CN102643346B - Antibiotic containing antibody simulant and pharmacy application for same - Google Patents

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a novel antibiotic containing an antibody simulant as well as a preparation method and an application for the same. The novel antibiotic containing the antibody simulant is composed of colicins E1, Ia, Ib, A, B and N or the waterborne pore structural domains thereof and the antibody simulant, wherein the antibody simulant is formed by connecting the carboxyl terminal of the V[H]CDR1 of an immunoglobulin with the amino terminal of V[H]FR2, and then connecting the carboxyl terminal of the V[H]FR2 with the amino terminal of V[L]CDR; and the immunoglobulin specifically recognizes a bacterial outer membrane protein. The sterilization capability of the antibiotic is a thousand times greater than that of common antibiotic, and pathogenic bacterium is hard in generation of medicine resistance due to the special action mechanism of the antibiotic, and the normal cells of a human body cannot be injured during a sterilization process; and the antibiotic can be used for preparing medicines resisting neisseria meningitides, resisting vancomycin-resistant enterococci, resisting methicillin-resistant staphylococcus orresisting multi-resistant pseudomonas aeruginosa.

Description

A kind of antibody analog and pharmaceutical applications thereof

The present invention is that application number is " 200910092128.4 ", and the applying date is " on September 2nd, 2009 ", and denomination of invention is divided an application for the patent application of " a kind of new antibiotic that contains antibody analog and preparation method thereof and use ".

Technical field

The present invention relates to biomedicine field, particularly a kind of new antibiotic that contains antibody analog and preparation method thereof and application.

Background technology

Since nineteen forty-four, the microbiotic such as penicillin came into operation, be not only meningococcus, other a lot of life-threatening pathogenic bacterium have all produced resistance to it such as staphylococcus aureus, streptococcus pneumoniae, Pseudomonas aeruginosa, tubercule bacillus.According to the relevant address prediction that the Center for Disease Control (CDC) is delivered over the years, after 10 years to 20 years, these microbiotic may complete failure.

At present microbiotic commonly used is mainly by suppressing Cell wall synthesis, inhibition or disturbing the nucleic acid of bacterium and protein metabolism and route of synthesis reach antibiotic purpose.Yet these antimicrobial modes are induced easily bacterium to undergo mutation and are produced resistance.Therefore people are being devoted to the microbiotic of development of new always.Imitating the mode of operation of killing and wounding mutually between the different strain bacterium of the same race, to come the development of new microbiotic be one of more promising direction.Occurring in nature has many bacteriotoxins directly to come killing bacteria at bacterial membrane formation ionic channel.Its type specimen is exactly a kind of bacteriotoxin-colicin of intestinal bacteria secretion.Wherein colicin Ia is after nineteen fifty-two is found by Jacob, effort through the several generations, finally disclosed open and cross-film three-dimensional arrangement (the Qiu et al when closing of colicin Ia institute's formation ionic channel on artificial bimolecular lipid membrane in 1996, Major transmemebrane movement soociated with colicin Ia channel gating.J.Gen.Physiology, 107:313-328 (1996)), established theoretical basis for designing and prepare novel antibiotic at molecular level.

As mentioned above, colicin is a kind of desirable ionic channel microbiotic prototype, but the wild-type colicin can only act on the intestinal bacteria of different strain of the same race, and therefore the essential targeting that changes it just can make colicin then other pathogenic bacterium of attack; Porin Porin is the channel protein on the adventitia of the adventitia, plastosome and the chloroplast(id) that are present in the bacterium plasma membrane, they allow larger molecule to pass through, has stronger immunogenicity, can in host, induce specific high-caliber monoclonal antibody, if utilize the antibody that resists bacterium surface Porin albumen as prototype, design the antibody analog with better recognition performance, changing the targeting of colicin with these stand-in, should be a kind of desirable microbiotic exploitation direction.

Summary of the invention

The present invention is directed to the blank in above-mentioned field, a kind of new antibiotic is provided, and this antibiotic sterilizing ability is that common antibiotics is more than thousand times, because its mechanism of action is special, therefore pathogenic bacterium are difficult to produce resistance, can not injure the human normal cell in the sterilization process.

