CN1296483C - Beta-lactamase/anminoglycosides modifying enzyme genes, their fusion gene and the expression products, the composition thereof, and their uses in medicine and environmental protection - Google Patents

Abstract

The present invention relates to separation of an antibiotic drug resistance enzyme gene, preparation of a fusion gene thereof, separation of drug resistance enzyme gene and a fusion gene expression product, a composition containing drug resistance enzyme protein and fusion protein and application thereof in medical science and environmental protection. More specifically, the present invention relates to separation of beta-lactamase and a coding gene of amino glucoside passivation enzyme, preparation of the beta-lactamase and a fusion gene of the amino glucoside passivation enzyme, separation of an expression product thereof, a composition containing the drug resistance enzyme protein and the fusion protein and application thereof in medical science and environmental protection.

Description

β-Nei Xiananmei/aminoglycoside modifying enzyme gene, its fusion gene and expression product, its composition, the application in medical science and environment protection

The application enjoys the right of priority that the application number of submitting on 02 11st, 2002 is 02105038.4 application.

Invention field

The present invention relates to the separation of antibiotics resistance enzyme gene, the preparation of its fusion gene reaches the separation of resistance enzyme gene and fusion gene expression product, contains the composition of this resistance zymoprotein and fusion rotein, and the application in medical science and environment protection.Concrete; the present invention relates to β-Nei Xiananmei; the separation of the encoding gene of aminoglycoside modifying enzyme; the preparation of β-Nei Xiananmei/aminoglycoside modifying enzyme fusion gene; and the separation of expression product; the composition that contains this resistance zymoprotein fusion rotein, and the application in medical science and environment protection.

Relate in the antibiotic therapy process; protect non-infection site; stability as little ecological zones such as enteron aisle, vagina, oral cavity, respiratory tracts; eliminate specific environment; as the medical space; the pollution of pharmaceutical factory Chinese traditional medicine, and as the pretreatment additive of animal-derived food, the antibacterials that wherein contained with degraded..More specifically, the present invention relates to β-Nei Xiananmei, the separation of the encoding gene of aminoglycoside acetyltransferase, the preparation of β-Nei Xiananmei/aminoglycoside acetyltransferase fusion gene, the separation of this gene and fusion gene expression product thereof, contain the composition and the application in medical science and environment protection thereof of this resistance zymoprotein and fusion rotein. particularly, the normal micro-ecological bacterial group who protects little ecological zone is (as vagina, enteron aisle, oral cavity etc.) do not killed by medicine, avoid antibiotic companion connection property disease due to the micro-ecological bacterial group imbalance (as pseudomembranous enteritis, vaginitis, stomatitis and other subclinical property disease), and prevent because of drug-induced generation Resistant strain, and the generation of other toxic side effect; Residual antibacterials in some specific environment (as medical space, pharmaceutical factory etc.) are degraded and removed, eliminate chemical that antibacterials caused and biotic pollution (as the increase of environment resistant organism and propagation etc.), simultaneously, also comprise with the pretreatment additive of above-mentioned zymin as animal-derived food, the antibacterials that wherein contained with degraded (as the pre-treatment of the animal-derived food of artificial breeding etc.).

Background technology

One, body micro-ecological bacterial group and resistance thereof

Normal microflora is lodged in the body surface and body cavity of body, has formed complete little ecosystem (Microeologic system).

Antibiotic therapy has been the most frequently used medical control measure.But in this course, the metabolism of antibacterials is generals, and promptly also there is the drug level that is enough to suppress or kill its sensitive strain little ecological zones such as enteron aisle, vagina, oral cavity, respiratory tract.These control medicines can suppress or kill the normal non-virulent flora of aforementioned region equally in a large number, thereby cause the microecological balance imbalance of part or whole body, bring out pseudomembranous enteritis, antibiotic companion connection property diarrhoea, vaginitis and some subclinical property disease etc. that various antibiotic companion connection property diseases such as difficult gemma clostridium (Clostridium difficile) excessive multiplication cause.These non-infection sites are killed in large quantities to the flora of antibiotic sensitivity, have stayed the resistance flora, in a single day autogenous infection takes place from now on, and increase of its treatment difficulty can't expect that this is one of difficult problem of serious at present puzzlement medical circle.In brief, during preventing with antibiotic and treating, antibacterials also have the effect that these regional microorganism species resistances are increased considerably to existing the killing in a large number of micro-ecological bacterial group of non-infection site and bring out the danger of various complication.

Two. environment micro-ecological bacterial group and resistance thereof

The micro-ecological bacterial group of external environment constitutes very complicated, and there is the different micro-ecological environment of formation in different zones, and the composition and the biological property of its microbial population are not quite similar.For example, the tolerance that the micropopulation in specific environments such as medical treatment and pharmaceutical factory shows medicine is higher than other external environment person, and major cause is because these regional antibacterial medicine residue amounts are higher than other environment.The pollution that prevents nosocomial infection and minimizing specific environment Chinese traditional medicine has become global public health and environmental issue.

With environmental correclation as food, feeds etc. also form with body and environment micro-ecological bacterial group's imbalance and resistance confidential relation, for example, all kinds of animals of artificial breeding (as bird, pig, ox, sheep, fish, soft-shelled turtle, shrimp, crab etc.) in the feed, the different antibacterials that have high level mostly, therefore, contain a large amount of resistant micro-organism flora and antibacterials in the movement of these animals, both caused biological and chemical contamination to environment, the endurance strain that has also caused among the environment micro-ecological bacterial group rolls up, moreover, in a single day these raising property animals are processed into food, because of it has the antibacterials of high level, the micro-ecological bacterial group of human body has also been constituted the potential threat of dysequilibrium and endurance strain increase.So the antibacterial medicine residue amount control of various food, feed has been the leading indicator that developed country formulates quality standard.

Three. antibiotics resistance enzyme and for the deactivation of antibiotic molecule

Yet any microbiotic uses soon, occurs immediately at the antibiotic resistance mechanism of this kind.Wherein the resistance enzyme is main mechanism.

Antibiotic resistance enzyme is the general designation with the various enzymes of its functional definition, by effects such as hydrolysis, esterification, acetylize, phosphorylated, nucleosidesization, makes the antibacterials molecule lose anti-microbial activity.This fermentoid can be by plasmid-mediated and chromosome coding, has different genotype, can have the coding or the mediation enzyme of different mechanisms in the same thalline simultaneously, can be " intracellular enzyme " that is positioned at the outer pericentral siphon of mycetocyte district, also can be " extracellular enzyme " of excretion to mycetocyte.

The antibiotic resistance enzyme that has been found that at present mainly contains: the β-Nei Xiananmei (β-lactamase that Beta-lactam medicine is had deactivation, BLA), some the deactivating enzyme (modifyingenzymes that aminoglycoside medicaments (Aminoglycosides) is had deactivation, AME), as Transacetylase (acetyltransferases, AAC), nucleotidyltransferase (adenylyltransferases or nucteolidyl transferases, AAD), phosphotransferase (Phosphotransferases, APH), to paraxin have deactivation Transacetylase (Chloramphenicol acetyltransferases, CAC); To erythromycin (Erythromycin), lincomycin (Lincomycin) etc. have deactivation the esterification enzyme (Macrolides esterase, MES) etc.

Four. the prior art situation

WO 88/07865, and A61K 35/74 discloses a kind of pharmaceutical composition, wherein adds the escherichia coli alive that can produce β-Nei Xiananmei sample active substance.This activity bacterium derives from the human feces strain isolated through vitro culture; used viable bacteria can produce β-Nei Xiananmei in vitro culture; do not destroyed by the beta-lactam medicine with protection intestinal tract normal flora clump, this patent has been ignored resistant organism to biological pollution problems such as resistant organism increase that human intestinal caused, resistance propagation.

WO 93/13795; A61K 37/54 discloses the β-Nei Xiananmei or the Ntn hydrolase (amidases) that produce with natural bacterial strain and has made the enteric solubility slow releasing capsule; during the 'beta '-lactam antibiotic treatment, in order to protection normal intestinal flora and the toxic side effect that prevents medicine.

Summary of the invention

One. summary of the invention and mechanism

The present invention relates to the screening of endurance strain, particularly possess β-Nei Xiananmei, or aminoglycoside modifying enzyme, the particularly screening of the endurance strain of aminoglycoside acetyltransferase activity.From the β-Nei Xiananmei or the aminoglycoside modifying enzyme of endurance strain, the particularly separation of the encoding gene of aminoglycoside Transacetylase; Contain this coding β-Nei Xiananmei; and aminoglycoside modifying enzyme expression of gene plasmid and contain the structure of expression plasmid of β-Nei Xiananmei/aminoglycoside modifying enzyme fusion gene and the expression in recipient cell thereof; the purifying of its expression product; this expression product and separate resistance enzyme from the natural resistance strain as toolenzyme; during the combination therapy of two kinds and two or more antibiotic; by of the hydrolysis of resistance enzyme to non-infection site antibacterials; acetylize; phosphorylation; nucleosidesization; effects such as esterification make the antibacterials that arrive these positions obtain reducing or removing.Be applied to specific external environment, to eliminate antibacterials (as specific regions such as medical space, pharmaceutical factories) wherein.Containing before the artificial animal-feed nutrition purposes, especially for feeding animals of high dosage antibacterials is processed into food, give antibiotic resistance zymin, to eliminate antibacterials wherein.

Antibacterials resistance enzyme involved in the present invention can be the extracellular enzyme that obtains of the engineering bacterium fermentation liquid of natural bacterium or reorganization, also can be the intracellular enzyme that bacterium obtains that concentrates after the fermentation; Can also be to be rich in the biological tissue of these resistance enzymes or the extraction enzyme of cell.The expression strain of resistance enzyme (or biological tissue or cell) can be natural screening strain, also can be by genetically engineered reorganization or transformation strain.The expressed enzyme of its encoding gene both can be karyomit(e) enzyme (Chromosomal enzymes), also can be plasmid enzyme (Plasmid enzymes), the enzyme protein expression form can be single purifying enzyme, also can be by the coded fusion zymoprotein form of recombination.

