CN105237630A - Pesticin and phage lysozyme fusion protein and encoding gene and application thereof - Google Patents

Abstract

The invention relates to a pesticin and phage lysozyme fusion protein and an encoding gene and an application thereof. The fusion protein sequentially contains a pesticin protein Pst of yersinia pestis and a lysozyme e of a phage Bp7 from an amino terminal to a carboxyl terminal; the fusion protein gene is composed of 990 nucleotides. The amino acid sequence of the fusion protein is encoded, so that the obtained fusion protein has a relatively good effect in preventing or/and treating staphylococcus aureus.

Description

Pesticin and bacterial virus bacteriolysis enzyme fusion proteins and encoding gene thereof and application

Technical field

the present invention relates to biological technical field, relate to a kind of recombination fusion protein and uses thereof, in particular to the gene of a kind of fusion rotein of being formed by pesticin albumen Pst and phage lysozyme e and coding thereof, and the application of this fusion rotein in anti-Staphylococcus aureus.

Background technology

phage be a class can bacterial infection, fungi, the microbial virus such as actinomycetes or spirochete general name, as antibiotic substitute, there is potential Development volue.Phage acts on the peptidoglycan composition in bacteria cell wall mainly through N,O-Diacetylmuramidase, cause bacteria lysis.N,O-Diacetylmuramidase results from the biosynthesizing late period in the bacterial virus bacteriolysis cycle, can be highly purified, have when killing and wounding bacterium action time short, not easy in inactivation, not by bacterial drug resistance restriction, to advantages such as humans and animals have no side effect, and the fragmentation pattern of N,O-Diacetylmuramidase generally than its source phage splitting wider.Due to the textural difference of bacteria cell wall, N,O-Diacetylmuramidase is different with the fragmentation effect of Gram-negative bacteria to gram-positive microorganism.Gram-positive bacteria cell wall forms primarily of peptidoglycan, and the N,O-Diacetylmuramidase of lower concentration can play obvious bactericidal effect, but also has one deck membrane bound outside the peptidoglycan layer of gram-negative bacteria cell wall, and N,O-Diacetylmuramidase can not produce destruction from outside to it.

pesticin is a kind of bacteriocin that yersinia pestis produces, and pesticin can some pathogenic colon bacillus of cracking, but does not have toxicity to zooblast.Pesticin sequence N holds 1-164 position to be membrane-spanning domain and receptor binding domains, can in conjunction with the outer membrane protein FyuA of Bacillus coli cells wall, and the energy system on triggering cell film, pesticin is transported into periplasmic space subsequently.Pesticin C holds 168-357 position to be active binding domain, the β-Isosorbide-5-Nitrae glycosidic link that can rupture on peptidoglycan chain, and degraded peptidoglycan, causes bacteria lysis.But because pesticin is as a kind of bacteriocin, its effect is more weak, has no the report of clinical application.

pesticin C holds active binding domain can the peptidoglycan of bacterium for degrading cell walls, thus cause bacteria lysis, the functional similarity of this and phage lysozyme, analyze sequence similarity both finding and only have 13%, but its space structure is very similar, show that its mode of action has similarity.Therefore in theory with the fusion rotein that N,O-Diacetylmuramidase sequence replacing pesticin C holds active binding domain sequence to be formed, the high reactivity of lysozyme lysis gram-positive microorganism can be possessed, the membrane-spanning domain simultaneously utilizing fusion rotein N to hold and receptor binding domains are through intestinal bacteria adventitia, thus cracking intestinal bacteria.There is not been reported in the current research for this kind of fusion rotein.

Summary of the invention

object of the present invention makes to provide a kind of pesticin and bacterial virus bacteriolysis enzyme fusion proteins and encoding gene thereof and application.

fusion rotein provided by the invention contains the pesticin albumen Pst of plague bacillus and the N,O-Diacetylmuramidase e of phage Bp7 from aminoterminal successively to carboxyl terminal.

the aminoacid sequence of described pesticin albumen Pst is the 1-167 position of sequence 1 in sequence table; The aminoacid sequence of described N,O-Diacetylmuramidase e is the 168-330 position of sequence 1 in sequence table.

described fusion rotein is specially (1) or (2) as follows:

(1) albumen be made up of the aminoacid sequence shown in sequence in sequence table 1;

(2) aminoacid sequence in sequence 1 is replaced and/or disappearance and/or add and have the albumen derived by sequence 1 of identical function through one or several amino-acid residue.

wherein, sequence 1 is made up of 330 amino acid, and 1-167 position is described pesticin albumen Pst, and 168-330 position is described N,O-Diacetylmuramidase e.

