external and body.

In sum, pCM-19 has definite anti-microbial effect, and its mechanism of action is the integrity that influences target mycetocyte film; The interior experiment confirm of external and body, the effect of its no lysed erythrocyte.Therefore, pCM-19 has the good prospect that develops into the bio-pharmaceutical with anti-microbial effect, can be widely used in the different field that needs antibacterial therapy and processing.

The hL52 of embodiment 1, endogenous abduction delivering is to the growth inhibition test of e. coli bl21

1, the structure of pET-22b-hL52

To not recombinate between the EcoR I and Xho I recognition site of prokaryotic expression carrier pET-22b (+) (available from Novagen company) with the hL52 gene (sequence 2) of any label, transformed into escherichia coli BL21, the upgrading grain is cut by enzyme and to be identified positive colony 1 and 8.To between the EcoR of pET-22b (+) I and Xho I recognition site, be inserted with the plasmid called after pET-22b-hL52 of hL52 gene.

2, the structure of control plasmid pET-22b (+)-UBF and pET-22b (+)-PTH

With UBF gene (GenBankUBF-fl AF294842; China's applied physiology magazine, 20:66,2004) and PTH gene (34 peptide gene sequences of Rat parathyroid hormone 1-34) (GenBank NM000315) multiple clone site that is cloned into pET-22b (+) respectively obtain control plasmid pET-22b (+)-UBF and pET-22b (+)-PTH.With pET-22b (+)-UBF and pET-22b (+)-PTH difference transformed into escherichia coli BL21, screening obtains containing the positive colony of pET-22b (+)-UBF, contains the positive colony of pET-22b (+)-PTH.

3, growth inhibition test

Picking contains the

positive colony

1 and 8 of pET-22b-hL52 respectively, the positive colony that contains pET-22b (+)-UBF, the positive colony that contains pET-22b (+)-PTH, be inoculated into respectively in the liquid LB substratum (wherein penbritin is 50 μ g/ml) of AMP resistance, in 37 ℃, the shaking table of 250rpm is cultivated 12h; Volume ratio with 1:100 inserts test tube, and continuing to be cultured to the OD value is 0.03 o'clock, and adding IPTG in every test tube is 0.5mM to final concentration, and negative control group then adds the PBS of respective volume; Shake bacterium in 30 ℃, 250rpm, took out 1 milliliter of bacterium liquid every 45 minutes and survey the OD600 value, survey continuously more than 10 times.With time is transverse axis, and the OD600 logarithmic value is that the longitudinal axis is done growth curve.Result such as Figure 1A, shown in Figure 1B and Fig. 1 C, Figure 1A shows that the bacterial strain of single empty carrier pET-22b of commentaries on classics (+) is adding and do not adding under two kinds of situations of IPTG, the growth curve of bacterium is identical substantially; Figure 1B shows single to be changeed under two known situations that do not have anti-microbial activity gene UBF and a PTH, adds and does not add under two kinds of situations of IPTG, and the growth of bacterium is unaffected equally; Fig. 1 C shows, after changing people hL52 gene over to, add with do not add two kinds of situations of IPTG under the growth curve of bacterium obviously different, the growth of bacterium obviously is suppressed in the previous case, from figure, it can also be seen that, along with incubation time prolongs the IPTG approach exhaustion, the growth of bacterium is again with recovery, and other test cdnas all do not have this phenomenon.The above results proves absolutely that this inhibitory effect is the effect of hL52 albumen itself.

The anti-microbial effect analysis of embodiment 2, synthetic different lengths hL52 protein polypeptide