(1) β-Nei Xiananmei and mechanism of action thereof

The enzyme of lactam nucleus is β-Nei Xiananmei in all energy catalytic hydrolysis 6-amino-penicillanic acids (6-APA), 7-amino-cephalosporanic acid (7-ACA) and the N-acyl derivative molecule thereof.So far, in the family of β-Nei Xiananmei, found about 200 surplus kind of hypotype (kind).Can classify to it by multiple criteria for classification, for example, based on substrate profile or substrate spectrum, genes encoding position, aminoacid sequence, iso-electric point (Isoelectric point, pI), classification such as physico-chemical property, various sorting techniques all have to be used and citation, for example, can be divided into according to its substrate spectrum: penicillinase (penicillnase), cephalosporinase (cephalosporinase), wide spectrum enzyme (broad-spectrum enzymes) and super wide spectrum enzyme (extended-spectrum enzymes); Ambler, RP etc. are according to the aminoacid sequence difference of enzyme, it is divided into four types of A, B, C, D: Bush, K etc. are according to substrate spectrum, molecule type, rejection characteristic etc., it is divided into 1,2,3,4 group, wherein, 2 groups and 3 groups can be divided into many subgroups again, and most super wide spectrum enzyme belongs to 2be group and AmblerA class.Profile is set forth in table 1.

Table 1 bacterium β-Nei Xiananmei classification schemes

Bush-Jacoby-Medeir os group	Molecule type	Preferential substrate	Be suppressed		Represent enzyme
						CA	EDTA
			1	C				Cynnematin	-	-	The AmpC enzyme that gram-negative bacteria produces; MIR 1, and MOX 1, and CMY 1, BIL 1LAT 1
2a	A	Penicillin			+	-	The penicillinase that gram positive organism produces
			2b	A				Penicillin, cynnematin	+	-	TEM 1，TEM 2，SHV 1
2be	A	Penicillin, narrow spectrum and super broad-spectrum cephalosporin and monoamide class			+	-	TEM 3-TEM 29, TEM 42, TEM 43, TEM 46-TEM 52, SHV 2-SHV 9, PER 1 CTX-M 1, acid-producing Klebsiella bacterium K1
			2br	A				Penicillin	±	-	TEM 3-TEM 41，TEM 44，TEM 45，TRC 1
2c	A	Penicillin, Gepcillin			+	-	PSE 1，PSE 3，PSE 4
			2d	D				Penicillin, cloxacillin	±	-	OXA 1-OXA 21，PSE 2(OXA 10)
2e	A	Cynnematin			+	-	Proteus vulgaris produces induces cephalosporinase
			2f	A				Penicillin, cynnematin, carbapenems	+	-	NMC 1 of enterobacter cloacae and IMI 1, the Sme 1 of serratia marcesens
3a	B	Most beta-lactam, comprise carbon			-	+	The L1 of Pseudomonas Maltophilia, the CcrA of bacteroides fragilis, serratia marcesens

		Penems			IMP 1
						3b	B	Carbapenems	-	+	The CphA of Aeromonas hydrophila, the PCM 1 of flavobacterium odoratum
4	ND	Penicillin	-		The penicillinase of pseudomonas cepacia

Annotate: CA: clavulanic acid; EDTA: ethylenediamine tetraacetic acid (EDTA); ND: undetermined;

Confirmed at present 200 surplus in kind of the β-Nei Xiananmei, show as on the physico-chemical property: molecular weight ranges is about 1.3-5.0 ten thousand dalton, iso-electric point (pI) scope is about 4.3-0.5, show the feature (only replacing glutamine by Methionin) of isozyme at the 39th as TEM1 and TEM2 type, the enzyme kinetics feature (speed of hydrolysis substrate submits to V=Vmax * [S]/Km+[S]), and the enzyme inhibitors feature etc.

(2) amino glucosides antibiotics and resistance enzyme thereof

An aminocyclitol and one or several aminosugar molecules are all arranged in the molecular structure of amino glucosides antibiotics, link to each other by glycosidic linkage.Such medicine can be obtained by microbial fermentation, the Streptomycin sulphate that fermentation produces as streptomyces, gentamicin that the micromonospora fermentation produces or the like, also have chemosynthesis as semi-synthetic derivative Amikacin Sulphate (Kanamycin A Sulfate) of kantlex etc., wide clinical application reach tens of kinds more than, as Streptomycin sulphate (Streptomycin), kantlex (Kanamycin), gentamicin (Gentamicin), tobramycin (Tobramycin), Amikacin Sulphate (Kanamycin A Sulfate), new mould (Neomycin), ribostamycin (Ribostamycin), paromycin (Paromomycin), fortimicin (Fortimicin), dibekacin (Dibekacin), isepamicin (Isepamicin), reach ground Mi Xing (Dactimicin), micronomicin (Micronomicin), spectinomycin (Spectinomycin) or the like.The stable in properties of this antibiotics, has a broad antifungal spectrum, antibacterial mechanisms are to act on the intravital rrna of bacterium, and arrestin matter is synthetic, and existing bacteriostatic action has powerful germicidal action again.This class medicine is called bactericide for rest period again.

In the mechanism of drug resistance of microorganism to such medicine, producing deactivating enzyme is major reason, mainly contains three fermentoids, and every fermentoid comprises multiple isomerase again, and sum is up to tens of kinds more than.There is certain difference in these enzymes at aspects such as the aminoacid sequence of substrate spectrum (or substrate profile), pI, molecular weight, activity center, encoding genes; but; all be to make drug molecule lose anti-microbial activity on function by free hydroxyl group in the passivation aminoglycoside quasi-molecule or free amine group; show as: Transacetylase (AAC)-the make free amine group acetylize in the drug molecule; nucleotidyltransferase (AAD)-the make free hydroxyl group nucleosidesization in the drug molecule, phosphotransferase (APH)-the make free hydroxyl group phosphorylation in the drug molecule.The kind of the main deactivating enzyme of aminoglycoside medicaments and substrate spectrum see Table 2, and the mechanism of above-mentioned two class resistance enzymes is summarized in table 3:

The kind of the main aminoglycoside deactivating enzyme of table 2 and effect substrate thereof

The deactivating enzyme kind	Enzyme labelling (isomery)	The substrate spectrum
			Transacetylase	AAC(2’) AAC(6’) AAC(3)	Gentamicin, tobramycin, netilmicin kantlex, tobramycin, Amikacin Sulphate, netilmicin gentamicin, tobramycin, netilmicin, kantlex, sisomicin
Nucleotidyltransferase	AAD(4’) AAD(2”) AAD(3”) AAD(6)	Tobramycin, kantlex, Amikacin Sulphate gentamicin, tobramycin, kantlex, sisomicin Streptomycin sulphate, spectinomycin Streptomycin sulphate
			Phosphotransferase	APH(3’)-I，II， III APH(3”) APH(2”) APH(5”) APH(6) APH(2’)	Kantlex, Xin Meisu Streptomycin sulphate gentamicin, kantlex, tobramycin, sisomicin ribostamycin Streptomycin sulphate gentamicin, Amikacin Sulphate

The mechanism of drug resistance of the main antibacterials of table 3

Drug type	Main resistance mechanism	The resistance enzyme for example
			Beta-lactam	Outer porin (channel protein) variation of the low-affinity of resistance enzyme-deactivating penicillin-binding protein (PBP)	The beta-lactam enzyme is as penicillinase, cephalosporinase, wide spectrum enzyme, super wide spectrum enzyme etc.
Aminoglycoside	Resistance enzyme-deactivating rrna (target position) tolerance drug transport is improper	Transacetylase phosphotransferase nucleotidyltransferase etc.

Two. detailed Description Of The Invention

Bacterial strain

An aspect of of the present present invention provides has chemical sproof bacterial strain.

The escherichia coli that the contriver obtains from clinical sample (E.coli), Klebsiella Pneumoniae (K.pneumoniae), Pseudomonas aeruginosa (P.aeruginose), streptococcus aureus (S.aureus), not surplus the labor ground citric acid bacterium (C.freundii) etc. 20 the strain multi-drug resistant strains, filter out beta-lactam enzymic activity or aminoglycoside modifying enzyme active bacterial strain, the particularly bacterial strain of aminoglycoside acetyltransferase activity with high expression level.

Polynucleotide

(1) target of the present invention provides the polynucleotide of coding beta-lactam enzyme polypeptide, and the named herein of particularly encoding is the polynucleotide of the polypeptide of ESBL.

In a particularly preferred embodiment according to the invention, described polynucleotide comprise that is set forth in a SEQ ID NO:1, sequence, perhaps its coding has the variant of the active active polypeptide of β-Nei Xiananmei substantially.

The polynucleotide of SEQ ID NO:1 are the polynucleotide from Klebsiella Pneumoniae bacterial strain coding extended spectrum (ESBL).

The encode polynucleotide of beta-lactam enzyme polypeptide of the present invention, for example be set forth in the polynucleotide sequence of SEQ IDNO:1, can obtain by clone and the screening method that uses standard, for example use Klebsiella Pneumoniae strain cell of the present invention as parent material, from bacterium, separate total DNA, use from the radiolabeled oligonucleotide of partial sequence and survey total dna clone library.Then, by hybridization conditions, can pick out the clone who carries with dna probe homologous DNA.By using the sequencing primer of pressing the polynucleotide sequence design, the single clone who identifies through hybridization is checked order, might increase to both direction to polynucleotide sequence, to determine full-length gene order.For example, for the purpose of making things convenient for, use the double-stranded DNA of the sex change for preparing by plasmid clone to check order.Maniatis, T., Fritsch, E.F. and Sambrook etc. are at molecular cloning: laboratory manual (MOLECULAR CLONE, A LABORATORY MANUAL), second edition; Cold spring port experiment press, the cold spring port, New York is described suitable method in (1989).

In addition, be set forth in the dna sequence dna of SEQ ID NO:1, comprise the open reading frame of coded protein, this protein has the about amino-acid residue number that is set forth among the SEQ ID NO:2, those skilled in the art's its molecular weight of can deriving easily.

A kind of isolated polynucleotide provided by the invention, these polynucleotide comprise or are made up of following polynucleotide sequence:

(a) a kind of polynucleotide sequence, this polynucleotide sequence and SEQ ID NO:1 are with regard to SEQ ID NO:1 total length, at least 70% identity is arranged respectively, preferably have 80% identity, 85% identity is more preferably arranged, more preferably at least 90% identity, even more preferably at least 95% identity, the more preferably identity of 97-99% or same fully at least; Perhaps

(b) a kind of polynucleotide sequence of coding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:2 has at least 70% identity respectively with regard to SEQ ID NO:2 total length, preferably have 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, the more preferably identity of 97-99% or 100% same at least.