A kind of new antibiotic that contains antibody analog, antibody analog by colicin E1, Ia, Ib, A, B, N or its water-based pore passage structure territory and covalently bound peptide chain carboxy-terminal in described colicin or its water-based pore passage structure territory consists of, and described antibody analog is the V by immunoglobulin (Ig) _HThe carboxyl terminal of CDR1 connects V _HThe aminoterminal of FR2, V _HThe carboxyl terminal of FR2 connects V again _LThe aminoterminal of CDR consists of; Described immunoglobulin (Ig) specific recognition porin.

Described bacterium refers to meningococcus, and described porin refers to the porin PorA on meningococcus surface.

Described immunoglobulin (Ig) refers to that having the PUBMED accession number be that the peptide chain of the aminoacid sequence put down in writing of 2MPA_H is the antibody protein that the peptide chain covalent attachment of the aminoacid sequence put down in writing of 2MPA_L gets with having accession number.

Described colicin is Ia.

A kind of peptide chain molecule, it is characterized in that being formed by connecting by the peptide chain in colicin E1, Ia, Ib, A, B, N or its water-based pore passage structure territory and the antibody analog peptide chain that covalently bind in its carboxyl terminal, described antibody analog peptide chain is by the Three regions V of immunoglobulin (Ig) _HCDR1, V _HFR2, V _LThe peptide chain of CDR3 connects the aminoterminal formation of next regional peptide chain in turn with carboxyl terminal; Described immunoglobulin (Ig) specific recognition porin.

Described peptide chain molecule has aminoacid sequence shown in the Seq ID No.6.

The encode nucleotide sequence of above-mentioned peptide chain molecule.

Described nucleotide sequence is shown in Seq ID No.5.

The recombinant expression vector that comprises above-mentioned nucleotide sequence.

The preparation method of above-mentioned new antibiotic refers to above-mentioned recombinant expression vector imported in the expression system and expresses; The polypeptide that separation and purification is expressed obtains microbiotic.

The application of described new antibiotic in the preparation anti-bacterial drug.

Described anti-bacterial drug refers to the medicine of anti-meningococcus, anti-vancomycin-resistant enterococcus, anti-Methicillin-resistant Staphylococcus aureus or anti-multidrug resistant Pseudomonas aeruginosa.

The present invention utilizes colicin to form ionic channel at the target bacteria film, the target cell endoplasm is leaked and dead, and the subregion of the structure that plays the targeting effect among the present invention to be antagonism bacterium surface porin the be immunoglobulin (Ig) of Porin albumen is reconstructed the analog antibody thing of rear acquisition, comprises the first complementary determining region V of the variable region of heavy chain of this immunoglobulin (Ig) _HThe second skeleton district V of CDR1, variable region of heavy chain _HThe 3rd complementary determining region V of FR2, variable region of light chain _LCDR3, Three regions link next regional aminoterminal with carboxyl terminal in turn and consist of V _HCDR1-V _HFR2-V _LThe linear molecule of CDR3; As everyone knows, the active region that immunoglobulin (Ig) plays recognition reaction is called complementary determining region, complementary determining region only is comprised of to tens amino acid several, with complete immunoglobulin (Ig) or there are at present the engineered antibodies such as single-chain antibody seFv, Fab of report to compare, molecular weight of the present invention is minimum, organize perviousness good, simultaneously because it is simple in structure, most of skeleton construction and the Fc section of complete immunoglobulin molecules have been removed, its immunogenicity to the patient is reduced greatly, be beneficial to very much and lead colicin to arrive pathogenic position identification pathogenic bacterium.In treatment, colicin is drawn towards the pathogenic bacterium surface of cell membrane, and colicin forms ionic channel at the target bacteria film, makes target cell endoplasm leakage and dead, also has identical sterilizing ability for having produced chemical sproof bacterial strain at present.Because its recognition site is the distinctive antigenic substance of bacterium surface, do not have such recognition site on human tissue cell's film, so it is safe for human body.Be easy to produce chemical sproof microbiotic with respect to other, microbiotic of the present invention, because its antibody analog that plays targeting only needs to guide colicin into pathogenic bacterium and has just finished task, we do not need by the deadly bacterium of porin site effect, but act on the microbial film of bacterium by colicin, making microbial film produce ionic channel causes cell to leak and dead, traditional bacterial drug resistance enters to microbiotic by the structure that changes porin generally that obstacles produces resistance in the bacterial cell, and the present invention only needs the activity in the antibody recognition site of porin can arrive the purpose of sterilization, can say, it is a kind of strategy of looking one way and rowing another, namely identify the porin site, but colicin is combined in the deadly bacterium of the another one site shape ionic channel of cytolemma, real action site is not at Porin albumen, therefore bacterium is difficult to by variation, evolve and lose or changes the necessary structure of this existence of porin, so bacterium is difficult to microbiotic generation resistance of the present invention.Since the present invention according to the diversity of different bacterium surface film porin and with the diversity of the immunoglobulin (Ig) of its identification, the new antibiotic that this design concept obtains according to the present invention also is various.