the encoding gene of described fusion rotein is also the scope of protection of the invention.

the encoding gene of described fusion rotein is specially arbitrary described gene in following (1)-(3):

(1) DNA molecular shown in sequence 2 in sequence table;

(2) DNA molecular limited with (1) has more than 90% homology, and the DNA molecular of encoding said fusion protein;

(3) DNA molecule hybridize limited with (1) or (2) under strict conditions, and the DNA molecular of encoding said fusion protein.

above-mentioned stringent condition can be the solution with 6 × SSC, 0.5%SDS, hybridizes, then use 2 × SSC under 65 DEG C of conditions, and 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

wherein, sequence 2 is made up of 990 Nucleotide, fusion rotein shown in encoding sequence 1.

recombinant vectors containing described gene, expression cassette, reconstitution cell or recombinant bacterium also belong to protection scope of the present invention.

described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.

in an embodiment of the present invention, described recombinant expression vector is the recombinant plasmid (pET28a-Pst-E) obtained after the multiple clone site place of Pet28a carrier inserts described gene.Described multiple clone site is specially EcoRI and XhoI.

described expression cassette by the promotor that can start described genetic expression, described gene, and transcription termination sequence composition.

described recombinant bacterium is described recombinant expression vector is poured into the recombinant bacterium that Host Strains obtains, and described Host Strains is preferably intestinal bacteria, and described intestinal bacteria are preferably BL21.

described fusion rotein, or described gene, or described recombinant expression vector, expression cassette, reconstitution cell or recombinant bacterium for the preparation of prevention or/and the biological products for the treatment of fowl source colibacillosis also belong to protection scope of the present invention.

another object of the present invention makes to provide a kind of prevention or/and the biological products for the treatment of streptococcus aureus disease.

prevention provided by the present invention is or/and the biological products for the treatment of streptococcus aureus disease, and its activeconstituents is described fusion rotein, described gene, described recombinant expression vector, expression cassette, reconstitution cell or described recombinant bacterium.

sequence 1:

MSDTMVVNGSGGVPAFLFSGSTLSSYRPNFEANSITIALPHYVDLPGRSNFKLMYIMGFPIDTEMEKDSEYSNKIRQESKISKTEGTVSYEQKITVETGQEKDGVKVYRVMVLEGTIAESIEHLDKKENEDILNNNRNRIVLADNTVINFDNISQLKEFLRRSVNIVMDIFDMLRQGEGLDLNLYKDTEGYWTIGIGQLVTKNPSKDVARAELDKLMGRVCNGRITMAEAEQLFNRSVENARRAILRNPKLKPVYDVLDEVRRCALINMVFQMGEAGVAGFTNSLRMLQQKRWNDAAVNLAQSRWYKQTPNRAKRVIATFKTGTWAAYR.

sequence 2:

ATGTCAGATACAATGGTAGTGAATGGTTCAGGTGGTGTTCCGGCTTTTCTCTTTTCCGGAAGTACATTAAGCAGTTACAGACCAAATTTTGAAGCTAATTCGATTACAATTGCATTACCACATTATGTGGATCTGCCTGGCCGGAGTAATTTTAAACTGATGTACATTATGGGGTTTCCGATTGATACGGAGATGGAGAAAGACAGTGAATATTCAAATAAGATCCGCCAGGAAAGTAAAATTTCAAAAACTGAAGGGACCGTGTCTTACGAACAGAAAATAACTGTTGAAACAGGTCAGGAAAAAGACGGTGTGAAAGTCTACCGTGTCATGGTTCTTGAGGGAACGATTGCCGAATCTATTGAACATCTCGATAAGAAAGAGAACGAAGACATTCTGAATAATAACCGAAATCGCATCGTCCTAGCGGACAACACTGTCATTAACTTTGACAATATTAGTCAACTGAAGGAATTTTTACGTCGTTCGGTAAATATTGTTATGGATATTTTTGATATGTTACGTCAAGGCGAAGGTCTTGATTTAAATCTTTATAAAGACACTGAAGGTTATTGGACTATTGGTATTGGCCAGTTAGTTACCAAGAACCCATCTAAAGATGTTGCTCGTGCTGAACTTGATAAGCTTATGGGTCGAGTTTGCAATGGTCGTATTACTATGGCTGAAGCCGAACAACTCTTTAACCGGTCGGTTGAAAATGCTCGTAGAGCTATTCTGCGTAATCCTAAATTGAAACCTGTTTATGATGTACTGGACGAAGTTCGTCGTTGTGCTTTAATTAACATGGTCTTCCAAATGGGTGAAGCAGGTGTAGCAGGGTTCACTAACTCTTTACGTATGCTCCAACAGAAACGTTGGAATGATGCGGCAGTTAACCTGGCCCAATCCCGTTGGTATAAACAAACTCCTAATCGTGCTAAGCGCGTTATCGCCACCTTTAAAACAGGTACTTGGGCTGCTTATAGATAA