According to the aminoacid sequence of hL52 gene, by the biochemical company limited of gill salvage the polypeptide of different lengths, the position of four peptide sections is respectively the 31-46 (H of hL52 aminoacid sequence ₃N ⁺-Met-Ala-Ala-Gly-Ala-Leu-Phe-Gly-Thr-Phe-Ser-Cys-Leu-Arg-Ile-Gly-COO ^-, pMG-16), and 42-60 (sequence 1, pCM-19), 53-59 (pMT-7, H ₃N ⁺-Met-Gly-Gly-Ile-Gly-Lys-Thr-COO ^-), 61-79 (pMG-19, H ₃N ⁺-Met-Gln-Ser-Gly-Gly-Thr-Phe-Gly-Thr-Phe-Met-Ala-Ile-Gly-Met-Gly-Ile-Arg-Cys-COO ^-) individual amino acid.The purity of institute's section of synthesized peptide is 85-90%.Be dissolved into 20mg/ml with aseptic deionized water.The method that antibacterial experiment adopts is Kirby-Bauer diskdiffusion (David T.Kingsbury et al.Microbiology, 2 ^NdEdition, ed by HarwalPublishing, 1990, page 37) method.To contain the glass dish that 1% agar LB substratum is tiled in 15cm diameter behind the autoclaving, after the agar condensation with OD ₆₀₀=4.0 30 μ l bacterium liquid (e. coli bl21s, HB101 or DH5 α) be diluted to 2ml through LB, be uniformly coated on LB agar solid culture primary surface respectively, in the grid of each delimitation, place the sterilization filter paper of a diameter 8mm, 15 μ l specimen are dripped on the filter paper.With plate be placed in 37 ℃ of incubators cultivate 4-10 hour after, take out the plate observations, with penbritin positive control and solvent (aseptic deionized water) negative control group relatively, with the big or small result of determination of inhibition zone.Result such as Fig. 2 A shown in Fig. 2 B and Fig. 2 C, show that above-mentioned four peptide species solution are under the identical condition of concentration, effect to three kinds of target bacterium is similar, the anti-microbial effect difference of different polypeptide solutions is bigger during with concentration, and wherein pCM-19 and pMT-7 have strong anti-microbial effect, and pCM-19 is stronger than pMT-7.Fig. 2 A, label 1-5 is respectively pCM-19 32,64,128,256 and 512 μ g among Fig. 2 B and Fig. 2 C, and label 6 is pMG-19 512 μ g, label 7 is pMG-16 512 μ g, label 8 is pMT-7 512 μ g, and label 9 is penbritin 512 μ g, and label 10 is a solvent control.

The anti-microbial effect of embodiment 3, synthetic pCM-19 polypeptide is measured

Direct point sample method: with peptide solution (solvent is an aseptic deionized water) 50 μ g/15 μ l, 100 μ g/15 μ l, 200 μ g/15 μ l, 300 μ g/15 μ l and 30 minutes the pCM-19 300 μ g/15 μ l of 80 ℃ of heating of the different pCM-19 content of equal-volume, and the aseptic deionized water solution of isopyknic aseptic deionized water and isopyknic penbritin 300 μ g/15 μ l, directly point has on the 1% agar LB culture plate of intestinal bacteria BL-21 growing respectively, cultivated 6-10 hour for 37 ℃, observe size and sharpness that pCM-19 drips the local inhibition zone of sample.The result as shown in Figure 3, experimental result shows, with group of solvents relatively, pCM-19 has the effect of the engineering bacteria that directly kills local growth, when low partial concn, act on fully inadequately, the point sample circle is interior unintelligible.Label 1-4 is respectively pCM-19 polypeptide solution 50 μ g/15 μ l among Fig. 3,100 μ g/15 μ l, 200 μ g/15 μ l and 300 μ g/15 μ l, label 5 is 30 minutes pCM-19 300 μ g/15 μ l of 80 ℃ of heating, and label 6 is a solvent control, and label 7 is penbritin 300 μ g/15 μ l.

Test tube method: get OD ₆₀₀=2.5 e. coli bl21 test bacterium liquid 8 μ l add the LB liquid nutrient medium and are diluted to 20 μ l, add the aseptic deionized water (H of 300 μ g/20 μ l pCM-19 peptide solutions (solvent is an aseptic deionized water) (pCM-19 group) or 20 μ l again ₂O group) or LB liquid nutrient medium (LB group) or the 300 μ g/20 μ l penbritin solution (solvent is an aseptic deionized water) (Amp group) of 20 μ l, 37 ℃ of effects 30 minutes, add LB nutrient solution 4ml again, at 37 ℃, 175rpm, shaking table was cultivated 3 hours, measured OD with Cary 50 BioUV-visible Spectrophotometer instrument ₆₀₀Absorbance value.The result shows and adds merely the LB nutrient solution or add group of solvents relatively as shown in Figure 4, and pCM-19 has obviously suppressed the growth of target bacteria.The LB group is the contrast of target bacterium normal growth condition; H ₂The O group is solvent (aseptic deionized water) control group; The Amp group is antibacterial growth control group.Above-mentioned each group has 3 samples.