Because the meningitis that meningococcus causes has caused great threat to baby both domestic and external and teen-age health, and its resistance performance to present common medicine is very obvious, make pharmaceutical quantities more and more higher for this bacterium of establishment, this serious harm patient health, therefore, the contriver adopts design of the present invention, antibody for the porin Porin A of meningococcus, the PUBMED accession number of its heavy chain peptide chain is 2MPA_H, the PUBMED accession number of light chain peptide chain is 2MPA_L, be reconstructed the acquisition antibody analog, i.e. aminoacid sequence shown in Seq ID No.2; And be operatively coupled on the carboxyl terminal of the peptide chain of colicin Ia, obtain to have the new antibiotic PMC-AM1 of the aminoacid sequence shown in Seq ID No.6.As shown in Figure 7, in the survival experiment of meningococcus lethal quantity infecting mouse, 8 days survival rates of injecting the mouse of antibiotic P MC-AM1 of the present invention are 90%, illustrate that microbiotic of the present invention has incomparable anti-microbial activity and protective effect in vivo than now with microbiotic penicillin, gentamicin etc.; Simultaneously, pass through Comparison of effect on sterilization, as shown in Figure 6A, antibiotic P MC-AM1 more of the present invention and CAZ, penbritin are to the minimum inhibitory concentration (MIC value) of meningococcus, experimental result shows the MIC 0.11nMol of PMC-AM1, the MIC 3.02nMol of CAZ, the MIC1.35nMol of penbritin; Illustrate that its sterilizing ability is apparently higher than the microbiotic that is usually used at present suppressing meningococcus.

The contriver produces at present the serious pathogenic bacterium of resistance with new antibiotic PMC-AM1 of the present invention to other and carries out exploratory experiment, find that PMC-AM1 also has extremely strong antibacterial efficacy to multidrug resistant Pseudomonas aeruginosa, vancomycin-resistant enterococcus, Methicillin-resistant Staphylococcus aureus, as shown in Figure 5, PMC-AM1 is existing stronger more than 127～3800 times with antibiotic than CAZ, levofloxacin, gentamicin etc. to the sterilization effect of multidrug resistant Pseudomonas aeruginosa; PMC-AM1 has obvious inhibition to vancomycin-resistant enterococcus, Methicillin-resistant Staphylococcus aureus, shown in Fig. 6 B, C.

Microbiotic of the present invention can be used for preparing antibacterials, particularly the application in the medicine of the anti-meningococcus of preparation, anti Bacillus pyocyaneu Flugge, anti-vancomycin-resistant enterococcus and anti-Methicillin-resistant Staphylococcus aureus.

Code book can be invented antibiotic Nucleotide and be cloned into and make up recombinant expression vector in the expression vector, these carriers can be in the host expressed fusion protein, a minute isolated fusion protein obtains avidin of the present invention.

Degenerate according to codon; the nucleotide sequence of microbiotic of the present invention or antibody analog of encoding is adjustable; can adjust nucleotide sequence to the Preference of codon according to host cell, as long as the amino acid of coding is constant, all belong to the scope of protection of present invention.

Description of drawings

Fig. 1 contains the structure of the recombinant plasmid pBHC-PorA1 of antibody analog and colicin Ia

Wherein the peptide chain of antibody analog is connected to the peptide chain carboxy-terminal of Ia, and the aminoacid sequence of antibody analog is shown in Seq ID No.2.

Fig. 2 contains the structure of the recombinant plasmid pBHC-PorA2 of antibody analog peptide chain and colicin Ia

Wherein the antibody analog peptide chain is connected to the carboxyl terminal of Ia, the antibody analog peptide chain is: the carboxyl terminal link heavy chain second skeleton district of the first complementary determining region of variable region of heavy chain, the peptide chain carboxy-terminal amino acid chain of the 3rd complementary determining region of variable region of light chain is connected on the carboxyl terminal amino acid in heavy chain the second skeleton district, shown in Seq ID No.4.