the pesticin gene Pst of plague bacillus and the lysozyme gene e of phage Bp7 is together in series by the present invention first, constructs recombinant expressed recombinant plasmid pET28a-Pst-E, and obtains fusion rotein Pst-E at expression in escherichia coli.Compared with pesticin Pst, there is higher bacteria lysis active.Test proves, after pesticin Pst and the mutual effect of bacterium liquid phase, S. aureus bacterium number declines 22%, intestinal bacteria O78 bacterial count decline 14%.After fusion rotein Pst-E and the mutual effect of bacterium liquid phase, S. aureus bacterium number 44%, the intestinal bacteria O78 bacterial count that declines declines 2.7%.The present invention provides theory and practice foundation for developing the biological products of anti-Staphylococcus aureus.

Accompanying drawing explanation

fig. 1 is pesticin gene Pst, the lysozyme gene e of phage Bp7 and the PCR primer of both tandem gene Pst-e.Wherein, swimming lane M is DNA molecular amount standard DL2000Marker; Swimming lane 1 is the PCR primer (501bp) of Pst gene; Swimming lane 2 is the PCR primer (489bp) of e gene; Swimming lane 1 is the PCR primer (990bp) of Pst-e tandem gene.

fig. 2 is the double digestion qualification result of recombinant plasmid pET28a-Pst-E.Wherein, swimming lane M is DNA molecular amount standard DL2000Marker; Swimming lane 1 is the double digestion result of EcoRI and XhoI of recombinant plasmid pET28a-Pst-E.

fig. 3 fusion rotein Pst-ESDS-PAGE and western-blot detected result.Wherein, swimming lane 1 is that fusion rotein Pst-E is expressing the expression in bacterium BL21 precipitation; Swimming lane 2 is that fusion rotein Pst-E is expressing the expression in bacterium BL21 supernatant; Swimming lane M is standard protein molecular weight Marker; Swimming lane 3 is for carrying the protein expression situation of the expression bacterium BL21 of empty carrier pET28a; Swimming lane 4 is the content of the fusion rotein Pst-E after purifying; Swimming lane 5 is the western-blot detected result of fusion rotein Pst-E.

the bacteriostatic activity detected result of Fig. 4 fusion rotein Pst-E.Wherein Fig. 4 A is the bacteriostatic action of fusion rotein Pst-E to streptococcus aureus 25923; Fig. 4 B is that fusion rotein Pst-E is to the colibacillary bacteriostatic action of O78 type.In figure, arrow is depicted as the inhibition zone that fusion rotein Pst-E is formed; Pst and kantlex are respectively protein control and drug control.

the Bacteriolytic activity measurement result of Fig. 5 fusion rotein Pst-E.

Embodiment

the experimental technique used in following embodiment if no special instructions, is ordinary method.

material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

carry the plasmid of pesticin gene, the public can obtain from Qingdao Agricultural University of Shandong Province Animal Science And Technology microorganism and immunization experiment room.

carry the plasmid of phage lysozyme gene, the public can obtain from Qingdao Agricultural University of Shandong Province Animal Science And Technology microorganism and immunization experiment room.

all primers in following embodiment are by the raw work synthesis in Shanghai; Examining order completes by Hua Da Gene Tech. Company Limited.

the construction and expression of embodiment 1. recombinant expression plasmid pET28a-Pst-E

1. the Design and synthesis of primer

according to the sequence of pesticin gene (Pst) and phage lysozyme gene (e), design and synthesize primer (table 1).Wherein primers F 1, F2 are for the ORF of the Pst gene that increases, and primers F 3, F4, for the ORF of the e gene that increases, carry EcoRI and XhoI restriction enzyme site (underscore part) respectively in F1, F4 primer sequence.

the primer sequence of table 1 pesticin gene (Pst) and phage lysozyme gene (e)