The invention provides a kind of polynucleotide sequence, the encoding sequence (open reading frame) of its total length and SEQ ID NO:1 is same.The present invention also provides one section encoding mature polypeptide or its fragments sequence itself, and another section encoding sequence arranged in the reading frame, for example encode leader sequence or secretion sequence, preceding protein sequence, or crude protein sequence, or the encoding mature polypeptide of the sequence of preceding crude protein sequence or its segmental encoding sequence.Polynucleotide of the present invention also comprise at least one section non-coding sequence, for example include but are not limited to: at least one section 5 ' and 3 ' non-coding sequence, the sequence of for example transcribing but not translating, termination signal (for example termination signal and the ind termination signal of rho-of rho-dependence), ribosome bind site, Kozak sequence, the sequence of stable mRNA, intron, and polyadenylation signal.

Polynucleotide sequence also can comprise the extra encoding sequence of the additional amino acid of encoding.For example, the one section flag sequence that helps the purifying fusion polypeptide of can encoding.In particular of the present invention, flag sequence is the 6xHN marker, the peptide sequence that helps purifying to merge with it.

Polynucleotide of the present invention also including, but not limited to, comprise the polynucleotide of the natural binding sequence that structure gene and controlling gene express.

The present invention also comprises such nucleotide sequence, the nucleotide sequence of its extended spectrum polypeptide of SEQ ID NO:2 that encodes because of the degeneracy of genetic code, in other words, it can be the sequence of the polypeptide of one section coding SEQ ID NO:2, and this sequence is the result of genetic code degeneracy.

(2) target of the present invention provides the coding aminoglycoside modifying enzyme, the polynucleotide of Transacetylase polypeptide particularly, and the named herein of particularly encoding is the polynucleotide of the polypeptide of aaC2.

In a particularly preferred embodiment according to the invention, described polynucleotide comprise that is set forth in a SEQ ID NO:3, sequence, perhaps its variant.

The polynucleotide of SEQ ID NO:3 are from escherichia coli bacterial strain coding Transacetylase polynucleotide.

The encode polynucleotide of Transacetylase polypeptide of the present invention, for example be set forth in the polynucleotide sequence of SEQ ID NO:3, can obtain by clone and the screening method that uses standard, for example use escherichia coli strain cell of the present invention as parent material, from bacterium, separate total DNA, use from the radiolabeled oligonucleotide of partial sequence and survey total dna clone library.Then, by hybridization conditions, can pick out the clone who carries with dna probe homologous DNA.By using the sequencing primer of pressing the polynucleotide sequence design, the single clone who identifies through hybridization is checked order, might increase to both direction to polynucleotide sequence, to determine full-length gene order.For example, for the purpose of making things convenient for, use the double-stranded DNA of the sex change for preparing by plasmid clone to check order.Maniatis, T., Fritsch, E.F. and Sambrook etc. are at molecular cloning: laboratory manual (MOLECULAR CLONE, A LABORATORY MANUAL), second edition; Cold spring port experiment press, the cold spring port, New York is described suitable method in (1989).

In addition, be set forth in the dna sequence dna of SEQ ID NO:3, comprise the open reading frame of coded protein, this protein has the about amino-acid residue number that is set forth among the SEQ ID NO:4, those skilled in the art's its molecular weight of can deriving easily.

A kind of isolated polynucleotide provided by the invention, these polynucleotide comprise or are made up of following polynucleotide sequence:

(a) a kind of polynucleotide sequence, this polynucleotide sequence and SEQ ID NO:3 are with regard to SEQID NO:3 total length, at least 70% identity is arranged respectively, preferably have 80% identity, 85% identity is more preferably arranged, more preferably at least 90% identity, even more preferably at least 95% identity, the more preferably identity of 97-99% or same fully at least; Perhaps

(b) a kind of polynucleotide sequence of coding one peptide species, the aminoacid sequence of this polypeptide and SEQ ID NO:4 has at least 70% identity respectively with regard to SEQ ID NO:4 total length, preferably have 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, the more preferably identity of 97-99% or 100% same at least.

The invention provides a kind of polynucleotide sequence, the encoding sequence (open reading frame) of its total length and SEQ ID NO:3 is same.The present invention also provides one section encoding mature polypeptide or its fragments sequence itself, and another section encoding sequence arranged in the reading frame, for example encode leader sequence or secretion sequence, preceding protein sequence, or crude protein sequence, or the encoding mature polypeptide of the sequence of preceding crude protein sequence or its segmental encoding sequence.Polynucleotide of the present invention also comprise at least one section non-coding sequence, for example include but are not limited to: at least one section 5 ' and 3 ' non-coding sequence, the sequence of for example transcribing but not translating, termination signal (for example termination signal and the ind termination signal of rho-of rho-dependence), ribosome bind site, Kozak sequence, the sequence of stable mRNA, intron, and polyadenylation signal.

Polynucleotide sequence also can comprise the extra encoding sequence of the additional amino acid of encoding.For example, the one section flag sequence that helps the purifying fusion polypeptide of can encoding.In particular of the present invention, flag sequence is the 6xHN marker, the peptide sequence that helps purifying to merge with it.

Polynucleotide of the present invention also including, but not limited to, comprise the polynucleotide of the natural binding sequence that structure gene and controlling gene express.

The present invention also comprises such nucleotide sequence, the nucleotide sequence of its aminoglycoside Transacetylase polypeptide of SEQ ID NO:4 that encodes because of the degeneracy of genetic code, in other words, it can be the sequence of the polypeptide of one section coding SEQ ID NO:4, and this sequence is the result of genetic code degeneracy.

Polypeptide

(1) one aspect of the present invention provides extended spectrum (ESBL) polypeptide of Klebsiella Pneumoniae, with and have the active variant of β-Nei Xiananmei substantially.

The present invention further provides:

(a) a kind of polypeptide separated, or it has the active fragment of β-Nei Xiananmei substantially, this polypeptide comprises one section aminoacid sequence, this section aminoacid sequence and SEQ ID NO:2 have at least 70% identity, preferably have 80% identity, at least 85% identity is more preferably arranged, at least 90% identity is more preferably arranged, more preferably at least 95% identity, the most preferably identity of 97-99% or same fully at least;

(b) a kind of polypeptide by isolated polynucleotide encoding, or it has the active fragment of β-Nei Xiananmei substantially, these polynucleotide comprise one section polynucleotide sequence, this section polynucleotide sequence and SEQ ID NO:1, with regard to SEQ ID NO:1 total length, 70% identity is arranged respectively, preferably have 80% identity, at least 85% identity is more preferably arranged, more preferably at least 90% identity, even more preferably at least 95% identity, the more preferably identity of 97-99% or same fully at least;

(c) variant of aforementioned polypeptides promptly passes through conservative amino acid replacement and the polypeptide different with former polypeptide, and a residue is replaced by the residue that another has similar characteristics thus.Typical this displacement occurs in L-Ala, and Xie Ansuan is between leucine and the Isoleucine; Between Serine and the Threonine; Between acidic residues aspartic acid and the L-glutamic acid; Between l-asparagine and the glutamine; And between alkaline residue Methionin and the arginine; Perhaps between aromatic residue phenylalanine and the tyrosine.

Preferred fragment comprises, for example comprises the polypeptide that blocks of part SEQ ID NO:2 aminoacid sequence or its variant, for example a series of amino-and/or residues of carboxyl-terminal amino acid sequence that comprise.Degraded form by host cell or the polypeptide of the present invention that produces in host cell also is preferred.

Particularly preferably be wherein several, 5-10,1-5,1-3,1-2 or 1 variant that amino acid is replaced, deleted or add with arbitrary combination.

Polypeptide of the present invention, can with " sophisticated " protein form exist or with its for example precursor forms exist.It usually is favourable comprising one section extra aminoacid sequence, and this extra aminoacid sequence comprises secretion or leader sequence, former sequence (pro-sequence).

Polypeptide of the present invention can prepare in any suitable manner.Such polypeptide comprises isolated naturally occurring polypeptide, the polypeptide that reorganization produces, and the synthetic polypeptide that produces, perhaps these methods are in conjunction with the polypeptide that produces.The method for preparing these polypeptide is known in this area.

Wherein several, minority, 5-10,1-5,1-3,2,1 amino acid or do not have amino acid is replaced, is modified with arbitrary combination, deletion and/or add.Wherein particularly preferably be the character and active reticent displacement, deletion and the interpolation that do not change the ESBL polypeptide.

(2) one aspect of the present invention provides the aminoglycoside Transacetylase polypeptide of escherichia coli bacterial strain, with and variant.

The present invention further provides:

(d) a kind of polypeptide separated, or it has the fragment of aminoglycoside acetyltransferase activity substantially, this polypeptide comprises one section aminoacid sequence, this section aminoacid sequence and SEQ ID NO:4 have at least 70% identity, preferably have 80% identity, at least 85% identity is more preferably arranged, at least 90% identity is more preferably arranged, more preferably at least 95% identity, the most preferably identity of 97-99% or same fully at least;

(e) a kind of polypeptide by isolated polynucleotide encoding, or it has the fragment of aminoglycoside acetyltransferase activity substantially, these polynucleotide comprise one section polynucleotide sequence, this section polynucleotide sequence and SEQ ID NO:3, with regard to SEQ ID NO:3 total length, 70% identity is arranged respectively, preferably have 80% identity, at least 85% identity is more preferably arranged, more preferably at least 90% identity, even more preferably at least 95% identity, the more preferably identity of 97-99% or same fully at least;

(f) variant of aforementioned polypeptides promptly passes through conservative amino acid replacement and the polypeptide different with former polypeptide, and a residue is replaced by the residue that another has similar characteristics thus.Typical this displacement occurs in L-Ala, and Xie Ansuan is between leucine and the Isoleucine; Between Serine and the Threonine; Between acidic residues aspartic acid and the L-glutamic acid; Between l-asparagine and the glutamine; And between alkaline residue Methionin and the arginine; Perhaps between aromatic residue phenylalanine and the tyrosine.

Preferred fragment comprises, for example comprises the polypeptide that blocks of part SEQ ID NO:4 aminoacid sequence or its variant, for example a series of amino-and/or residues of carboxyl-terminal amino acid sequence that comprise.Degraded form by host cell or the polypeptide of the present invention that produces in host cell also is preferred.