The structure of Fig. 3 new antibiotic of the present invention

Wherein T and R are that colicin Ia is positioned at N-terminal two signal recognition structure territories; Channel-forming is the formation ionic channel structural domain that colicin Ia is positioned at carboxyl terminal; AM is antibody analog.

Fig. 4 is that new antibiotic PMC-AM1 of the present invention is to the inhibition experiment of meningococcus

Curve from left to right is followed successively by contrast among the figure, 5 μ g/ml penbritins, 5 μ g/ml PMC-AM2,5 μ g/ml PMC-AM1,10 μ g/ml PMC-AM1.

This figure abscissa is the bacterial growth time, and unit is hour; Ordinate is nutrient solution 600nm optical density(OD), shows bacterial growth quantity.

Fig. 5 agar doubling dilution is measured the minimum inhibitory concentration (MIC) of new antibiotic

Plate shows: (Con), blank, (A), CAZ is 16 μ g/ml, (B), levofloxacin is 8 μ g/ml, (C), gentamicin is greater than 512 μ g/ml, (D), and new antibiotic (PMC-AM1) is 8 μ g/ml to the minimum inhibitory concentration of multidrug resistant Pseudomonas aeruginosa.

Fig. 6 new antibiotic of the present invention and antibiotic commonly used are to the comparative experiments of Methicillin-resistant Staphylococcus aureus (ATCC BAA-42), vancomycin-resistant enterococcus (ATCC 700802), multidrug resistant Pseudomonas aeruginosa (West China Hospital clinical separation strain 13578) and meningococcus (Chinese bacterial classification is preserved center 29332) minimal inhibitory concentration

Ordinate is minimal inhibitory concentration (nMol);

Wherein A figure is meningococcus, (1) PMC-AM1, MIC=0.11nMol, (2) CAZ, MIC=3.02 nMol, (3) penbritin, MIC=1.35nMol;

B figure is vancomycin-resistant enterococcus, (1) PMC-AM1, MIC=0.23nMol, (2) vancomycin, MIC=21.54nMol, (3) penbritin, MIC=10.78nMol;

C figure is Methicillin-resistant Staphylococcus aureus, (1) PMC-AM1, MIC=0.06nMol, (2) penbritin, MIC=21.55nMol, (3) Oxazacillin, MIC=14.1nMol;

D figure is the multidrug resistant Pseudomonas aeruginosa, (1) PMC-AM1, MIC=0.91nMol, (2) levofloxacin, MIC=43.2nMol, (3) CAZ, MIC=29.3nMol, (4) gentamicin, MIC＞889.4nMol.

Fig. 7 new antibiotic of the present invention and wild-type colicin, anti-golden Portugal's bacterium polypeptide (ZL01128836.1) to Methicillin-resistant Staphylococcus aureus (ATCC BAA-42), vancomycin-resistant enterococcus (ATCC 700802), the inhibiting ratio of multidrug resistant Pseudomonas aeruginosa (West China Hospital clinical separation strain 13578) than survival curve

Ordinate is minimal inhibitory concentration (nMol);

Wherein A figure is vancomycin-resistant enterococcus, (1) anti-golden Portugal bacterium polypeptide, MIC=0.91nMol, (2) wild-type colicin Ia, MIC=0.91nMol, (3) PMC-AM1, MIC=0.23nMol;

B figure is Methicillin-resistant Staphylococcus aureus, (1) anti-golden Portugal bacterium polypeptide, MIC=0.06nMol, (2) wild-type colicin Ia, MIC=0.23nMol, (3) PMC-AM1, MIC=0.06nMol;

C figure is the multidrug resistant Pseudomonas aeruginosa, (1) anti-golden Portugal bacterium polypeptide, MIC=0.91nMol, (2) wild-type colicin Ia, MIC=0.91nMol, (3) PMC-AM1, MIC=0.23nMol.

Fig. 8 new antibiotic of the present invention is the mouse survival time to the survival curve abscissa of the In vivoprotective test of meningococcus infection animal, and unit is the sky; Ordinate is the animals survived number, and unit is every mouse

1) PMC-AM1, new antibiotic of the present invention; 2) Gen, gentamicin; 3) PEN, penicillin; 4) Con., contrast; All drug injection concentration are 1.5mg/kg.

Embodiment

By reference to the accompanying drawings, the description by preferred embodiment of the present invention specifies the present invention.