2. the amplification of goal gene

respectively to carry the plasmid Pst-T of pesticin gene and to carry the plasmid e-T of phage lysozyme gene for template, adopt primer pair F1/F2 and F3/F4 of design in step 1 to carry out PCR reaction, increase pesticin gene Pst and phage lysozyme gene e respectively.Reaction conditions is 94 DEG C of denaturation 5min, then starts circulation: 94 DEG C of sex change 30s, and 52 DEG C of annealing 30s, 72 DEG C extend 40s, 30 circulations, last 72 DEG C of total elongation 5min.1.0% agarose gel electrophoresis checks PCR primer, and result as shown in Figure 1, obtains two bands that size is about 500bp, conforms to expected results.The PCR primer obtained increasing reclaims test kit with agarose gel purification respectively and carries out purifying recovery, confirms through order-checking the lysozyme gene being respectively pesticin gene and phage Bp7, by these two PCR primer called after Pst and e respectively.

the PCR primer Pst that obtains and e that increases being reclaimed test kit with agarose gel purification respectively and carries out purifying, with Pst and the e product after purifying for template, is that upstream and downstream primer carries out second and takes turns PCR reaction, the series connection fragment Pst-E of amplification Pst and e gene with F1/F4.Reaction conditions is 94 DEG C of denaturation 5min, then starts circulation: 94 DEG C of sex change 1min, and 52 DEG C of annealing 1min, 72 DEG C extend 1min, 30 circulations, last 72 DEG C of total elongation 10min.1.0% agarose gel electrophoresis checks PCR primer, and result as shown in Figure 1, obtains the object band that size is about 990bp, conforms to expected results.The PCR primer agarose gel purification obtained increasing reclaims test kit and carries out the object fragment that purifying obtains purifying, turns out to be the series connection fragment of the lysozyme gene of pesticin gene and phage Bp7, by this PCR primer called after Pst-E through order-checking.

3. the structure of recombinant expression plasmid pET28a-Pst-E

carry out double digestion with the PCR primer Pst-E of restriction enzyme EcoRI and XhoI to expression vector pET28a and purifying, after agarose gel electrophoresis checking, reclaim carrier segments and the object fragment linearly with sticky end.With T4DNA ligase enzyme, object fragment is connected with carrier pET28a, product conversion bacillus coli DH 5 alpha competent cell will be connected, screening positive clone, upgrading grain, and carry out double digestion qualification, order-checking.Double digestion result as shown in Figure 2, correctly insert between EcoRI and the XhoI recognition site of pET28a by Pst-E fragment; Sequencing result is consistent with expected results, by this recombinant expression plasmid called after pET28a-Pst-E.

the structrual description of recombinant expression plasmid pET28a-Pst-E is: the recombinant plasmid obtained after sequence 2 fragment in insertion sequence table between the multiple clone site EcoRI and XhoI of pET28a carrier.

sequence 2 is made up of 990 Nucleotide altogether, and wherein 1-501 position is described pesticin Pst gene, and 502-990 position is described phage lysozyme e gene.

albumen shown in sequence 1 in sequence 2 polynucleotide.Sequence 1 is made up of 330 amino acid altogether, and 1-167 position is described pesticin Pst albumen, and 168-330 position is described phage lysozyme e albumen.

sequence 1:

MSDTMVVNGSGGVPAFLFSGSTLSSYRPNFEANSITIALPHYVDLPGRSNFKLMYIMGFPIDTEMEKDSEYSNKIRQESKISKTEGTVSYEQKITVETGQEKDGVKVYRVMVLEGTIAESIEHLDKKENEDILNNNRNRIVLADNTVINFDNISQLKEFLRRSVNIVMDIFDMLRQGEGLDLNLYKDTEGYWTIGIGQLVTKNPSKDVARAELDKLMGRVCNGRITMAEAEQLFNRSVENARRAILRNPKLKPVYDVLDEVRRCALINMVFQMGEAGVAGFTNSLRMLQQKRWNDAAVNLAQSRWYKQTPNRAKRVIATFKTGTWAAYR.

sequence 2:

