Antibiotic preparation method and platform system based on same
10071358 ยท 2018-09-11
Assignee
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
C07K16/00
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
G01N2500/04
PHYSICS
B01J19/0046
PERFORMING OPERATIONS; TRANSPORTING
C07K16/085
CHEMISTRY; METALLURGY
International classification
C40B30/04
CHEMISTRY; METALLURGY
B01J19/00
PERFORMING OPERATIONS; TRANSPORTING
C07K16/00
CHEMISTRY; METALLURGY
A61K39/395
HUMAN NECESSITIES
Abstract
Provided are a novel antibiotic preparation method and platform system based on the method, belonging to a novel drug development method. The method is based on a fixed structural formula: FR, wherein F is an effect area, and R is an identification area. At the prior art level, the present invention can quickly develop a specific novel antibiotic for most pathogenic microorganisms or biological cells. Also provided is a platform for implementing the method, ensuring that the novel antibiotic is developed in an efficient streamlined process.
Claims
1. A method of identifying an agent that inhibits growth, proliferation, and/or survival of a virus or cell, wherein the agent comprises a recognition moiety and an effector moiety, the method comprising: (a) providing a first library of polynucleotides each encoding an antibody mimetic having the structure V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 from N-terminal to C-terminal which is constituted by regions V.sub.HCDR1, V.sub.HFR2, and V.sub.LCDR3 on Fab short arm of an immunoglobulin, wherein the amino acid sequence of the antibody mimetic has a sequence selected from the group consisting of SEQ ID NOs: 8-13, SEQ ID NOs: 14-20, SEQ ID NOs: 27-33, and a combination thereof, wherein (i) the antibody mimetic is a recognition moiety for a target molecule on the surface of a virus or cell, and (ii) regions of V.sub.HCDR1, V.sub.HFR2, and V.sub.LCDR3 are from an antibody that specifically recognizes a target molecule on the surface of the virus or cell; (b) providing a second library of polynucleotides each encoding an effector moiety that inhibits a property of the virus or cell, wherein (i) the effector moiety is selected from the group consisting of colicin Ia, colicin Ib, colicin A, colicin B, and colicin N, and (ii) the property of the virus or cell is selected from the group consisting of growth, proliferation, survival and a combination thereof; (c) constructing a third library of recombinant polynucleotides, each recombinant polynucleotide of the third library of recombinant polynucleotides comprising a polynucleotide of the first library operably linked to a polynucleotide of the second library; (d) expressing the third library of recombinant polynucleotides in a biological expression system to generate a fourth library of fusion polypeptides; (e) screening the fourth library of fusion polypeptides to identify a fusion polypeptide that specifically recognizes the target molecule on the surface of the virus or cell and inhibits a property of the virus or cell, thereby identifying an agent comprising the identified fusion polypeptide as one that inhibits growth, proliferation, and/or survival of the virus or cell.
2. The method according to claim 1, wherein the second library comprises a polynucleotide comprising the sequence set forth in SEQ ID NO: 1.
3. The method according to claim 1, wherein the agent protects a mammal from an infection, a toxin, and/or death.
4. The method according to claim 3, wherein the infection is a Bacillus anthracis infection and the toxin is a necrosin or an edema factor.
5. The method according to claim 3, wherein the recognition moiety of the agent recognizes a wild-type Bacillus anthracis protective antigen (PA), and the effector moiety of the agent comprises a mutant Bacillus anthracis PA that forms a heterozygous PA heptamer with the wild-type Bacillus anthracis PA, and the heterozygous PA heptamer, in comparison to a wild-type PA heptamer, has no or a reduced ability to transport a Bacillus anthracis toxin across a cell membrane.
6. The method according to claim 1, wherein said antibody is generated by immunizing an animal with the cell or the target molecule or a portion thereof as an immunogen.
7. The method according to claim 1, wherein said antibody is generated by immunizing an animal with a plurality of target molecules or portions thereof as immunogens.