Particularly preferably be wherein several, 5-10,1-5,1-3,1-2 or 1 variant that amino acid is replaced, deleted or add with arbitrary combination.

Polypeptide of the present invention, can with " sophisticated " protein form exist or with its for example precursor forms exist.It usually is favourable comprising one section extra aminoacid sequence, and this extra aminoacid sequence comprises secretion or leader sequence, former sequence (pro-sequence).

Polypeptide of the present invention can prepare in any suitable manner.Such polypeptide comprises isolated naturally occurring polypeptide, the polypeptide that reorganization produces, and the synthetic polypeptide that produces, perhaps these methods are in conjunction with the polypeptide that produces.The method for preparing these polypeptide is known in this area.

Wherein several, minority, 5-10,1-5,1-3,2,1 amino acid or do not have amino acid is replaced, is modified with arbitrary combination, deletion and/or add.Wherein particularly preferably be the character and active reticent displacement, deletion and the interpolation that do not change the AAC polypeptide.

Fusion rotein

On the other hand, what the present invention relates to genetic engineering preparation comprises polypeptide of the present invention or its segmental fusion rotein, these protein can Chemical bond, also can be used as fusion protein expression, fusion rotein is compared with non-fusion rotein, can be increased in the output in the expression system, fusion rotein sequence of the present invention is seen SEQ.ID.No:6 and 8.

In an embodiment of the present invention, this fusion rotein prepares by genetic engineering, particularly, the contriver has made up the expression vector that comprises aminoglycoside Transacetylase and extended spectrum encoding gene, by with this carrier transformed host cell, the method of knowing by the art technology expert prepares recombinant polypeptide of the present invention, and the nucleotide sequence of the fusion rotein of the present invention of encoding is seen SEQ.ID.No:5 and 7.

Composition

The present invention also relates to such composition, said composition comprises antibiotics resistance bacterium discussed herein and/or polynucleotide and/or polypeptide.For example, this composition comprises, antibiotic resistant bacteria of the present invention and/or polypeptide and/or polynucleotide and the pharmaceutically acceptable carrier or the vehicle of treatment significant quantity.

Pharmaceutical composition of the present invention can be with the administration of any mode effectively easily, for example comprising local application, oral, the anus medication, vagina medicinal, the intravenously medication, intraperitoneal administration, the intramuscular medication, subcutaneous medication, Nasacort or intracutaneous route of administration, its form can be that the avirulent pharmaceutically acceptable carrier used always is mixed and made into tablet, pill, capsule, suppository, solution, emulsion, suspensoid, injection, ointment, basting agent, eye drop, lotion, gel, creme and other formulation that is suitable for or the like arbitrarily.This carrier can include, but are not limited to salt solution, damping fluid, glucose, water, glycerine, ethanol and their mixture.Preparation should be fit to the mode of medication.。

Environmental protection preparation of the present invention comprises liquid state, solid-state, pulvis, microcapsule, granular preparation.With regard to its attribute, comprise sterilizing agent, clean-out system etc.

Compound of the present invention can make up pharmaceutically acceptable sustained-release matrix biological example degradability polymkeric substance to form sustained release preparation.Sustained-release matrix is selected from for example liposome of biological compatibility material, polylactide (poly(lactic acid)), poly-glycollide (polymkeric substance of oxyacetic acid), polylactide-co-glycolide (multipolymer of lactic acid and oxyacetic acid) gathers acid anhydrides, poe, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, lipid acid, phosphatide, polysaccharide, amino acids, amino acid is phenylalanine for example, tyrosine, Isoleucine, polynucleotide, the polyvinyl propylene, polyvinylpyrrolidone and silicone.Preferred biodegradability matrix is one of polylactide and poly-glycollide or is polylactide-co-glycolide (multipolymer of lactic acid and oxyacetic acid).

The topical mode comprises the surface that is administered to skin, mucous membrane and lung and eyes.Topical drug delivery composition comprises inhalation, can be prepared into dry powder, and described dry powder can high pressure seal, also can not pressurize.If to the eye topical, compound of the present invention discharges in carrier at pharmaceutically useful eye, and pharmaceutically useful comprises for example ointment, vegetables oil or capsule encapsulation material with carrier.

Compound of the present invention can pressure seal, and contain pressurized gas for example nitrogen and night the oxidizing gases propellent.Liquefaction propellent matrix and the real preferred such form of total composition: activeconstituents does not dissolve on very large degree.The composition of this pressure seal can also contain for example liquid or solid nonionogenic tenside or can be the solid anion surfactant of tensio-active agent.The solid nonionogenic tenside of advantageous applications sodium-salt form.

Rectum or the preferred suppository of vagina administration composition, its preparation method is: with compound of the present invention with suitable non-irritating carrier or auxiliary material for example theobroma oil, polyoxyethylene glycol or suppository wax, they at room temperature are solid, but under body temperature liquid, therefore in rectum or vagina, dissolve, discharge active compound.

The also preferred effervescent of vagina administration composition.Its preparation method is: with suitable non-irritating carrier or auxiliary material and whipping agent, the medicament that produces bubble as sodium bicarbonate etc. under vaginal environment mixes with compound of the present invention.

Composition of the present invention also contains auxiliary agent, stablizer, thickening material, solubilizing agent and tinting material and spices.Preferably, contain enzyme stabilizers.

Description of drawings

Fig. 1 describes body and environment micro-ecological bacterial group's protection, resistance flora, and relation each other.

Fig. 2 describes technological line involved in the present invention.

The explanation of Fig. 3 expression vector plasmid map: pPROTe t.E 6 * HN is a Clontech company product, is fusion protein prokaryotic expression system.Plteto-1: heterozygous promotor-operon; RBS: ribosome bind site; 6 * HN: epi-position flag sequence; EK site: enteropeptidase restriction enzyme site; T1: transcription termination sequence; ColE1: replication initiation; T0: transcription termination sequence; Cmr: paraxin drug resistant gene.This system advantage: have potent cis-acting elements Plteto-1; The immunoaffinity chromatography reagent separation and purification that 6 * HN tag in the fusion rotein can easily become commercialized; Can utilize enteropeptidase to obtain native protein; Has the Cmr gene.

The sds gel electrophoresis photo of Fig. 4, expression product.

The full fragment cloning electrophoretic analysis of Fig. 5, ESBL figure.

The full fragment cloning electrophoretic analysis of Fig. 6, AAC figure.

Fig. 7, fusion gene cloning electrophoretic analysis figure.

The sds gel electrophoresis figure of Fig. 8 E SBL/AAC fusion gene expression product.

Fig. 9 detects fragment PCR electrophoretic analysis figure.

Figure 10 recombinant PCR gene fusion construct synoptic diagram.

Embodiment

Following example carries out with standard technique, these technology, except other detailed description, all be to those skilled in the art know with routine.Embodiment is illustrative, but does not limit the present invention.

Embodiment 1

1, the screening that has beta-lactam enzymic activity Klebsiella Pneumoniae

With E-Test method (the E-test kit is a Sweden AB Biodisc.Co. product) 40 strain β-Nei Xiananmeis and the strain of Transacetylase male multi-drug resistant clinical isolates are screened, be separated to Klebsiella Pneumoniae (K.pneumoniae) bacterial strain from patient's sample, fermenting and amplifying has also been separated partly BLA (reference: Edited by V.Lorian.AntibioticinLaboratory Medicine.2 ^Nd.Williams ﹠amp; Wilkins, Baltimore, 1986, Lu Yongsui, Chen Xiushu etc., six kinds of β-Nei Xiananmei qualitative test methods are estimated and clinical application Chinese journal of medical examination 1993.16:328; Sun Changgui, Chen Hanmei etc. estimate three kinds of screening methods and detect extended spectrums and clinical application thereof, Chinese journal of medical examination 1999.22:228)), estimate the ratio vigor of its substrate spectrum and enzyme.The result proves that same bacterial strain can be expressed different enzyme types, and there are certain difference in substrate spectrum and hydrolysis ability thereof between the different enzyme types.See Table 6.

The beta-lactam enzyme substrates spectrum and the vigor of table 6, the name of four class substrates spectrum

Substrate	Penicillinase	Cephalosporinase	The wide spectrum enzyme	Super wide spectrum enzyme
					Penicillin Oxacillin Carbenicillin Cephalothin Cefuroxime Cefotaxime Moxalactam Aztreonam Imipenem	2+ 1+ 1+ 1+ - - - - -	1+ - - 3+ - - - - -	3+ 1+ 1+ 2+ 1+ 1+ - 1+ -	3+ 3+ 3+ 2+ 2+ 2+ 2+ 1+ 3+

Annotate: " 1+ ": representative is that substrate, enzyme activity are approximately 1,000IU/mg with various medicines _ZymoproteinInternational unit is defined as under 37 ℃, the condition of pH 4.0-8.5, and per minute hydrolysis or deactivation 1 μ mol substrate are a unit.

Embodiment 2

Enzyme is deactivation beta-lactam medicine and protect the effectiveness of normal microorganism species in vivo,

9 Japanese white big ear rabbits divide three groups (3/group).Blank group: not administration gives isopyknic stroke-physiological saline solution; Control group and test group vein heavy dose (0.3 gram/kg body weight) respectively give cefotaxime (Cefotaxime), every day twice, continuous three days; Test group is when using antibiotic, and rectum gives the beta-lactam zymin and (is immediately Wide spectrum and The mixed preparation of super wide spectrum enzyme); Three groups are tried the rabbit similarity condition and are raised and nursing, medication same day, adopt the quantitative analysis that the excrement sample is made enteron aisle anerobe sum and enterobacteria sum every day, continuous four days; The result proves: flora sum quantitative result difference not significantly (P＞0.05) between test group and the blank group; And control group flora sum quantitatively obviously reduces, and compares difference highly significant (P＜0.01) with test group with blank group; The above results proves that the β-Nei Xiananmei mixing enzyme preparation has the effect of significant protection intestinal microflora and microecological balance in the pharmacological agent phase.

Embodiment 3. has the separation of the active escherichia coli bacterial strain of aminoglycoside medicaments deactivating enzyme

With the E-Test method multi-drug resistant clinical isolates strain of 40 strain β-Nei Xiananmeis and aminoglycoside enzyme positive is screened, from clinical sample, separate the escherichia coli bacterial strain, fermenting and amplifying, separate and obtained second enzyme transferring enzyme (AAC), nucleotidyltransferase (AAD) and phosphotransferase (APH), ability to its external such antibacterials of deactivation experimentizes, and the results are shown in Table 7.