[embodiment 1] expresses structure and the new antibiotic preparation of the plasmid of new antibiotic

Original plasmid is the pSELECT that has loaded colicin and immunity protein gene ^TM-1 plasmid (8.3kb).Through double-stranded oligonucleotide point mutation technology (QuickChange ^TMKit, Strategene company) with encoding antibody stand-in gene, be inserted into respectively on 626 sites of colicin allosteric polypeptide gene such as Seq ID No.1 and the described nucleotide sequence of Seq ID No.3, prepared mutant plasmid pBHC-PorA1, the pBHC-PorA2 (such as Fig. 1-shown in Figure 2) of new antibiotic.Mutant plasmid is transfected in the E.coli BL-21 engineering bacteria and prepares new antibiotic.

The sudden change program is undertaken by Strategene QuickChange SiteDirected Mutagenesis Kit (catalog#200518) medicine-chest handbook: namely

1. prepare the point mutation reactant:

5ul 10X buffer

2ul (10ng) has loaded the original plasmid of colicin allosteric polypeptide and immunity protein gene

1.25ul (125ng) 5 '-3 ' oligonucleotide primer (seeing listed primer sequence) of design

1.25ul (125ng) 3 '-5 ' oligonucleotide primer (seeing listed primer sequence) of design

1ul dNTP

Distilled water 50ul

1ul pfu

(except plasmid, primer and distilled water, being the standby reagent of medicine-chest institute)

2. carry out pcr amplification, amplification condition: 95 ℃ of sex change, 35 seconds, anneal 53 ℃, 70 seconds, extend 68 ℃, 17 minutes, totally 20 circulations;

3. add (37 ℃, 1 hour) behind the Dpn 1 restriction endonuclease 1ul digestion mother body D NA chain, get 1ul reactant and XL1-Blue competent cell 50ul and ice and incubated 30 minutes, 42 ℃ of thermal shockings 45 seconds, were inserted in the ice 2 minutes again;

4. add NZY and train basic 0.5ml, 220rpm, 37 ℃ were shaken bacterium 1 hour, got 50-100ul reactant bed board (LB training base adds 1% agar, adds the 50ug/ml penbritin, and 37 ℃ are spent the night);

5.18 choose bacterium after hour, order-checking is determined to suddenly change successfully behind the extraction plasmid;

6. the BL-21 engineering bacteria competent cell 40ul ice of mutant plasmid 100ng and preparation was incubated 5 minutes, 42 ℃ of thermal shockings, 30 seconds, inserted again in the ice 2 minutes, and added SOC and train basic 160ul, 220rpm, 37 ℃ are shaken bacterium bed board after 1 hour (LB training base add 1% agar, add the 50ug/ml penbritin, 37 ℃ are spent the night), picking mono-clonal bacterium colony increases bacterium in a large number;

7. increase in a large number bacterium, 8-10 rises FB training base, 250rpm, 30 ℃, 3-4 hour; Be warming up to 42 ℃, 250rpm growth 0.5 hour; Be cooled to 37 ℃, 250rpm growth 1.5 hours; 4 ℃, 6000g, 20 minutes centrifugation thalline, get 4 ℃, 50mM borate buffer (pH9.0,2mMEDTA) 80-100ml suspension thalline, ultrasonication thalline behind the adding PMSF 50ug (4 ℃, 400W, 1 minute, repeat 4-5 time, intermittently guaranteed the bacterium liquid temp in 2-3 minute), (4 ℃ of the thalline of high speed centrifugation precipitation fragmentation, 75,000g, 90 minutes), get supernatant and add the precipitation DNA of Vetstrep 5,000,000 units (4 ℃ were stirred 1 hour), 10,000g, 4 ℃, after the centrifugation in 10 minutes, get supernatant and pack molecular weight 15,000 dialysis tubings in 4 ℃, after 10 liters of dialysed overnight of 50mM borate buffer, again 10,000g, 4 ℃, centrifugation in 10 minutes, get supernatant and be splined on the CM ion exchange column, fully after the flushing, 0.3 M NaCl+50mM borate buffer wash-out can obtain prepared new antibiotic.Corresponding to above 2 kinds of plasmids, can obtain respectively two kinds of microbiotic of PMC-AM1 and PMC-AM2, its aminoacid sequence is respectively shown in Seq ID No.6, Seq ID No.8.