8. The method according to claim 1, wherein said antibody comprises a plurality of monoclonal antibodies, and the V.sub.HCDR1 and V.sub.LCDR3 are from the same monoclonal antibody or from different monoclonal antibodies.
9. The method according to claim 1, wherein the cell is a Gram-negative bacterium or a Gram-positive bacterium.
10. The method according to claim 1, wherein the biological expression system for the third library is an Escherichia coli (E. Coli) pET expression system.
11. The method according to claim 10, wherein the E. coli pET system is BL-21(DE3) or B834(DE3).
12. The method according to claim 1, further comprising separating and/or purifying the identified fusion polypeptide.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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MODES OF CARRYING OUT THE INVENTION
(12) The method and platform of this invention will be described by the following currently-completed development examples.
Example 1 Preparation of Novel Antibiotics Against Virus-Induced Tumor
(13) (1) determining targets: to determine lethal novel antibiotics against EB virus-induced tumor cells.
(14) (2) designing molecular structure of novel antibiotics: the following designing work was performed according to general formula
(15) ##STR00003##
wherein F is effect region; R is recognition region.
(16) Establishing recognition region molecular structure library: monoclonal antibodies specifically-recognizing EB virusanti-EB virus envelope glycoprotein antibodies gp320, i.e., monoclonal antibodies secreted by ATCC HB-168 hybridoma cells and amino acid sequences information thereof which had existed in prior art were found by searching in database.
(17) Based on said monoclonal antibody, inventors designed a series of antibody mimetic structures as shown in Table 1, and obtained a series of mutamers through random point mutation on the first 5 and the last 9 amino acids of antibody mimetics with structures listed in Table 1.
(18) TABLE-US-00001 TABLE 1 The designed antibody mimetic structures V.sub.HCDR1-V.sub.HFR2-V.sub.HCDR3 V.sub.LCDR1-V.sub.HFR2-V.sub.LCDR3 V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 V.sub.HCDR2-V.sub.HFR2-V.sub.LCDR3 V.sub.LCDR1-V.sub.HFR2-V.sub.HCDR3 V.sub.LCDR2-V.sub.HFR2-V.sub.HCDR3
(19) Establishing effect region molecular structure library: because the preparation goal was lethal novel antibiotics against EB-virus induced tumor cells, colicin could form lethal ion channel through cell membrane of Escherichia coli of the same species but different strains by itself to cause death of Escherichia coli of the same species but different strains, and it was a competent candidate substance for effect region. Thus, colicins Ia, Ib, A, B and N or mutant sequence were selected as substances of effect region library and offered to laboratory system.
(20) Preliminarily obtaining the designed molecular structure library of recombinants: from amino terminal to carboxyl terminal: colicin or mutamers thereof +28 peptides mimetic recognizing EB virus envelop glycoprotein, and molecular structures of some recombinants are shown in Table 2:
(21) TABLE-US-00002 TABLE 2 effect region Recombinant molecule (amino No. Recognition region molecule molecule terminal-carboxyl terminal) 1 V.sub.HCDR1-V.sub.HFR2-V.sub.HCDR3 Ia Ia-V.sub.HCDR1-V.sub.HFR2-V.sub.HCDR3 2 V.sub.LCDR1-V.sub.HFR2-V.sub.LCDR3 Ia Ia-V.sub.LCDR1-V.sub.HFR2-V.sub.LCDR3 3 V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 Ia Ia-V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 4 V.sub.HCDR2-V.sub.HFR2-V.sub.LCDR3 Ia Ia-V.sub.HCDR2-V.sub.HFR2-V.sub.LCDR3 5 V.sub.LCDR1-V.sub.HFR2-V.sub.HCDR3 Ia Ia-V.sub.LCDR1-V.sub.HFR2-V.sub.HCDR3 6 V.sub.LCDR2-V.sub.HFR2-V.sub.HCDR3 