Table 7, three kinds of other substrate spectrums of aminoglycoside medicaments deactivating enzyme different shaped

The enzyme type	Gentamicin (Geniamicin)	Antibacterials
					Kantlex (Kanangcin)	Tobramycin (Tobramycin)	Amikacin Sulphate (Aamikacin)
		Transacetylase AAC (2 ') AAC (6 ') AAC (3 ') nucleotidyltransferase AAD (4 ') AAD (2 ") phosphotransferase APH (3 ') APH (2 ')	2+ - 3+ - 2+ - 2+	- 2+ 2+ 2+ 2+ 2+ +				+ + 2+ 2+ 2+ - -	- 2+ 2+ 2+ ± + 2+

Annotate: " 1+ ": representative is that substrate, enzyme activity are approximately the 500IU/mg zymoprotein with various medicines; Unit definition is with table 6.

Embodiment 4

Enzyme is deactivation aminoglycoside medicaments and protect the effectiveness of normal microorganism species in vivo,

In order to estimate this fermentoid ability of deactivation aminoglycoside medicaments in vivo, we with Three groups The mixing enzyme preparation of enzyme (AAC, AAD, APH)(comprising dissimilar), 9 Japanese white big ear rabbits are carried out parallel check experiment, divide three groups (3/group).If blank group: not administration gives isopyknic physiological saline; Control group: administration, but do not give zymin; Test group: after the administration immediately rectum give zymin; Giving medicine is Amikacin Sulphate (Kanamycin A Sulfate), intravenously administrable, and each 2, dosage is 0.3 gram/kg body weight, continuous three days, medication same day, gathers the excrement sample every day and does enterobacteria sum quantitative counting, continuous four days.The result proves: control group is compared with blank group, test group, and the enterobacteria sum reduces (P＜0.01) very significantly, illustrates: intravenously administrable has caused the enterobacteria of enteron aisle to be killed in a large number; And the enterobacteria sum differs not significantly (P＞0.05) between test group and the blank group, illustrates: the intestinal microflora of zymin after to intravenous administration has provide protection significantly.

Embodiment 5: from resistant organism Klebsiella Pneumoniae strains separation extended spectrum ESBL gene

1. extract total DNA (reference: " fine works molecular biology experiment guide ", Science Press, 1999:39 page or leaf) from resistant organism Klebsiella Pneumoniae (Klebsiella pneumoniae) bacterial strain.

2. the primer design of extended spectrum (ESBL) gene is used to increase:

According to the information that U.S. gene bank of 027199 Kebsiella pneumoniae plasmidpkk50-2 EXTEND3ED SPECTRUM BETA-LACTAMATASE (BLAtem-52) genecompletete cds announces, designed following a pair of primer:

Upstream primer: 218-239

5’GGT GGATCCGGTGGCGGCGGATCTAGTATTCAACATTTTCGTGTCG 3’

(g gatcc) BamHI intermediate head (link)

Downstream primer: 1040-1062

3’CGACTCTATCCACGGAGTGACTA TCTAGAGCA 5’

XbaI(t ctaga)

Wherein, the base of underscore is represented restriction endonuclease sites.

3. obtain extended spectrum (ESBL) gene through pcr amplification

Get in the 1 microgram step 1 DNA that obtains, according to Maniatis, T., Fritsch, E.F. and Sambrook etc. are at molecular cloning: laboratory manual (MOLECULAR CLONE, ALABORATORY MANUAL), second edition; Cold spring port experiment press, cold spring port, the method amplification ESBL gene of New York (1989).

Embodiment 6: from escherichia coli (Escherichia coli.) strains separation aminoglycoside Transacetylase aacC2-Link gene.

1. from the total DNA of escherichia coli (Escherichia coli.) strains separation,

2. according to U.S. gene bank Gi|45769|emb|X54723.1|PRAACC2E.coliR-plasmid aacC2 gene for

aminoglycoside-(3)-N-acetyltransferase

Be designed for the primer of amplification aminoglycoside Transacetylase aacC2 gene:

aacC2-Link

Upstream primer: 822-843:5 ' ACGC GTCGACCATACGCGGAAGGCAATAACGG3 '

SalI(g tcgac)

Downstream primer: 1657-1679:

3’CGAGAACGCTCGGAAGTCCAATCCCACCGCCACCTAGACCGCCACCA CCTAGGCCA 5’

Intermediate head (link) BamHI

Wherein, connect aacC ₂With the intermediate head of ESBL is one to contain 15 amino acid whose small peptides (Gly4Ser) 3, at its encoding sequence 5 ' GGTGGCGGTGGATCTGGCGGTGGT GGATCCGGTGGCGGCGGATCT3 ' postmedian.Design a BamHI site, (utilizing degeneracy) aacC ₂Synthetic this site that all originates in of-Link downstream primer and ESBL upstream primer

4. obtain aminoglycoside Transacetylase aacC2-Link gene through pcr amplification

Get in the 1 microgram step 1 DNA that obtains, according to Maniatis, T., Fritsch, E.F. and Sambrook etc. are at molecular cloning: laboratory manual (MOLECULAR CLONE, ALABORATORY MANUAL), second edition; Cold spring port experiment press, cold spring port, the method amplification aacC2 gene of New York (1989).

Embodiment 7: the production with fusion rotein of extended spectrum and aminoglycoside acetyltransferase activity

1. the structure of plasmid pPaacC2-L

The SalI/BamHI enzyme of aacC2-link is cut product and the BamHI/SalI enzyme of pPROTet.E to be cut product (big fragment) and is connected by correct reading frame.

2 make up plasmid pPALE

Recombinant plasmid pPaacC2-L is carried out the BamHI/XbalI enzyme again cut, its big fragment is cut product with the BamHI/XbalI enzyme of ESBL be connected, make up plasmid pPALE.

Embodiment 8: the prokaryotic expression of fusion rotein

With the pPALE plasmid transform host bacterium DH5aPRO E.coli (Clontech company product)-, LB+ paraxin (20 mcg/ml) carries out resistance screening. choosing obtains and efficiently expresses bacterium, called after CWL888.By the fermentation, (the product TALONTMPurification Kit of Clontech company (#K1253-1) obtains pure product (see figure 8) to chromatogram purification, and the molecular weight of purified fusion protein is that 65KD, iso-electric point are (pI) 7.8, aminoacid sequence such as following.This regroup merge enzyme with penbritin, Amikacin Sulphate be the ratio work of substrate be respectively 〉=2,000IU/mg _ZymoproteinWith 〉=1,000IU/mg _Zymoprotein

Embodiment 9: the order-checking of extended spectrum ESBL gene

ABI PRISM377 DNA Sequencer, 5 ′ ﹠amp are adopted in order-checking; 3 ' PROTetSequencing Primers is a sequencing primer, and sequence is seen SEQ ID No:1.

The order-checking of embodiment 10:AAC gene

ABI PRISM377 DNA Sequencer, 5 ′ ﹠amp are adopted in order-checking; 3 ' PROTetSequencing Primers is a sequencing primer, and sequence is seen SEQ ID No:3.

Embodiment 11: the order-checking of fusion gene of the present invention

ABI PRISM377 DNA Sequencer, 5 ′ ﹠amp are adopted in order-checking; 3 ' PROTetSequencing Primers is a sequencing primer, and sequence is seen SEQ ID No:5.

The enteric solubility capsule of embodiment 12. 500mg, each capsule contains:

Fusion rotein (BLA+AAC) 4mg

Trypsin inhibitor 120mg

Oligofructose (FOS) 350mg

Magnesium Stearate 26mg

The vagina effervescence of embodiment 13. 500mg, every contains:

Fusion rotein (BLA+AAC) 2mg

Trypsin inhibitor 18mg

Boric acid 120mg

Sodium bicarbonate 60mg

Oligofructose (FOS) 200mg

Magnesium Stearate 100mg

The vagina effervescence of embodiment 14. 500mg, every contains:

BLA 1mg

AME 1mg

Trypsin inhibitor 18mg

Boric acid 120mg

Sodium bicarbonate 60mg

Oligofructose (FOS) 200mg

Magnesium Stearate 100mg

Embodiment 15. 20% microcapsule (microcapsulation) granules (particle diameter 1.0-2.5mm) contain:

Fusion rotein (BLA+AAC) 20g

Protease enzyme inhibitor 30g

Magnesium Stearate 50g

Embodiment 16. 20% microcapsule (microcapsulation) granules (particle diameter 1.0-2.5mm) contain:

AME 20g

Protease enzyme inhibitor 30g

Magnesium Stearate 50g

Embodiment 17. 20% microcapsule (microcapsulation) granules (particle diameter 1.0-2.5mm contains:

BLA 10g

AME 10g

Protease enzyme inhibitor 30g

Magnesium Stearate 50g

Embodiment 18. acute oral toxicity tests

One, material and animal

1. tried the vagina effervescence of thing: embodiment 14, sample is shallow milk yellow powdery.

2. animal: 20 of ICR small white mouses, body weight 18-22g economizes emphasis natural drug Pharmacological Experiment Room by unming Medical College (animal conformity certification number: No. the 9806th, the real moving card in Yunnan) is provided.

Two, method

1. test basis: " disinfection technology standard " third edition first fascicle 3.4 acute oral toxicity tests.

2. test method: get 20 animals, male and female half and half are irritated animal fasting 12h before the stomach, and one time per os is irritated stomach, observes record animal toxicity symptom and death condition continuously 14 days.

Three, test-results:

Vagina effervescence is to chmice acute per os toxicity test result

The animal sex	Dosage (mg/kg.bw)	Number of animals (only)	Dead animal number (only)	Mortality ratio (%)
					Male and female	5000 5000	10 10	0 0	0 0

Animal subject does not see that at viewing duration animal has obvious toxicity symptom, does not see death.Medium lethal dose LD ₅₀＞5000mg/kg.bw

Four, conclusion

Under this experiment condition, tried the thing vagina effervescence to animal subject small white mouse acute oral toxicity test by acute toxicity LD ₅₀The dosage grade scale is judged to be actual nontoxic.