Wherein AM1 is the first complementary determining region, heavy chain the second skeleton district, variable region of light chain the 3rd complementary determining region of variable region of heavy chain, and Three regions links next regional aminoterminal with carboxyl terminal in turn, and aminoacid sequence is shown in Seq ID No.2; AM2 is the carboxyl terminal link heavy chain second skeleton district of the first complementary determining region of variable region of heavy chain, the peptide chain carboxy-terminal amino acid chain of the 3rd complementary determining region of variable region of light chain is connected on the carboxyl terminal amino acid in heavy chain the second skeleton district, and aminoacid sequence is shown in Seq ID No.4.

With the contrast of PMC-AM2 as PMC-AM1, the antibiotic function that under different mode of connection, produces between the amino acid section of the antibody analog of checking the present invention design.

Oligonucleotide primer sequence designed in the above-mentioned preparation plasmid is as follows: 5 '-3 ' (SEQ ID NO.9)

gcg aat aag ttc tgg ggt att TCT TAT TGG CTG CAT TGG ATT AAA CAG taa ata aaa tat aag aca ggc

3’-5’(SEQ ID NO.10)

gcc tgt ctt ata ttt tat tta CTG TTT AAT CCA ATG CAG CCA ATA AGA aat acc cca gaa ctt att cgc

5’-3’(SEQ ID NO.11)

tgg ctg cat tgg att aaa cag AGA CCT GGT CAG GGA CTG TGG ATC GGA taa ata aaa tat aag aca ggc

3’-5’(SEQ ID NO.12)

gcc tgt ctt ata ttt tat tta TCC GAT CCA CAG TCC CTG ACC AGG TCT ctg ttt aat cca atg cag cca

5’-3’(SEQ ID NO.13)

ggt cag gga ctg tgg atc gga TCT CAG TCC ACG CAT GTG CCG AGA ACC taa ata aaa tat aag aca ggc

3’-5’(SEQ ID NO.14)

gcc tgt ctt ata ttt tat tta GGT TCT CGG CAC ATG CGT GGA CTG AGA tcc gat cca cag tcc ctg acc

pBHC-PorA 2

5’-3’(SEQ ID NO.15)

gcg aat aag ttc tgg ggt att TCT TAT TGG CTG CAT TGG ATT AAA CAG taa ata aaa tat aag aca ggc

3’-5’(SEQ ID NO.16)

gcc tgt ctt ata ttt tat tta CTG TTT AAT CCA ATG CAG CCA ATA AGA aat acc cca gaa ctt att cgc

5’-3’(SEQ ID NO.17)

tgg ctg cat tgg att aaa cag AGA CCT GGT CAG GGA CTG TGG ATC GGA taa ata aaa tat aag aca ggc

3’-5’(SEQ ID NO.18)

gcc tgt ctt ata ttt tat tta TCC GAT CCA CAG TCC CTG ACC AGG TCT ctg ttt aat cca atg cag cca

5’-3’(SEQ ID NO.19)

ggt cag gga ctg tgg atc gga ACC AGA CCG GTG CAT ACG TCC CAG TCT taa ata aaa tat aag aca ggc

3’-5’(SEQ ID NO.20)

gcc tgt ctt ata ttt tat tta AGA CTG GGA CGT ATG CAC CGG TCT GGT tcc gat cca cag tcc ctg acc

[embodiment 2] new antibiotic is to the restraining effect of meningococcus

Bacterium is that Chinese bacterial classification is preserved center 29332 meningococcus strains, bacterium liquid 2 microlitres (10 ⁵CFU/ml) add the chocolate nutrient solution of rabbit blood 10 milliliters of (beef extract 50mg, Tryptones 100mg, KH ₂PO ₄30mg, NaCl 50mg, defiber rabbit blood 0.5-0.8ml) in, prepare altogether 5 groups, first group adds 0.3M NaCl+50mM borate buffer (is the blank preservation liquid of new antibiotic, amount is identical with the new antibiotic amount of liquid that adds in the experimental group) in contrast, second group adds 5 μ g/ml penbritins, the 3rd group adds 5 μ g/ml PMC-AM1, and the 4th group adds 5 μ g/ml PMC-AM2, and the 5th group adds 10 μ g/ml PMC-AM1.

The above-mentioned liquid of respectively organizing places respectively 100 milliliters of Erlenmeyer flasks, 200rpm, 37 ℃ of growths, 100 microlitres of per hour sampling add in the 96 hole enzyme dash boards through spectrophotometer (A 595nm) colorimetric bacteria tested growth turbidity, draw the inhibitory effect that growth curve of bacteria comes the comparison new antibiotic, the result shows that trial meningococcus can only be suppressed by PMC-AM1 as shown in Figure 4.