ATGTCAGATACAATGGTAGTGAATGGTTCAGGTGGTGTTCCGGCTTTTCTCTTTTCCGGAAGTACATTAAGCAGTTACAGACCAAATTTTGAAGCTAATTCGATTACAATTGCATTACCACATTATGTGGATCTGCCTGGCCGGAGTAATTTTAAACTGATGTACATTATGGGGTTTCCGATTGATACGGAGATGGAGAAAGACAGTGAATATTCAAATAAGATCCGCCAGGAAAGTAAAATTTCAAAAACTGAAGGGACCGTGTCTTACGAACAGAAAATAACTGTTGAAACAGGTCAGGAAAAAGACGGTGTGAAAGTCTACCGTGTCATGGTTCTTGAGGGAACGATTGCCGAATCTATTGAACATCTCGATAAGAAAGAGAACGAAGACATTCTGAATAATAACCGAAATCGCATCGTCCTAGCGGACAACACTGTCATTAACTTTGACAATATTAGTCAACTGAAGGAATTTTTACGTCGTTCGGTAAATATTGTTATGGATATTTTTGATATGTTACGTCAAGGCGAAGGTCTTGATTTAAATCTTTATAAAGACACTGAAGGTTATTGGACTATTGGTATTGGCCAGTTAGTTACCAAGAACCCATCTAAAGATGTTGCTCGTGCTGAACTTGATAAGCTTATGGGTCGAGTTTGCAATGGTCGTATTACTATGGCTGAAGCCGAACAACTCTTTAACCGGTCGGTTGAAAATGCTCGTAGAGCTATTCTGCGTAATCCTAAATTGAAACCTGTTTATGATGTACTGGACGAAGTTCGTCGTTGTGCTTTAATTAACATGGTCTTCCAAATGGGTGAAGCAGGTGTAGCAGGGTTCACTAACTCTTTACGTATGCTCCAACAGAAACGTTGGAATGATGCGGCAGTTAACCTGGCCCAATCCCGTTGGTATAAACAAACTCCTAATCGTGCTAAGCGCGTTATCGCCACCTTTAAAACAGGTACTTGGGCTGCTTATAGATAA

4. the Expression and purification of recombinant plasmid in intestinal bacteria

(1) expression of recombinant protein

recombinant plasmid heat shock method is proceeded in e. coli bl21 competent cell, in being inverted overnight incubation containing on the LB flat board of Kana under 37 DEG C of conditions.The single bacterium colony of random picking, inoculation is containing the LB liquid nutrient medium of Kana, 37 DEG C of shaking culture are about 2h makes the OD600 of nutrient solution be about 0.6-0.8, adding final concentration is that 0.8mmol/LIPTG inducible protein is expressed, collected by centrifugation thalline after 37 DEG C of shaking culture 5h, SDS-PAGE electrophoresis detection expression of recombinant proteins situation, sets the BL21 carrying pET28a empty plasmid of abduction delivering under the same terms as contrast simultaneously.As shown in Figure 3, fusion rotein Pst-E is mainly with inclusion bodies correction in expression bacterium BL21 for electrophoresis detection result; Contrast thalline is negative, proves do not have expressing fusion protein.

(2) qualification of recombinant protein

obtain fusion rotein Pst-E with step (1) and carry out SDS-PAGE electrophoresis, carry out western-blot detection subsequently.Western-blot detects and makes primary antibodie used be the monoclonal antibody of mouse-anti HIS label, and two resist the goat anti-rabbit igg antibody for HRP mark.As shown in Figure 3, fusion rotein Pst-E obtains correction to western-blot detected result.

(3) purifying of recombinant protein

bacterium liquid collected by centrifugation thalline after the IPTG induction that step (1) is obtained, with 50mL lysis buffer (the Tris-HCL damping fluid that composition is 1mol/L, pH value is 8.0,5mol/LNaCl, 0.5mol/LEDTA, 5% glycerine) resuspended thalline, 30 DEG C of water-bath effect 10min, the ultrasonication of ice bath interval, condition is: ultrasonic 3s, interval 1s, altogether 30min, stop ultrasonic when bacterium liquid is more limpid, the centrifugal 10min of 12000r/min obtains bacterial cell disruption thing precipitation, and this precipitation is inclusion body crude extract.Add in inclusion body crude extract 9 times of volumes inclusion body washings I (composition to be 50mmol/LpH value be 8.0 Tris-HCl damping fluid, 100mmol/LNaCl, 2mmol/LEDTA, 0.5%TritionX-100) resuspended precipitation, normal temperature effect 15min after mixing gently, 4 DEG C of centrifugal 20min of 12000r/min, then by similar steps, precipitation is resuspended in inclusion body washings II (composition is add the urea that final concentration is 2mol/L in above-mentioned inclusion body washings I) and inclusion body washings III (composition is add the urea that final concentration is 4mol/L in above-mentioned inclusion body washings I) successively, wash inclusion body successively, in precipitation, the solubilization of inclusion bodies liquid (composition be add urea that final concentration is 8mol/L in above-mentioned inclusion body washings I and final concentration is the DTT of 5mmol/L) of 9 times of volumes is added after 4 DEG C of 12000r/min centrifugal 20min supernatant discarded, normal temperature vibration 2h, 4 DEG C of centrifugal 20min of 12000r/min get supernatant and add in the dialysis tubing handled well that (dialysis tubing 2%NaHCO3 soaks and boils 10min, steep in 1mmol/LEDTA again after cleaning with distilled water and boil 10min, use after cooling), gradient dialysis renaturation is carried out under 4 DEG C of conditions, namely dialysis tubing puts into urea concentration successively after sealing is 6mol/L, 4mol/L, 2mol/L, 1mol/L, in the renaturing inclusion bodies damping fluid of 0mol/L (pH value is the urea adding EDTA that final concentration is 1mmol/L and above-mentioned different final concentration in the PBS damping fluid of 7.2 respectively), the renaturing inclusion bodies damping fluid effect 12h of each gradient, finally take out the centrifugal 20min of liquid 4 DEG C of 12000r/min in dialysis tubing and collect supernatant, obtain the Pst-E albumen of purifying.The Pst-E protein solution of purifying is carried out SDS-PAGE electrophoresis, and as shown in Figure 3, object product is near 41KDa, consistent with expected results, and purity reaches more than 90% for result.