Similar approach is used to measure the amount-result relation between pCM-19 and the target bacteria.In 15 test tubes, add OD respectively ₆₀₀The e. coli bl21 bacterium liquid 5 μ l of nm=0.3 also are diluted to 20 μ l with the LB liquid nutrient medium, these 15 test tubes are divided into 5 groups, add the aseptic deionized water solution (0,5,10,20,50 μ g/20 μ l) of the pCM-19 of 20 μ l different concns respectively, each dose point is 3 repetitions.37 ℃ act on 1 hour, get 0.2ml bacterium liquid then respectively and are diluted to 1.0ml with sterilized water, coat 1% agar plate surface, cultivate 12 hours at 37 ℃ again, count formed colony number.The result shows when pCM-19 concentration is low as shown in Figure 5, and bacterium colony generates between the polypeptide amount of number and adding amount-result relation preferably, and as pCM-19 in the system during greatly to 20 μ g, formed colony number is reduced to suddenly almost asepticly is born.This experimental result confirms that pCM-19 has definite anti-microbial effect, but only in low dose of scope amount-result relation is arranged.

Further, containing 1 * 10 ⁴The pCM-19 polypeptide solution that adds 200 μ l aseptic deionized waters (negative control), 100 μ g/200 μ l and 200 μ g/200 μ l in the every test tube of CFU/200 μ l attenuation plague bacillus (epidemiological study institute of Military Medical Science Institute) respectively, 37 ℃ act on 1 hour, whole mixed solutions are uniformly coated on 1% agar plate surface, continue to cultivate 3 days.The result shows that the contrast flat board has the growth of a large amount of plague bacilluses as shown in Figure 6, and has lost energy for growth fully with attenuation plague bacillus after 100 μ g or the 200 μ g pCM-19 polypeptide effects.Among Fig. 6, A is the aseptic deionized water contrast; B is 100 μ g effect groups; C is 200 μ g effect groups.

Embodiment 4, pCM-19 anti-microbial effect Mechanism analysis

Utilize PI (propidium iodide) fluorescence dye to mix the principle of the imperfect cell of cytolemma, in pCM-19 and e. coli bl21 effect, add the PI fluorescence dye, at different time sampling flush away free PI dyestuff and carry out flow cytometer (FACS) analysis, observe fluorescence dye and transfect cell number and pCM-19 and the Changing Pattern of target bacterium interaction time then.Get OD ₆₀₀=0.3 e. coli bl21 bacterium liquid 50 μ l add in the testing tube, add physiological saline 50 μ l, add PI dyestuff 100 μ l again, and 37 ℃, the 160rpm effect was organized in 30 minutes in contrast.As experimental group, get OD ₆₀₀=0.3 e. coli bl21 bacterium liquid 50 μ l add respectively in 12 testing tubes, be divided into 5,10,20 and 30 minutes 4 time points, each time point is established 3 testing tubes, contain bacterium liquid 50ul in each test tube, pCM-19 peptide solution 50ul (50 μ g) and 100 μ, 1 PI, 37 ℃, the 160rpm shaking table acts on different time respectively.Each sample to the time is all washed twice to remove free PI with physiological saline, again with 500 μ l physiological saline suspension bacteria liquid again and carry out facs analysis.The result is shown in Fig. 7 A-E, show that adding 30 minutes PI positive bacteria ratio of brinish control group only is 1.7%, and the pCM-19 effect promptly can be observed PI in 5 minutes and the transfect cell ratio and increase, and reached the peak to acting on 20 minutes, and 30 minutes PI the transfect cell ratio and reduce on the contrary.PI positive bacteria percentage ratio is the mean number of 3 samples among Fig. 7 A-E, and M1 is illustrated in the cell count in this fluorescence intensity scope.This experimental result has pointed out pCM-19 that the integrity of target mycetocyte film is had destruction.

Adopt and use the same method, tested pCM-19 the BL21 bacterium that obtains amicillin resistance (with pET-22b (+) transformed into escherichia coli BL21, has been screened the influence of e. coli bl21-pET22b (+)/BL21) cell membrane integrity that contains pET-22b (+) that obtains.Get OD ₆₀₀=0.5 o'clock amicillin resistance bacterium liquid (pET22b (+)/BL21) 100 μ l/ pipe adds penbritin or the equivalent pCM-19 peptide solution of 100 μ l (200 μ g) respectively, add again PI to final concentration be 25 μ g/ml.37 ℃ of shaking tables, 160rpm acts on 5,10,20 and 30 minutes respectively.3 samples of each time point.FACS specimen preparation and test the same.The result is shown in Fig. 8 A-H, and the result shows in pCM-19 effect group (Fig. 8 E-H), and PI the transfect cell ratio with prolonging action time and significantly increasing, and acts on 20 minutes and reaches the peak, and downtrending was arranged in 30 minutes.On the contrary, penbritin effect group (Fig. 8 A-D) is in the process of effect in 30 minutes, and PI almost no change of transfect cell ratio.Each time point is 3 samples among Fig. 8 A-H, and PI positive bacteria percentage ratio is the mean number of 3 samples, and M1 is illustrated in the cell count in this fluorescence intensity scope.This experimental result confirms that penbritin is obviously different with the anti-microbial effect mechanism of pCM-19, and pCM-19 has obviously influenced the integrity of target bacteria film.