Ia Ia-V.sub.LCDR2-V.sub.HFR2-V.sub.HCDR3 7 V.sub.HCDR1-V.sub.HFR2-V.sub.HCDR3 mIa (SEQ ID NO: 1) mIa-V.sub.HCDR1-V.sub.HFR2-V.sub.HCDR3 8 V.sub.LCDR1-V.sub.HFR2-V.sub.LCDR3 mIa mIa-V.sub.LCDR1-V.sub.HFR2-V.sub.LCDR3 9 V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 mIa mIa-V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 10 V.sub.HCDR2-V.sub.HFR2-V.sub.LCDR3 mIa mIa-V.sub.HCDR2-V.sub.HFR2-V.sub.LCDR3 11 V.sub.LCDR1-V.sub.HFR2-V.sub.HCDR3 mIa mIa-V.sub.LCDR1-V.sub.HFR2-V.sub.HCDR3 12 V.sub.LCDR2-V.sub.HFR2-V.sub.HCDR3 mIa mIa-V.sub.LCDR2-V.sub.HFR2-V.sub.HCDR3 Note* monoclonal antibodies secreted by ATCC HB-168 hybridoma cells, V.sub.LCDR1, V.sub.LCDR2, V.sub.HCDR1, V.sub.HCDR2 V.sub.HFR2, V.sub.LCDR3, V.sub.HCDR3, colicin Ia and amino acid sequence thereof as well as nucleotide sequence thereof are known, accordingly amino acid sequence and nucleotide sequence of recombinants can be deduced, and they will take too much space. Thereby, such sequence information will not be listed in this description.
(22) (3) Laboratory system: recombinant library was obtained by binding the provided effect region and recognition region; gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector; a batch of recombinant polypeptides were obtained by transforming said recombinant expression vector into engineered bacteria.
(23) Anti-target verification experiment was conducted on the obtained recombinants (verification method and experimental design were the same as recorded in ZL2004/10081446.8). Recombinant 3 and 9 in Table 2 had the best killing effect against EB-virus induced tumor cells, and their results of in vitro killing experiment on EB-virus induced Burkitt's lymphoma are shown in
(24) Recombinants prepared by taking the mutants of antibody mimetics in recombinants 3 and 9 as recognition region were verified that, in Table 3, recombinants with SEQ ID NO: 2-6 as recognition region has basically equivalent killing effects against EB virus induced tumor cells as that of recombinant 3 or 9.
(25) TABLE-US-00003 TABLE3 aminoacidsequencesof28anti-EBvirus inducedtumorpeptidemimetic V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3andmutamersthereof V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 NO. andpointmutantsthereof SEQIDNO:2 SFGMHWVRQAPEKGLEWVAGQGYSYPYT SEQIDNO:3 SYGMHWVRQAPEKGLEWVAGQGYSYPYT SEQIDNO:4 SFGMHWVRQAPEKGLEWVAQQWSSNPYT SEQIDNO:5 SFGMHWVRQAPEKGLEWVALQGTHQPYT SEQIDNO:6 SFGMHWVRQAPEKGLEWVAQQLHFYPHT SEQIDNO:7 RQGMHWVRQAPEKGLEWVAGQGYSYPYT
(26) It took less than 6 months for this preparation, and a batch of candidate novel antibiotics with specific killing effect against targets were obtained successfully.
(27) Experimental methods and materials adopted to obtain each recombinant in this example were exactly the same as recorded in Patent No. ZL2004/10081446.8, except for the inserted gene sequences when constructing vectors, so they are not repeated here.
Example 2 Preparation of Novel Antibiotics Against Diplococcus intracellularis
(28) (1) determining targets: diplococcus intracellularis.
(29) (2) designing molecular structure of novel antibiotics: the following designing work was performed according to general formula
(30) ##STR00004##
wherein F is effect region; R is recognition region.
(31) Establishing recognition region molecular structure library:
(32) porin is one outer membrane protein which is common in gram-positive bacteria, like staphylococcus, streptococcus, enterococcus, gram-negative bacteria, like Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Bacillus breslaviensis, Serratia marcescens, Aeromonas, Vibrio, Myxococcus, and Mycobacterium tuberculosis; it is an ideal antigen protein, and PorA is one kind of porin.