the Activity determination of embodiment 2. fusion rotein Pst-E

(1) bacteriostatic activity of fusion rotein Pst-E detects

by streptococcus aureus ATCC25923(purchased from China Veterinary Drugs Supervisory Inst.) be that O78 type intestinal bacteria (being separated preservation with immunization experiment room by Qingdao Agricultural University Animal Science And Technology microorganism) are cultured to logarithmic phase (bacterial concentration reaches 108cfu/mL) respectively with serotype, get 200 μ L bacterium liquid at plain agar planar surface even spread, sterilizing Oxford cup (diameter 8mm) is positioned over the surface of substratum, at the Pst-E protein solution that its 0.3mg/mL embodiment 1 adding 200 μ L obtains, with pesticin albumen Pst solution (expressed by Qingdao Agricultural University Animal Science And Technology microorganism and immunization experiment room and preserve) for protein control, be that microbiotic contrasts with kantlex, about 18h is cultivated in 37 DEG C of incubators, measure inhibition zone size.As shown in Figure 4, fusion rotein Pst-E produces fungistatic effect to streptococcus aureus ATCC25923 and O78 type intestinal bacteria to result, and antibacterial circle diameter is respectively 19mm and 13mm; Pesticin Pst albumen is to streptococcus aureus ATCC25923 and O78 type intestinal bacteria all without fungistatic effect, and this shows that fusion rotein Pst-E all has bacteriostatic activity to streptococcus aureus ATCC25923 and O78 type intestinal bacteria.

(2) Bacteriolytic activity of fusion rotein Pst-E detects

by streptococcus aureus ATCC25923(purchased from China Veterinary Drugs Supervisory Inst.) be that O78 type intestinal bacteria (being separated preservation with immunization experiment room by Qingdao Agricultural University Animal Science And Technology microorganism) are cultured to logarithmic phase (bacterial concentration reaches 108cfu/mL) respectively with serotype, 104cfu/mL is reached to 10-5(bacterial concentration) with LB meat soup successively doubling dilution, the fusion rotein Pst-E that the 100 μ L embodiments 1 of getting 0.3mg/mL obtain and the mutual effect of 200 μ L different dilution bacterium liquid phase, establish pesticin Pst protein control and PBS contrast simultaneously, after effect 2h, take out 100 μ L, with LB meat soup successively doubling dilution to 10-3 and 10-2, getting 400 μ L adds in sterilized petri dishes, pour the ordinary culture medium of about 50 DEG C into, fully shake up, each extent of dilution repeats 3 times, solidify rear inversion and cultivate 24h, carry out enumeration.As shown in Figure 5, after pesticin Pst albumen and the mutual effect of bacterium liquid phase, compared with PBS control group, S. aureus bacterium number 22%, the O78 type E. coli bacteria number that declines declines 15% to result; After fusion rotein Pst-E and the mutual effect of bacterium liquid phase, compared with PBS control group, S. aureus bacterium number decline 44%, O78 type E. coli bacteria number declines 2.7%, and this shows that fusion rotein Pst-E all has Bacteriolytic activity to streptococcus aureus ATCC25923 and O78 type intestinal bacteria.