[embodiment 3] new antibiotic compares the minimum inhibitory concentration of multidrug resistant Pseudomonas aeruginosa and the minimum inhibitory concentration of now using antibiotic.

Adopt the agar doubling dilution to measure the minimum inhibitory concentration (MIC) of new antibiotic.It is surperficial in the agar plate that contains different pharmaceutical concentration with microbionation to inoculate instrument (Deneley A400) with multiple spot, and every some bacteria containing amount is 10 ⁵CFU/ml is hatched 18-24 hour observations for 37 ℃, take without the minimum concentration of contained drug in the bacterial growth plate substratum as the minimum inhibitory concentration (MIC value) of medicine to this bacterium.

Used bacterial classification is multidrug resistant Pseudomonas aeruginosa (West China Hospital clinical separation strain 13578), and substratum is MH substratum (per hundred milliliters: beef extract 500mg, caseinic acid hydrolyzate 1.75g, Zulkovsky starch 150mg, agar 1.7g).

The result as shown in Figure 5, new antibiotic (D) is that 8 μ g/ml, CAZ (A) are that 16 μ g/ml, levofloxacin (B) are that 8 μ g/ml, gentamicin (C) are greater than 512 μ g/ml to the minimum inhibitory concentration of multidrug resistant Pseudomonas aeruginosa (PMC-AM1).If with the molecular weight mark, PMC-AM1 is that 0.23nMol, CAZ are that 29.3nMol, levofloxacin are that 43.2nMol, gentamicin are greater than 890nMol to the minimum inhibitory concentration of multidrug resistant Pseudomonas aeruginosa; That is, PMC-AM1 now uses more than the antibiotic 127-3800 doubly strong CAZ, levofloxacin, the gentamicin etc. crossed of the antibacterial efficacy of multidrug resistant Pseudomonas aeruginosa.

The antibacterial activity in vitro of [embodiment 4] new antibiotic and the comparison of now using antibiotic

Adopt the agar doubling dilution to measure the minimum inhibitory concentration (MIC) of new antibiotic.It is surperficial in the agar plate that contains different pharmaceutical concentration with microbionation to inoculate instrument (Deneley A400) with multiple spot, and every some bacteria containing amount is 10 ⁵CFU/ml is hatched 18-24 hour observations for 37 ℃, take without the minimum concentration of contained drug in the bacterial growth plate substratum as the minimum inhibitory concentration (MIC value) of medicine to this bacterium.

Used bacterial classification is multidrug resistant Pseudomonas aeruginosa (West China Hospital clinical separation strain 13578), MH substratum (per hundred milliliters contain beef extract 500mg, caseinic acid hydrolyzate 1.75g, Zulkovsky starch 150mg, agar 1.7g); Methicillin-resistant Staphylococcus aureus (ATCC BAA-42), (per hundred milliliters contain Tryptones 1g, yeast 0.5g, glucose 0.1g, NaCl 1g, KH to the BM substratum ₂PO ₄100mg, agar 1g); Vancomycin-resistant enterococcus (ATCC 700802), the MH substratum; Meningococcus (Chinese bacterial classification is preserved center 29332), the training base is with embodiment 2 (adding in addition columbia blood agar base 3.9g).

The result as shown in Figure 6, A figure is meningococcus, (1) PMC-AM1, MIC=0.11nMol, (2) CAZ, MIC=3.02nMol, (3) penbritin, MIC=1.35nMol; B figure is vancomycin-resistant enterococcus, (1) PMC-AM1, MIC=0.23nMol, (2) vancomycin, MIC=21.54nMol, (3) penbritin, MIC=10.78nMol; C figure is Methicillin-resistant Staphylococcus aureus, (1) PMC-AM1, MIC=0.06nMol, (2) penbritin, MIC=21.55nMol, (3) Oxazacillin, MIC=14.1nMol; D figure is the multidrug resistant Pseudomonas aeruginosa, (1) PMC-AM1, MIC=0.91nMol, (2) levofloxacin, MIC=43.2nMol, (3) CAZ, MIC=29.3nMol, (4) gentamicin, MIC＞889.4nMol.