(33) Monoclonal antibody specifically-recognizing porin PorA which had existed in prior art was found by searching in database; PUBMED ID of its heavy chain peptide is 2 MPA_H, and PUBMED ID of its light chain peptide is 2 MPA_L. Based on said monoclonal antibody, inventors designed a series of antibody mimetic molecular structures as shown in Table 1 of Example 1.
(34) Establishing effect region molecular structure library: because the preparation goal was lethal novel antibiotics against Diplococcus intracellularis, colicin was a competent candidate substance for effect region. Thus, colicins Ia, Ib, A, B and N were selected as substances of effect region library, and colicins Ia, Ib, A, B and N, ion channel domain molecules thereof and mutant molecules thereof constitute effect region molecular structure library.
(35) The preliminarily designed molecular structure of recombinant library was: from amino terminal to carboxyl terminal: colicin or ion channel domain thereof or mutamers thereof +anti-PorA antibody mimetic and molecular structures of some recombinants are shown in Table 4:
(36) TABLE-US-00004 TABLE 4 Recognition region effect region Recombinant molecule (amino terminal NO. molecule molecule to carboxyl terminal) 1 V.sub.HCDR1-V.sub.HFR2-V.sub.HCDR3 Ia Ia-V.sub.HCDR1-V.sub.HFR2-V.sub.HCDR3 2 V.sub.LCDR1-V.sub.HFR2-V.sub.LCDR3 Ia Ia-V.sub.LCDR1-V.sub.HFR2-V.sub.LCDR3 3 V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 Ia Ia-V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 4 V.sub.HCDR2-V.sub.HFR2-V.sub.LCDR3 Ia Ia-V.sub.HCDR2-V.sub.HFR2-V.sub.LCDR3 5 V.sub.LCDR1-V.sub.HFR2-V.sub.HCDR3 Ia Ia-V.sub.LCDR1-V.sub.HFR2-V.sub.HCDR3 6 V.sub.LCDR2-V.sub.HFR2-V.sub.HCDR3 Ia Ia-V.sub.LCDR2-V.sub.HFR2-V.sub.HCDR3 Note* monoclonal antibodies specifically-recognizing porin PorA, and PUBMED ID of its heavy chain peptide is 2MPA_H; PUBMED ID of its light chain peptide is 2MPA_L, which are all-known. Thus, V.sub.LCDR1, V.sub.LCDR2, V.sub.HCDR1, V.sub.HCDR2 V.sub.HFR2, V.sub.LCDR3, V.sub.HCDR3 are known, accordingly amino acid sequence and nucleotide sequence of recombinant molecules can be deduced exactly. Thereby, such sequence information will not be listed in this description.
(37) (3) Laboratory system: gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector; a batch of recombinant polypeptides were obtained by transforming said recombinant expression vector into engineered bacteria.
(38) Anti-target verification experiment of the obtained recombinants was carried out. Verification experiment was conducted on the killing effects of the obtained recombinants against multi-drug resistant Pseudomonas aeruginosa, vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae and Mycobacterium tuberculosis (verification method and experimental design were the same as recorded in ZL2009/10092128.4). Recombinant 3 in Table 4 had the best killing effect against said pathogenic bacteria; comparison of survival curves about inhibition of novel antibiotics prepared by this invention on methicillin-resistant Staphylococcus aureus (ATCC BAA-42), vancomycin-resistant enterococci (ATCC 700802), multi-drug resistant Pseudomonas aeruginosa (clinical isolated strain 13578 in West China Hospital) is shown in
(39) Recombinants prepared by taking the mutants of antibody mimetics in recombinant 3 as recognition region and taking colicin Ia as effect region were verified that, in Table 5, recombinants with SEQ ID NOS: 9-13 as recognition region has basically equivalent killing effects against the above-mentioned pathogenic bacteria as that of recombinant 3.