SEQUENCELISTING

<110> Qingdao Agricultural University

<120> pesticin and bacterial virus bacteriolysis enzyme fusion proteins and encoding gene thereof and application

<130>2015

<160>2

<170>PatentInversion3.5

<210>1

<211>329

<212>PRT

<213>EnterobacteriaphageBp7

<400>1

MetSerAspThrMetValValAsnGlySerGlyGlyValProAlaPhe

151015

LeuPheSerGlySerThrLeuSerSerTyrArgProAsnPheGluAla

202530

AsnSerIleThrIleAlaLeuProHisTyrValAspLeuProGlyArg

354045

SerAsnPheLysLeuMetTyrIleMetGlyPheProIleAspThrGlu

505560

MetGluLysAspSerGluTyrSerAsnLysIleArgGlnGluSerLys

65707580

IleSerLysThrGluGlyThrValSerTyrGluGlnLysIleThrVal

859095

GluThrGlyGlnGluLysAspGlyValLysValTyrArgValMetVal

100105110

LeuGluGlyThrIleAlaGluSerIleGluHisLeuAspLysLysGlu

115120125

AsnGluAspIleLeuAsnAsnAsnArgAsnArgIleValLeuAlaAsp

130135140

AsnThrValIleAsnPheAspAsnIleSerGlnLeuLysGluPheLeu

145150155160

ArgArgSerValAsnIleValMetAspIlePheAspMetLeuArgGln

165170175

GlyGluGlyLeuAspLeuAsnLeuTyrLysAspThrGluGlyTyrTrp

180185190

ThrIleGlyIleGlyGlnLeuValThrLysAsnProSerLysAspVal

195200205

AlaArgAlaGluLeuAspLysLeuMetGlyArgValCysAsnGlyArg

210215220

IleThrMetAlaGluAlaGluGlnLeuPheAsnArgSerValGluAsn

225230235240

AlaArgArgAlaIleLeuArgAsnProLysLeuLysProValTyrAsp

245250255

ValLeuAspGluValArgArgCysAlaLeuIleAsnMetValPheGln

260265270

MetGlyGluAlaGlyValAlaGlyPheThrAsnSerLeuArgMetLeu

275280285

GlnGlnLysArgTrpAsnAspAlaAlaValAsnLeuAlaGlnSerArg

290295300

TrpTyrLysGlnThrProAsnArgAlaLysArgValIleAlaThrPhe

3053103320

LysThrGlyThrTrpAlaAlaTyrArg

325

<210>2

<211>990

<212>DNA

<213>EnterobacteriaphageBp7

<400>2

atgtcagatacaatggtagtgaatggttcaggtggtgttccggcttttctcttttccgga60

agtacattaagcagttacagaccaaattttgaagctaattcgattacaattgcattacca120

cattatgtggatctgcctggccggagtaattttaaactgatgtacattatggggtttccg180

attgatacggagatggagaaagacagtgaatattcaaataagatccgccaggaaagtaaa240

atttcaaaaactgaagggaccgtgtcttacgaacagaaaataactgttgaaacaggtcag300

gaaaaagacggtgtgaaagtctaccgtgtcatggttcttgagggaacgattgccgaatct360

attgaacatctcgataagaaagagaacgaagacattctgaataataaccgaaatcgcatc420

gtcctagcggacaacactgtcattaactttgacaatattagtcaactgaaggaattttta480

cgtcgttcggtaaatattgttatggatatttttgatatgttacgtcaaggcgaaggtctt540

gatttaaatctttataaagacactgaaggttattggactattggtattggccagttagtt600

accaagaacccatctaaagatgttgctcgtgctgaacttgataagcttatgggtcgagtt660

tgcaatggtcgtattactatggctgaagccgaacaactctttaaccggtcggttgaaaat720

gctcgtagagctattctgcgtaatcctaaattgaaacctgtttatgatgtactggacgaa780

gttcgtcgttgtgctttaattaacatggtcttccaaatgggtgaagcaggtgtagcaggg840

ttcactaactctttacgtatgctccaacagaaacgttggaatgatgcggcagttaacctg900

gcccaatcccgttggtataaacaaactcctaatcgtgctaagcgcgttatcgccaccttt960

aaaacaggtacttgggctgcttatagataa990

Claims (9)

1. a fusion rotein, is characterized in that, described fusion rotein contains the pesticin albumen Pst of plague bacillus and the N,O-Diacetylmuramidase e of phage Bp7 from aminoterminal successively to carboxyl terminal.

2. fusion rotein according to claim 1, is characterized in that, the aminoacid sequence of described fusion rotein is made up of 330 amino acid, and 1-167 position is described pesticin albumen Pst, and 168-330 position is described N,O-Diacetylmuramidase e.