The comparison of the antibacterial activity in vitro of [embodiment 5] new antibiotic and anti-golden Portugal bacterium polypeptide and wild-type colicin Ia

Adopt the agar doubling dilution to measure the minimum inhibitory concentration (MIC) of new antibiotic.Take without the minimum concentration of contained drug in the bacterial growth plate substratum as the minimum inhibitory concentration (MIC value) of medicine to this bacterium.

Used bacterial classification is multidrug resistant Pseudomonas aeruginosa (West China Hospital clinical separation strain 13578), Methicillin-resistant Staphylococcus aureus (ATCC BAA-42), vancomycin-resistant enterococcus (ATCC 700802), MH substratum; Meningococcus (Chinese bacterial classification is preserved center 29332), the training base is with embodiment 4.

The result as shown in Figure 7, A figure is vancomycin-resistant enterococcus, (1) anti-golden Portugal bacterium polypeptide, MIC=0.91nMol, (2) wild-type colicin Ia, MIC=0.91nMol, (3) PMC-AM1, MIC=0.23nMol; B figure is Methicillin-resistant Staphylococcus aureus, (1) anti-golden Portugal bacterium polypeptide, MIC=0.06nMol, (2) wild-type colicin Ia, MIC=0.23nMol, (3) PMC-AM1, MIC=0.06nMol; C figure is the multidrug resistant Pseudomonas aeruginosa, (1) anti-golden Portugal bacterium polypeptide, MIC=0.91nMol, (2) wild-type colicin Ia, MIC=0.91nMol, (3) PMC-AM1, MIC=0.23nMol.

The In vivoprotective test of the meningococcus infection animal of [embodiment 6] new antibiotic

One, experiment material

(1) medicine

PMC-AM1, gentamicin, penicillin.

(2) bacterium

Meningococcus (Chinese bacterial classification center (examining institute in the Bureau of Drugs Supervision of Beijing the Temple of Heaven country) 29332).

Two, experimental technique

As shown in Figure 7, experimental group is totally 40 kunming mices, is divided into 4 experimental group, 10 every group.Abdominal injection glucose ferrous iron solution (20mg/kg) is injected 0.5ml bacterium liquid after 1 hour again.(CFU is 2.36x10 to this bacterium liquid by 1 part of meningococcus nutrient solution ⁹) than 1.5 parts of deactivation 5% dry yeast solution compositions.The bacterium of abdominal injection lethal dose is after 1 hour, tail vein injection medicine and contrast physiological saline (all drug injection concentration are 1.5mg/kg), and per 2 hours observationss, continuous 8 days, with the positive result of dead mouse.

As shown in Figure 7: 1PMC-AM1, new antibiotic of the present invention; 2Gen, gentamicin; 3PEN, penicillin; 4Con., contrast.

Three, result

Mouse survival curve as shown in Figure 7, behind the abdominal injection lethal dose meningococcus, 1), control group is all dead in 2 days, and 2), the penicillin group is all dead in 2 days, 3), 8 days survival rates of gentamicin group are 50%, 4), 8 days survival rates of new antibiotic of the present invention are 90%.

The result shows that to the meningococcus lethal infection, new antibiotic PMC-AM1 of the present invention has shown the trial incomparable anti-microbial activity of microbiotic of now using.

Claims (6)

1. antibody analog, its aminoacid sequence is shown in Seq ID No.2, by the V of immunoglobulin (Ig) _HThe carboxyl terminal of CDR1 connects V _HThe aminoterminal of FR2, V _HThe carboxyl terminal of FR2 connects V again _LThe aminoterminal of CDR consists of; Described immunoglobulin (Ig) specific recognition porin.

2. the gene of coding claim 1 described antibody analog.

3. described gene according to claim 2, its nucleotide sequence is shown in Seq ID No.1.

4. the pharmaceutical applications of antibody analog claimed in claim 1, the application of finger in preparing take meningococcus, vancomycin-resistant enterococcus, Methicillin-resistant Staphylococcus aureus or multidrug resistant Pseudomonas aeruginosa as the medicine of target substance, it is characterized in that: the molecule of described medicine is made of recognition unit and functional unit; Described recognition unit is described antibody analog polypeptide, plays the identification target substance in the molecular structure of medicine, and described functional unit refers to the colicin polypeptide, the covalently bound peptide chain carboxy-terminal at described colicin polypeptide of described antibody analog.

5. pharmaceutical applications according to claim 4, described colicin polypeptide refers to colicin E1, Ia, Ib, A, B, N or its water-based pore passage structure territory.

The described pharmaceutical applications of claim 4 prepared and medicine.

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