The hemolytic action test of embodiment 5, pCM-19 polypeptide

In vitro method: get fresh anti-freezing human blood 100 μ l and join in the serial test tube, add stroke-physiological saline solution 100ul respectively or 10 μ l deionized waters add 90ul physiological saline as negative control group, or add aseptic deionized water solution 100 μ l (the 300 μ g) group of making comparisons of penbritin, or the 0.1%TritonX-100 that adds 100 μ l is as the haemolysis positive controls.Experimental group adds the aseptic deionized water solution that volume is the pCM-19 peptide of 100 μ l respectively, and the content of pCM-19 is 10,20,50,100,200,300 μ g.Each dose point is 3 samples.After 1 hour, centrifugal 1000g 5 minutes, collects supernatant and measures OD with Cary 50Bio UV-visible Spectrophotometer instrument all samples through 37 ℃ of insulations ₅₇₀The absorption value of nm.The result shows that pCM-19 does not have obvious hemolytic action to HRBC as shown in Figure 9.Among Fig. 9, Control (NS) is a 100ul stroke-physiological saline solution (quantity of solvent that is equivalent to peptide solution); H2O is that 10 μ l deionized waters add 90ul physiological saline (quantity of solvent that is equivalent to peptide solution).

Intracorporal method: at first, female Balb/c mouse is divided into three groups at random, 3/group.Negative control group tail vein injection 0.2ml physiological saline, two experimental group are injected the aseptic deionized water solution (dosage is 5mg/kg or 10mg/kg) of the pCM-19 of 0.2ml respectively.Injected back 20 minutes and 2 hours, and got tail vein 10 μ l respectively, require the dilute sample promoting circulation of blood picture analysis of going forward side by side according to Sysmex F-820 hemocyte automatic analyser.Animal is not found untoward reaction behind injection peptide solution or physiological saline.Analysis on hemogram result as shown in Table 1 and Table 2, showing between RBC, content of hemoglobin and other composition test value of three treated animals does not have significant difference, no haemolysis.

20 minutes blood picture measured value behind the pCM-19 of table 1. mouse tail vein injection 5mg/kg or 10mg/kg body weight

Annotate: M ± SD is the arithmetical av ± standard deviation of 3 mouse samples

^*Compare 0.01＜P＜0.05 with control group, all the other organize P〉0.05

2 hours blood picture measured value behind the pCM-19 of table 2. mouse tail vein injection 5mg/kg or 10mg/kg body weight

^*Compare 0.01＜P＜0.05 with control group, all the other organize P〉0.05

Sequence table

<160>3

<210>1

<211>19

<212>PRT

＜213〉Genus Homo people (Homo sapiens)

<400>1

<210>2

<211>240

<212>DNA

＜213〉Genus Homo people (Homo sapiens)

<400>2

<210>3

<211>79

<212>PRT

＜213〉Genus Homo people (Homo sapiens)

<400>3

Claims

1, the active fragments of Homo sapiens Glyrichin is SEQ ID № in the sequence table: the polypeptide of 1 amino acid residue sequence.

2, the encoding gene of the active fragments of the described Homo sapiens Glyrichin of claim 1.

3, the expression vector of encoding gene that contains the active fragments of the described Homo sapiens Glyrichin of claim 2.

4, the transgenic cell line of encoding gene that contains the active fragments of the described Homo sapiens Glyrichin of claim 2.

5, the host bacterium of encoding gene that contains the active fragments of the described Homo sapiens Glyrichin of claim 2.

6, the active fragments of the described Homo sapiens Glyrichin of claim 1 is as the application in the antibacterial peptide.

7, the active fragments of the described Homo sapiens Glyrichin of claim 1 has application in the bio-pharmaceutical of anti-microbial effect in preparation.

8, the application of active fragments encoding gene in antibiotic of the described Homo sapiens Glyrichin of claim 2.