Embodiment 19 eye irritant tests

One, material and animal

1. tried the vagina effervescence of thing: embodiment 14, sample is shallow milk yellow powdery.

2. animal: 4 of Japan large ear rabbits, male and female half and half, body weight 2.4kg-2.8kg is provided by unming Medical College, conformity certification number: No. the 9903rd, the moving pipe of cloud.

Two, method

1. test basis: " disinfection technology standard " third edition first fascicle 3.7 eye irritant tests.

2. method: get and tried that thing 0.1g is moistening to be placed in the animal subject one branch hole conjunctival sac, make the passive closed 4s of eye, use normal saline flushing 5min.6h, 24h, 48h, 4d, the 7d damage and the recovery situation of animal eye conjunctiva, iris and cornea that detect by an unaided eye behind eye drip.Another branch hole is as normal control.

Three, experimental result

Vagina effervescence is to White Rabbit eye irritation test-results

Number of animals	Look-out station	Eye irritation reaction integration
														6h		24h		48h		72h		4d		7d
		Sample	Contrast	Sample	Contrast	Sample	Contrast	Sample	Contrast	Sample	Contrast	Sample	Contrast
														1	Cornea iris conjunctiva total points	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0
2	Cornea iris conjunctiva total points	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0
														3	Cornea iris conjunctiva total points	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0
4	Cornea iris conjunctiva total points	0 0 4 4	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0
														Each time integral index		1	0	0	0	0	0	0	0	0	0	0	0
The preceding 7 days top indexes 1 in contamination back

Four, conclusion

Under this test conditions, tried the thing vagina effervescence White Rabbit eye irritant test is judged to be nonirritant by eye stimulus intensity judgement criteria.

Embodiment 20 skin irritations test

One, material and animal

1. tried the vagina effervescence of thing: embodiment 14, sample is shallow milk yellow powdery.

2. animal: 4 of Japan large ear rabbits, male and female half and half, body weight 2.1kg-2.4kg is provided by unming Medical College, animal conformity certification number: No. the 9903rd, the moving pipe of cloud.

Two, method

1. test basis: " disinfection technology standard " third edition first fascicle 3.6 skin irritations test.

2. 24h before the test cuts the about 3 * 3cm of unhairing scope with the hair of backbone both sides, White Rabbit back ²Get and tried thing 0.5g and directly be applied on the wetting in advance side skin.Cover with clean gauze, use the nonirritant immobilization with adhesive tape again.Opposite side is a blank.Take down gauze behind the 4h, remove the residual thing that tried with warm water, in remove tried thing after 1h, 24h, 48h observe local skin and react, carry out the irritant reaction scoring.

Three, experimental result

Vagina effervescence is to an irritant reaction scoring of rabbit skin

Number of animals		Body weight kg	1h				24h				48h
															Sample		Contrast		Sample		Contrast		Sample		Contrast
			Erythema	Oedema	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
															1	♀	2.1	0	0	0	0	0	0	0	0	0		0	0	0
2	♀	2.4	0	0	0	0	0	0	0	0	0	0
															3	♂	2.1	0	0	0	0	0	0	0	0	0	0
4	♂	2.2	0	0	0	0	0	0	0	0	0	0
															The total mark average			0	0	0	0	0	0	0	0	0	0

At viewing duration, experimental animal stands to try not show spot of thing site of action, oedema and other toxic action.

Four, conclusion

Under this implementation condition, tried the thing vagina effervescence animal subject White Rabbit skin irritation test is judged to be nonirritant by the skin irritation strength grading.

Embodiment 21 mouse marrow polychromatic erythrocyte micronucleus tests

One, material and animal

1. tried the vagina effervescence of thing: embodiment 14, sample is shallow milk yellow powdery.

2. positive thing: endoxan.Produce by Hualian Pharmaceutical Co., Ltd., Shanghai.

3. animal: Kunming kind small white mouse is provided (animal conformity certification number: No. the 9712nd, the real moving card in Yunnan) by Chinese Academy of Sciences's Kunming zooscopy.

Two, method

1. test basis: " disinfection technology standard " third edition first fascicle 3.11.4 mouse polychromatic erythrocytes micronucleus test.

2. test method is selected 50 of body weight 25-30g small white mouses for use, is divided into 5 groups at random, and 10 every group, male and female half and half.If 3 dosage groups and negative control group and positive controls (endoxan 40mg/kg.bw), adopt 30h secondary administration by gavage, 6h draws materials after contamination for the second time, wash medullary space with calf serum, conventional film-making dyeing microscopic examination, micronucleated cell number in every mouse 1000 polychromatic erythrocytes of counting (PCE) calculates different sexes animal micronuclear rates.

Three, test-results:

Vagina effervescence mouse polychromatic erythrocytes micronucleus test result

Dosage mg/kg.bw	Experimental animal number (only)	Sex	Observe PCE cell count (individual)	Micronucleated cell number (individual)	Micronucleated cell rate ‰
						Negative control	10	♂	5000	6	0.12
♀	5000	6	0.12
				1250	10			♂	5000	4	0.08
♀	5000	2	0.04
						2500	10	♂	5000	5	0.10
♀	5000	4	0.08
				5000	10			♂	5000	2	0.04
♀	5000	2	0.04
						Positive control	10	♂	5000	127	2.54
♀	5000	121	2.42

Adopt X ²Check is added up the result, and each dosage group micronucleated cell rate and negative control group comparing difference do not have significance, and p＞0.05 does not have dose-response relationship between each dosage group.

Four, conclusion

Under this test conditions, tried the thing vagina effervescence mouse polychromatic erythrocytes is not had the micronucleus of causing effect.

Embodiment 22

1. material and method

1.1. material

1.1.1. susceptibility reagent is conventional reagent

1.1.2.PCR and clone reagent Taq archaeal dna polymerase, Pfu archaeal dna polymerase, T4DNA ligase enzyme, dNTP, carrier pUCm-T all available from the living worker in Shanghai company; The DNA restriction enzyme is available from Dalian Bao Sheng biotech firm; Reagent such as DNA extraction, purifying and recovery reagent are analytical pure; Competent cell DH-5 α is preserved by this chamber.

1.1.3, utilize Primer Primier5.0 software to design detection primer respectively: AAC1 (5 ' GGGATACGCATCGTGGGACC 3 '), AAC2 (5 ' CCAAGCATCGGCATCTCATA 3 ') at aminoglycoside-resistant medicine and ESBL encoding gene conserved regions according to the sequence of Gene Bank; ESBL1 (5 ' TGGTGCGGTATTATCCCGTGTTG 3 '), ESBL2 (5 ' CGCTCGTCGTTTGGTATGGCTTC 3 '); Amplification complete encoding sequence primer is respectively: AAC1 (5 ' CATACGCGGAAGGCAATAACG 3 '), AAC2 (5 ' CTAACCGGAAGGCTCGCAAGA 3 '); ESBL1 (5 ' TTACCAATGCTTAATCAGTGA 3 '), ESBL2 (5 ' ATGAGTATTCAACATTTTCGTG 3 '); And according to joint (Gly ₄Ser) ₃Base sequence: 5 ' GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGC 3 ', design fusion gene PCR primer: F1 (5 ' GTATGGCTGCCGCCACCGCCGCTTCCGCCACCGCCGCTTCCACCGCCACCCCAATG CTTAATCAGTGA 3 '), F2 (5 ' CATTGGGGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGCCATAC GCGGAAGGCAATAACG 3 ').Primer is given birth to worker company by Shanghai and is synthesized.

1.2. method

1.2.1. drug sensitivity test (K-B method) is screened in clinical enteron aisle and the vagina aminoglycoside and the drug-fast clinical strains of cephalosporins medicine wide spectrum; The confirmatory test method that all adopts NCLLS (the stdn council of American National clinical labororatory) to recommend.

1.2.2.PCR detect the reaction conditions of screening positive strain

(add 3 unit archaeal dna polymerases, 0.25mmol/L Mg in the PCR reaction system of 50 μ L in 50 μ L reaction systems respectively with primer AAC1, AAC2 and ESBL1, ESBL2 ²⁺, 0.15mmol/L dNTP, each 0.2pmol of primer.) in 94 ℃ of 55sec (second), 60 ℃ of 50sec, 72 ℃ of 50sec circulations 30 times and 94 ℃ of 55sec, 58 ℃ of 50sec, 72 ℃ of 50sec circulations 30 times, gel electrophoresis is observed.

1.2.3 complete encoding sequence clone

Add TaqDNA polysaccharase and pfuDNA polysaccharase in proportion, respectively at circulation under 94 ℃ of 60sec, 58 ℃ of 55sec, 72 ℃ of 60sec and 94 ℃ of 60sec, 54 ℃ of 55sec, the 72 ℃ of 60sec conditions 30 times, gel electrophoresis is observed with primer AAC1, AAC2 and ESBL1, ESBL2.

After the purifying PCR reaction product, be connected with the T carrier respectively and change DH-5 over to, called after pUCm-T-ESBL and pUCm-T-AAC clone.

After pUCm-T-ESBL and pUCm-T-AAC cloned plasmids extract purifying, with EcoR I and HindIII carry out double digestion, HindIII carries out single endonuclease digestion, pcr analysis etc. and verifies.

1.2.4 recombinant PCR gene fusion construct clone

The structure of fusion gene as shown in figure 10.

With pUCm-T-ESBL, pUCm-T-AAC plasmid is template, and increasing with F1, ESBL2 primer obtains product pFE, and increasing with F2, AAC 2 primers obtains product pFA.

Obtain merging segment pFN with primer ESBL1, aac1 amplification behind the purifying, and change DH-5 over to, called after pUCm-T-E and A cloned plasmids after the T carrier is connected.

PUCm-T-E and A cloned plasmids carry out double digestion and HindIII carries out single endonuclease digestion, PCR analyzes with EcoR I, HindIII after extracting purifying.

1.2.5 cloned plasmids order-checking

Dna sequencing is with the M13 universal primer, carries out on ABI377 type automated DNA sequenator.

1.2.6 sequential analysis

2 results

2.1 detect PCR

Drug sensitivity test is according to the inhibition zone size, in accordance with regulations the standard determination result; Positive strain extracts after the bacteria total DNA with respective detection primer ESBL1, ESBL2 and AAC1, AAC2 through pcr amplification after product pE, pA respectively in 408bp, the visible (see figure 9) in 302bp place, big or smallly conforms to design.