(40) TABLE-US-00005 TABLE5 aminoacidsequencesofanti-diplococcus intracellularisantibodymimetic V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3andmutamersthereof V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 No. andpointmutantsthereof SEQIDNO:8 SYWLHWIKQRPGQGLWIGSQSTHVPRT SEQIDNO:9 SYGMHWIKQRPGQGLWIGSQSTHVPRT SEQIDNO:10 SYWIEWIKQRPGQGLWIGSQSTHVPRT SEQIDNO:11 NYWMHWIKQRPGQGLWIGSQSTHVPRT SEQIDNO:12 SYWLHWIKQRPGQGLWIGMQNIGLPWT SEQIDNO:13 SYWLHWIKQRPGQGLWIGQQFTSSPYT
(41) It took less than 6 months for this preparation, and a batch of candidate novel antibiotics with broad-spectrum antibacterial effect were obtained successfully.
(42) Experimental methods and materials adopted to obtain each recombinant in this example were exactly the same as recorded in Patent No. ZL2009/10092128.4, except for the inserted gene sequences when constructing vectors, so they are not repeated here.
Example 3 Preparation of Novel Antibiotics Against Bacillus anthracis
(43) (1) Goal proposing system determined anthrax toxin or bacillus anthracis as targets; lethal infection diseases caused by anthrax toxin or bacillus anthracis have been posing a huge threat against human health; in terrorist attacks, anthrax toxin is also the most horrible pathogen or toxin as weapon.
(44) The goal of this preparation is to provide a novel antibiotic to destroy the toxicity of bacillus anthracis or anthrax toxin, i.e., to inhibit or interfere anthrax toxin PA antigen from forming active PA heptamer.
(45) (2) Designing Novel Antibiotics:
(46) The following designing work was performed according to general formula
(47) ##STR00005##
wherein F is effect region; R is recognition region.
(48) The general characteristic of anthrax toxin is that, anthrax toxin is a binary toxin with high harmfulness to organisms, and consists of protein antigen PA, necrosin and edema factor (LF/EF); protein antigen PA is a transport structure and can recognize target cells, and it transports necrosin and edema factor (LF/EF) into target cells. It is illustrated by animal experiments that, a combination of protein antigen and necrosin can immediately lead to cell death, while no reaction will be caused as applying said two components separately. The novel antibiotic was designed preliminarily that recognition region of said novel antibiotic can recognize anthrax PA antigen, and effect region of said novel antibiotic can inhibit or interfere anthrax toxin PA antigen from forming active PA heptamer.
(49) Establishing recognition region molecular structure library: anti-bacillus anthracis protein antigenlethal factor complex antibody (National Center for Biotechnology Information (NCBI) CAL58671) generated in cynomolgus and anti-bacillus anthracis protein antibody (NCBI ABF69350) generated in house mouse were found by searching database, and they are competent to specifically recognize protein antigen PA of anthrax toxin. According to amino acid information of said antibodies, a series of antibody mimetic structures and mutants thereof with a structure of V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 which can recognize wild-type anthrax toxin were designed to build recognition region molecular structure library, and provided to laboratory system.
(50) Establishing effect region molecular structure library: because preparation goal is to inhibit anthrax toxin PA antigen from forming PA heptamer, in accordance with infection mechanism of anthrax toxin, in this experiment, some mutant anthrax toxin PA antigens (see SEQ ID NO:10 recorded in ZL2008/10045212.6) obtained by artificial mutation on anthrax toxin PA antigen were conducted as member of effect region molecular structure library, and PA lost recognition capacity to corresponding receptor on target cells; said mutant anthrax toxin PA antigen and wild-type anthrax toxin PA antigen constituted heterozygous PA heptamer, which lost transmembrane activity completely or partially, accordingly interfered with infection ability of anthrax toxin.
(51) (3) Laboratory system: recombinant library was obtained by binding the provided effect region and recognition region; gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector; a batch of recombinant polypeptides were obtained by transforming said recombinant expression vector into engineered bacteria.
(52) A batch of recombinants with amino acid sequence listed in Table 6 as recognition region and mutant anthrax toxin PA antigens (see SEQ ID NO:10 recorded in ZL2008/10045212.6) as effect region were obtained through verification, and they could protect mice infected by bacillus anthracis. Verification experiment and results thereof were similar to the effects of pCHCA-PA1 recorded in ZL2008/10045212.6.