3. fusion rotein according to claim 1, it is characterized in that, the aminoacid sequence of described fusion rotein is: MSDTMVVNGSGGVPAFLFSGSTLSSYRPNFEANSITIALPHYVDLPGRSNFKLMYI MGFPIDTEMEKDSEYSNKIRQESKISKTEGTVSYEQKITVETGQEKDGVKVYRVMV LEGTIAESIEHLDKKENEDILNNNRNRIVLADNTVINFDNISQLKEFLRRSVNIVM DIFDMLRQGEGLDLNLYKDTEGYWTIGIGQLVTKNPSKDVARAELDKLMGRVCNGR ITMAEAEQLFNRSVENARRAILRNPKLKPVYDVLDEVRRCALINMVFQMGEAGVAG FTNSLRMLQQKRWNDAAVNLAQSRWYKQTPNRAKRVIATFKTGTWAAYR or above-mentioned aminoacid sequence replace and/or disappearance and/or add and have the derivative albumen of the above-mentioned aminoacid sequence of identical function through one or several amino-acid residue.

4. the encoding gene of a pesticin and bacterial virus bacteriolysis enzyme fusion proteins, it is characterized in that, the encoding gene of described fusion rotein is selected from arbitrary described gene in following (1)-(3): (1), albumen coded sequence is as follows: ATGTCAGATACAATGGTAGTGAATGGTTCAGGTGGTGTTCCGGCTTTTCTCTTTTC CGGAAGTACATTAAGCAGTTACAGACCAAATTTTGAAGCTAATTCGATTACAATTG CATTACCACATTATGTGGATCTGCCTGGCCGGAGTAATTTTAAACTGATGTACATT ATGGGGTTTCCGATTGATACGGAGATGGAGAAAGACAGTGAATATTCAAATAAGAT CCGCCAGGAAAGTAAAATTTCAAAAACTGAAGGGACCGTGTCTTACGAACAGAAAA TAACTGTTGAAACAGGTCAGGAAAAAGACGGTGTGAAAGTCTACCGTGTCATGGTT CTTGAGGGAACGATTGCCGAATCTATTGAACATCTCGATAAGAAAGAGAACGAAGA CATTCTGAATAATAACCGAAATCGCATCGTCCTAGCGGACAACACTGTCATTAACT TTGACAATATTAGTCAACTGAAGGAATTTTTACGTCGTTCGGTAAATATTGTTATG GATATTTTTGATATGTTACGTCAAGGCGAAGGTCTTGATTTAAATCTTTATAAAGA CACTGAAGGTTATTGGACTATTGGTATTGGCCAGTTAGTTACCAAGAACCCATCTA AAGATGTTGCTCGTGCTGAACTTGATAAGCTTATGGGTCGAGTTTGCAATGGTCGT ATTACTATGGCTGAAGCCGAACAACTCTTTAACCGGTCGGTTGAAAATGCTCGTAG AGCTATTCTGCGTAATCCTAAATTGAAACCTGTTTATGATGTACTGGACGAAGTTC GTCGTTGTGCTTTAATTAACATGGTCTTCCAAATGGGTGAAGCAGGTGTAGCAGGG TTCACTAACTCTTTACGTATGCTCCAACAGAAACGTTGGAATGATGCGGCAGTTAA CCTGGCCCAATCCCGTTGGTATAAACAAACTCCTAATCGTGCTAAGCGCGTTATCG CCACCTTTAAAACAGGTACTTGGGCTGCTTATAGATAA,

(2) DNA molecular, with (1) limited has more than 90% homology, and the DNA molecular of encoding said fusion protein;

(3) DNA molecule hybridize limited with (1) or (2), under strict conditions, and the DNA molecular of encoding said fusion protein; Described stringent condition can be the solution with 6 × SSC, 0.5%SDS, hybridizes, then use 2 × SSC under 65 DEG C of conditions, and 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

5. a recombinant expression vector, is characterized in that, it is in the multiple clone site of pET28a carrier ecorI and xhoobtain after inserting fragment according to claim 4 between I.

6. an expression cassette, is characterized in that, described expression cassette by the promotor that can start genetic expression described in claim 4, gene according to claim 4, and transcription termination sequence composition.

7. a recombinant bacterium, is characterized in that, described recombinant bacterium obtains for pouring recombinant expression vector according to claim 5 into Host Strains; Described Host Strains is intestinal bacteria, and described intestinal bacteria are preferably BL21.

8. a prevention is or/and the biological products for the treatment of streptococcus aureus disease, it is characterized in that, its activeconstituents is the fusion rotein described in any one of claim 1-3, gene according to claim 4, recombinant expression vector according to claim 5, expression cassette according to claim 6 or recombinant bacterium according to claim 7.

9. the fusion rotein described in any one of claim 1-3, or gene according to claim 4, or recombinant expression vector, expression cassette or recombinant bacterium described in any one of claim 5-7 for the preparation of prevention or/and the purposes for the treatment of streptococcus aureus disease.