2.2AAC gene and ESBL gene clone

861bp when detection PCR positive sample obtains product pESBL, pAAC with design with corresponding total length coding primer ESBL1, ESBL2 and AAC1, AAC2 amplification conforms to 858bp.The pUCm-T-ESBL cloned plasmids is 3634bp, is that 3634bp, PCR are the 861bp (see figure 5) that conforms to the size of expection through EcoRI and HindIII double digestion size for 1008bp and 2626bp, HindIII single endonuclease digestion.The pUCm-T-AAC cloned plasmids is 3634bp, is that 3634bp, PCR are the 861bp (see figure 6) that conforms to the size of expection through EcoR I and HindIII double digestion size for 1008bp and 2626bp, HindIII single endonuclease digestion.

2,3AAC and ESBL fusion gene cloning

PCR product pFE is that 908bp, pFA are 909bp, merging segment pFN is 1761bp, the pUCm-T-EandA cloned plasmids is 4534bp, it is 4534bp for 1908bp and 2626bp and HindIII carry out single endonuclease digestion that EcoRI, HindIII carry out double digestion size, and plasmid PCR is that the 1761bp (see figure 7) all conforms to expected results.

2.4 sequential analysis

Sequencing result and GeneBank do homology analysis and find that the base sequence of pESBL and Tem107 is in full accord; The sequence of pAAC has three bases to take place to have a mind to suddenly change.The fusion gene order of connection, in the right direction.

The construction of recombinant plasmid of this extended spectrum and aminoglycoside Transacetylase II (ESBL/AAC2 ') also checks order.Its nucleotide sequence and aminoacid sequence are seen SEQ.ID.NO:7 and 8 respectively, in SEQ.ID.NO:7, and ESBL:28-882; Linker:883-927; AAC:928-1785.

Its prokaryotic expression recombinant enzyme purification product is to the specific activity 〉=500IU/mg zymoprotein of penbritin, and to the specific activity 〉=300IU/mg zymoprotein of gentamicin, the sds gel electrophoresis of expression product is seen Fig. 8.

Sequence table

＜110〉Chen Xiushu

＜120〉β-Nei Xiananmei/aminoglycoside modifying enzyme gene, its fusion gene and expression product,

Its composition, the application in medical science and environment protection

<130>

<140>

<141>2002/02/11

<160>6

<170>

<210>1

<211>1620

<212>DNA

＜213〉Klebsiella Pneumoniae (K.pneumoniae)

<220>

<221>CDS

<222>

<400>1

ataaaattct tgaagacgaa agggcctcgt gatacgctta tttttatagg ttaatgtcat 60

gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 120

tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 180

gtaaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt ttcgtgtcgc 240

ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 300

gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 360

caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 420

ttttaaagtt ctgctatgtg gtgcggtatt atcccgtgtt gacgccgggc aagagcaact 480

cggtcgccgc atacactatt ctcagaatga cttggttaag tactcaccag tcacagaaaa 540

gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 600

taacactgct gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 660

tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 720

agccatacca aacgacgagc gtgacaccac gacgcctgca gcaatggcaa caacgttgcg 780

caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 840

ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 900

tgctgataaa tctggagcca gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 960

agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 1020

tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 1080

<210>2

<211>268

<212>PRT

＜213〉Klebsiella Pneumoniae (K.pneumoniae)

<400>2

Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala

1 5 10 15

Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys

20 25 30

Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile

35 40 45

Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro

50 55 60

Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys

65 70 75

Gly Ala Val Leu Ser Arg Val Asp Ala Gly Gln Glu Gln Leu Gly

80 85 90

Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Lys Tyr Ser Pro

95 100 105

Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu

110 115 120

Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu

125 130 135

Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu

140 145 150

His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro

155 160 165

Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Thr

170 175 180

Pro Ala Ala Mat Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu

185 190 195

Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu

200 205 210

Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala

215 220 225

Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Ser Glu Arg Gly Ser

230 235 240

Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg

245 250 255

Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu

280 265 270

Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His

275 280 285

Trp

<210>3

<211>1620

<212>DNA

＜213〉escherichia coli (E.coli)

<220>

<221>CDS

<222>

1 tttggtactc tcagaatacc cttttgagtt cgtttttgtg ccaacagaac agcccgtagg 60

taaatctcgg agcatttcag gaagtgtctg tgtgagttga atctgaatgg cagttaatat 120

tttagtggtc aaggtcttag gcgagttgaa tgcagggcga cctcgttttt tggcaggcgg 180

ttttacaaca ggagggagaa ctattttctc aacaccttcg cgtgctggaa tcatggttgc 240

atcacggact aatatgaccg attaacgtat ccgcaagatt ggacttcacc atctgctcat 300

gaacgcgagt ggttagttta cgcgctgcaa attcagcaaa aacacggcta aatgtccctt 360

cgctgggtag ttttttatac agattgaagc cgcaaattcg ccgcaatcta cgatcaactt 420

ctaggcgttc aatcaagccc cgtgtggtga cgataccaag ttcagctttt gcaataaaag 480

gcacatgcca aagcgcagcg gtcagcagga ggtcttccca cagcacaccc ttgatattgg 540

tataccaagg attcgatgtc actccactcg agcacacgaa taatgcgttc gagctttgag 600

ctgatgccgt cctaaatcgg ctgcgactaa aggaataagt tcgtattgta aggcttgaaa 660

ccgttgtttg agaaaagggc ttaatgtagt attcatgctg tagatgttag ggtgttggtt 720

tagaagctca ttttaacatc tacaactttt ttcaaaaaat gttaattcag gctgtttgtg 780

gagttttgca agtgcctcga tttagaggag atatcgcgat gcatacgcgg aaggcaataa 840

cggaggcgct tcaaaaact cggagtccaaa ccggtgacct attgatggtg catgcctcac 900

ttaaagcgat tggtccggtc gaaggaggag cggagacggt cgttgccgcg ttacgctccg 960

cggttgggcc gactggcact gtgatgggat acgcatcgtg ggaccgatca ccctacgagg 1020

agactcgtaa tggcgctcgg ttggatgaca aaacccgccg tacctggccg ccgttcgatc 1080

ccgcaacggc cgggacttac cgtgggttcg gcctgctgaa tcagtttctg gttcaagccc 1140

ccggcgcgcg gcgcagcgcg caccccgatg catcgatggt cgcggttggt ccactggctg 1200

aaacgctgac ggagcctcac aagctcggtc acgccttggg ggaagggtcg cccgtcgagc 1260

ggttcgttcg ccttggcggg aaggccctgc tgttgggtgc gccgctaaac tccgttaccg 1320

cattgcacta cgccgaggcg gttgccgata tccccaacaa acggcgggtg acgtatgaga 1380

tgccgatgct tggaagcaac ggcgaagtcg cctggaaaac ggcatcggat tacgattcaa 1440

acggcattct cgattgcttt gctatcgaag gaaagccgga tgcggtcgaa actatagcaa 1500

atgcttacgt gaagctcggt cgccatcgag aaggtgtcgt gggctttgct cagtgctacc 1560

tgttcgacgc gcaggacatc gtgacgttcg gcgtcaccta tcttgagaag catttcggaa 1620

ccactccgat cgtgccagca cacgaagtcg ccgagtgctc ttgcgagcct tcaggttaga 1680

ggccgtcgac aatgataatc tggatcaacg gacctttcgg cgcgggaaag acgacgctcg 1740

ctgagcggtt gcgcgatcgg cgttccaaat cgctgatctt tgaccccgag gaaatcgggt 1800

tcgttgtgaa agaaacggtc cccatgccgg cgagcggaga ctatcaggat ctcctattat 1860

gcaattaatc caaaaactgc ccagaaagtt aataatctag aattctttcc cttgagtatt 1920

caaaggaact tctaataaat attattcaag aaaataaaca atctattcaa aaaattgaag 1980

aaatattaca taccataata cctgttcagt tatctgaaga ttctgaaaat gaatatcaac 2040

gagtgctgtc aaaatcaata aatgcagcat ttaaaaactg cggagccaaa gaaggagaaa 2100

ttattcaagg gcaacacata aataagttag tagaatgtct actagaagaa ttaactcctt 2160

ggataaatca taacatcaag aaatagacca ttaattgaac caatttctta aagttctagt 2220

agtcacatct agctgctttg aaatttcaat attccccata ccctgctttt taa

<210>4

<211>491

<212>PRT

＜213〉escherichia coli (E.coli)

Met His Thr Arg Lys Ala Ile Thr Glu Ala Leu Gln Lys Leu Gly

1 5 10 15

Val Gln Thr Gly Asp Leu Leu Met Val His Ala Ser Leu Lys Ala

20 25 30

Ile Gly Pro Val Glu Gly Gly Ala Glu Thr Val Val Ala Ala Leu

35 40 45

Arg Ser Ala Val Gly Pro Thr GHy Thr Val Met Gly Tyr Ala Ser

50 55 60

Trp Asp Arg Ser Pro Tyr Glu Glu Thr Arg Asn Gly Ala Arg Leu

65 70 75

Asp Asp Lys Thr Arg Arg Thr Trp Pro Pro Phe Asp Pro Ala Thr

80 85 90

Ala Gly Thr Tyr Arg Gly Phe Gly Leu Leu Asn Gln Phe Leu Val

95 100 105

Gln Ala Pro Gly Ala Arg Arg Ser Ala His Pro Asp Ala Ser Met

110 115 120

Val Ala Val Gly Pro Leu Ala Glu Thr Leu Thr Glu Pro His Lys

125 130 135

Leu Gly His Ala Leu Gly Glu Gly Ser Pro Val Glu Arg Phe Val

140 145 150

Arg Leu Gly Gly Lys Ala Leu Leu Leu Gly Ala Pro Leu Ash Ser

155 160 165

Val Thr Ala Leu His Tyr Ala Glu Ala Val Ala Asp Ile Pro Asn

170 175 180

Lys Arg Arg Val Thr Tyr Glu Met Pro Met Leu Gly Ser Asn Gly

180 185 190

Glu Val Ala Trp Lys Thr Ala Ser Asp Tyr Asp Ser Asn Gly Ile

195 200 205

Leu Asp Cys Phe Ala Ile Glu Gly Lys Pro Asp Ala Val Glu Thr

210 215 220

Ile Ala Asn Ala Tyr Val Lys Leu Gly Arg His Arg Glu Gly Val

225 230 235

Val Gly Phe Ala Gln Cys Tyr Leu Phe Asp Ala Gln Asp Ile Val

240 245 250

Thr Phe Gly Val Thr Tyr Leu Glu Lys His Phe Gly Thr Thr Pro

255 260 265

Ile Val Pro Ala His Glu Val Ala Glu Cys Ser Cys Glu Pro Ser

270 275 280

Gly

<210>5

<211>1748

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

catacgcgga aggcaataac ggaggcgctt caaaaactcg gagtccaaac cggtgaccta 60

ttgatggtgc atgcctcact taaagcgatt ggtccggtcg aaggaggagc ggagacggtc 120

gttgccgcgt tacgctccgc ggttgggccg actggcactg tgatgggata cgcatcgtgg 180

gaccgatcac cctacgagga gactcgtaat ggcgctcggt tggatgacaa aacccgccgt 240

acctggccgc cgttcgatcc cgcaacggcc gggacttacc gtgggttcgg cctgctgaat 300

cagtttctgg ttcaagcccc cggcgcgcgg cgcagcgcgc accccgatgc atcgatggtc 360

gcggttggtc cactggctga aacgctgacg gagcctcaca agctcggtca cgccttgggg 420

gaagggtcgc ccgtcgagcg gttcgttcgc cttggcggga aggccctgct gttgggtgcg 480

ccgctaaact ccgttaccgc attgcactac gccgaggcgg ttgccgatat ccccaacaaa 540

cggcgggtga cgtatgagat gccgatgctt ggaagcaacg gcgaagtcgc ctggaaaacg 600

gcatcggatt acgattcaaa cggcattctc gattgctttg ctatcgaagg aaagccggat 660

gcggtcgaaa ctatagcaaa tgcttacgtg aagctcggtc gccatcgaga aggtgtcgtg 720

ggctttgctc agtgctacct gttcgacgcg caggacatcg tgacgttcgg cgtcacctat 780

cttgagaagc atttcggaac cactccgatc gtgccagcac acgaagtcgc cgagtgctct 840

tgcgagcctt caggttaggg tggcggtgga tctggcggtg gtggatccgg tggcggcgga 900

tctagtattc aacattttcg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 960

gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 1020

cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 1080

gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggtgc ggtattatcc 1140

cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 1200

gttaagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 1260

tgcagtgctg ccataaccat gagtgataac actgctgcca acttacttct gacaacgatc 1320

ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 1380

gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgacg 1440

cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 1500

tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 1560

tcggcccttc cggctggctg gtttattgct gataaatctg gagccagtga gcgtgggtct 1620

cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 1680

acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 1740

tcactgat

<210>6<211>583

<212>PRT

＜213〉artificial sequence

＜223〉the italic underscore is Link

His Thr Arg Lys Ala Ile Thr Glu Ala Leu Gln Lys Leu Gly Val

1 5 10 15

Gln Thr Gly Asp Leu Leu Met Val His Ala Ser Leu Lys Ala Ile

20 25 30

Gly Pro Val Glu Gly Gly Ala Glu Thr Val Val Ala Ala Leu Arg

35 40 45

Ser Ala Val Gly Pro Thr Gly Thr Val Met Gly Tyr Ala Ser Trp

50 55 60

Asp Arg Ser Pro Tyr Glu Glu Thr Arg Asn Gly Ala Arg Leu Asp

65 70 75

Asp Lys Thr Arg Arg Thr Trp Pro Pro Phe Asp Pro Ala Thr Ala

80 85 90

Gly Thr Tyr Arg Gly Phe Gly Leu Leu Asn Gln Phe Leu Val Gln

95 100 105

Ala Pro Gly Ala Arg Arg Ser Ala His Pro Asp Ala Ser Met Val

110 115 120

Ala Val Gly Pro Leu Ala Glu Thr Leu Thr Glu Pro His Lys Leu

125 130 135

Gly His Aln Leu Gly Glu Gly Ser Pro Val Glu Arg Phe Val Arg

140 145 150

Leu Gly Gly Lys Ala Leu Leu Leu Gly Ala Pro Leu Asn Ser Val

155 160 165

Thr Ala Leu His Tyr Ala Glu Ala Val Ala Asp Ile Pro Asn Lys

170 175 180

Arg Arg Val Thr Tyr Glu Met Pro Met Leu Gly Ser Asn Gly Glu

185 190 195

Val Ala Trp Lys Thr Ala Ser Asp Tyr Asp Ser Asn Gly Ile Leu

200 205 210

Asp Cys Phe Ala Ile Glu Gly Lys Pro Asp Ala Val Glu Thr Ile

215 220 225

Ala Asn Ala Tyr Val Lys Leu Gly Arg His Arg Glu Gly Val Val

230 235 240

Gly Phe Ala Gln Cys Tyr Leu Phe Asp Ala Gln Asp Ile Val Thr

245 250 255

Phe Gly Val Thr Tyr Leu Glu Lys His Phe Gly Thr Thr Pro Ile

260 265 270

Val Pro Ala His Glu Val Ala Glu Cys Ser Cys Glu Pro Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln

290 295 300

His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu

305 310 315

Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala

320 325 330

Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu

335 340 345

Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe

350 355 360

Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu

365 370 375

Ser Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His

380 385 390

Tyr Ser Gln Asn Asp Leu Val Lys Tyr Ser Pro Val Thr Glu Lys

395 400 405

His Lcu Thr Asp Cly Met Thr Val Arg Glu Leu Cys Ser Ala

410 415 420

Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr

425 430 435

Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met

440 445 450

Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn

455 460 465

Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Thr Pro Ala Ala

470 475 480

Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr

485 490 495

Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys

500 505 510

Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe

515 520 525

Ile Ala Asp Lys Ser Gly Ala Ser Glu Arg Gly Ser Arg Gly Ile

530 535 540

Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val

545 550 555

Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg

560 565 570

Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp

575 580

<210>7

<211>1748

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

AGTATTCAACATTTTCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCC

TTCCTGTT

TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTT

GGGTGCACGA

GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTT

CGCCCCGAA

GAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTAT

TATCCCGT

ATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAAT

GACTTGGTT

GAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGACTGACAGTAAG

AGAATTATGC

AGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA

ACGATCGGA

GGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACT

CGCCTTGAT

CGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACAC

CACGATGCCT

GTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACT

CTAGCTTCC

CGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTT

CTGCGCTCG

GCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGT

GGGTCTCGC

GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTA

TCTACACG

ACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGAT

AGGTGCCTCA

CTGATTAAGCATTGGG

CATACGCGGAAGGCAATAACGGAGGCGCTTCAAAAACTCGGAGTCCAAAC

CGGTGACCTC

TTGATGGTGCATGCCTCACTTAAAGCGATTGGTCCGGTCGAAGGAGGAGCG

GAGACGGTC

GTTGCCGCGTTACGCTCCGCGGTTGGGCCGACTGGCACTGTGATGGGATAC

GCGTCGTGG

GACCGATCACCCTACGAGGAGACTCTGAATGGCGCTCGGCTGGATGACGA

AGCCCGCCGT

ACCTGGCTGCCGTTCGATCCCGCAACAGCCGGGACTTACCGTGGGTTCGGC

CTGCTGAAT

CAATTTCTGGTTCAAGCCCCCGGCGCGCGGCGCAGCGCGCACCCCGATGC

ATCGATGGTC

GCGGTTGGTCCGCTGGCTGAAACGCTGACGGAGCCTCACGAACTCGGTCA

CGCCTTGGGG

GAAGGATCGCCCGTCGAGCGGTTCGTTCGCCTTGGCGGGAAGGCCCTGCT

GTTGGGTGCG

CCGCTAAACTCCGTTACCGCATTGCACTACGCCGAGGCGGTTGCCGATATC

CCCAACAAA

CGGTGGGTGACGTATGAGATGCCGATGCTTGGAAGAGACGGTGAAGTCGC

CTGGAAAACG

GCATCGGATTACGATTCAAACGGCATTCTCGATTGCTTTGCTATCGAAGGAA

AGCCGGAT

GCGGTTGAAACTATAGCAAATGCTTACGTGAAGCTCGGTCGCCATCGAGAA

GGTGTCGTG

GGCTTTGCTCAGTGCTACCTGTTCGACGCGCAGGACATCGTGACGTTCGGC

GTCACCTAT

CTTGAGAAGCATTTCGGAACCACTCCGATCGTGCCTCCGCACGAGGCCGTC

GAGCGCTCT

TGCGAGCCTTCCGGTTAG

<210>8<211>583

<212>PRT

＜213〉artificial sequence

＜223〉the italic underscore is Link

SIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILE

SFRPE

ERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDG

MTVRELC

SAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPND

ERDTTMP

VAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKS

GAGERGSR

GIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW

HTRKAITEALQKLGVQTGDLLMVHASLKAIGPVEGGAETVVAALRSAVGPTG

TVMGYASW

DRSPYEETLNGARLDDEARRTWLPFDPATAGTYRGFGLLNQFLVQAPGARRS

AHPDASMV

AVGPLAETLTEPHELGHALGEGSPVERFVRLGGKALLLGAPLNSVTALHYAEA

VADIPNK

RWVTYEMPMLGRDGEVAWKTASDYDSNGILDCFAIEGKPDAVETIANAYVKL

GRHREGVV

GFAQCYLFDAQDIVTFGVTYLEKHFGTTPIVPPHEAWERSCEPSG

Claims (8)

1. beta lactamase/aminoglycoside modifying enzyme fusion gene, it is SEQ ID NO:7.

2. fusion rotein, it is by the fusion gene coding of claim 1, and it is SEQ.ID.NO 8.

3. pharmaceutical composition, it contains the albumen of claim 2, and pharmaceutically acceptable carrier.

4. the composition of claim 3 is used for the application of the preparation of inactivation beta-lactam class and aminoglycoside antibiotics in preparation.

5. the application of claim 4, said composition are the vagina effervescences that is used for inactivation beta-lactam class and aminoglycoside antibiotics.

6. claim 4 or 5 application, wherein, said preparation is used to protect the normal microorganism species of non-infection site to avoid killing.

7. the application of claim 4, wherein, said preparation is used for the removing of the residual antibacterials of specific environment.

8. the application of claim 4, wherein, said preparation is used for the pre-treatment of animal-derived food, with the wherein contained microbiotic of degraded.

Images