(53) TABLE-US-00006 TABLE6 aminoacidsequencesofantibody mimeticsandmutamersthereof recognizingwild-typeanthraxtoxinPA V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 NO. anditspointmutants SEQIDNO:14 STALHWRQAPGKGLEWVPRYDEFPYT SEQIDNO:15 SFGMHWRQAPGKGLEWVPRYDEFPYT SEQIDNO:16 NYWMHWRQAPGKGLEWVPRYDEFPYT SEQIDNO:17 STALHWRQAPGKGLEWVFQGSHVPFT SEQIDNO:18 STALHWRQAPGKGLEWVYCHQWSMYT SEQIDNO:19 STALHWRQAPGKGLEWVQQWSSNPYT SEQIDNO:20 STALHWRQAPGKGLEWVQQFTSSPYT
(54) It took less than 6 months for this preparation, and a batch of novel antibiotics which have protection effects against bacillus anthracis infection were obtained successfully.
(55) Experimental methods (e.g., vector construction, transformation, verification experiment, etc.) and materials adopted to obtain each recombinant in this example were exactly the same as examples recorded in Patent No. ZL2008/10045212.6, except for the inserted gene of novel antibiotics, so they are not repeated here.
Example 4 Preparation of Novel Antibiotics Against Fungi
(56) (1) Goal proposing system determined Candida albicans as targets, and the goal was determined to prepare novel antibiotics killing agricultural fungusCandida albicans.
(57) (2) Designing novel antibiotics:
(58) The following designing work was performed according to general formula
(59) ##STR00006##
wherein F is effect region; R is recognition region.
(60) Establishing recognition region molecular structure library: the great progress in fungus basic research had been achieved in recent years; amino acid sequence (SEQ ID NO:21) of Candida albicans pheromone consists of 14 amino acid residues. It can move around freely in biological media, and has biological activity of automatically searching the corresponding receptor on cell membranes of the same species of fungi cells. Thus, based on such automatically searching activity, it is available to utilize fungus pheromone as recognition region to induce effect region like bacterial exotoxin such as colicin to kill these fungi by forming ion channel through cell membranes, and a batch of novel biological biocides were constructed accordingly.
(61) Therefore, Candida albicans pheromone represented by SEQ ID NO:21 was selected as recognition region.
(62) Establishing effect region molecular structure library: because the preparation goal was lethal novel antibiotics against target of agricultural fungus-Candida albicans, colicin was a competent candidate substance for effect region owing to its characteristics. Thus, colicins Ia, Ib, A, B and N, and ion channel domains thereof were selected as members of effect region molecular structure library, and were provided to laboratory system.
(63) The preliminarily designed molecular structure of novel antibiotic was: from amino terminal to carboxyl terminal: colicin or ion channel domain thereof or mutamers thereof +Candida albicans pheromone.
(64) (3) Laboratory system: recombinant library was obtained by binding the effect region and recognition region; gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector; recombinant polypeptides were expressed by transforming said recombinant expression vector into engineered bacteria, which realized operable binding between effect region and recognition region.
(65) The obtained recombinants were verified that, they all have protection effects on rice (Oryza sativa) infected by fungi like Pyricularia oryzae, Aspergillus flavus, and their protection effect on rice blast infection is thousands of times higher than that of current agricultural antifungal.
(66) Experiments and data (e.g., vector construction, transformation) related to obtaining each recombinant in this preparation was recorded in examples of Patent No. ZL2007/10050926.1 or based on the record in prior art, person skilled in the art can obtain the experimental methods required for the preparation of this invention by a limited number of experiments, so they are not repeated here.
(67) It took 6 months for this preparation, and cost only 1.5-2 years including field experiments, whose efficiency and success rate are far higher than that of current preparation for a new drug.
Example 5 Preparation of Novel Antibiotics Against Drug-Resistant Staphylococcus aureus
(68) (1) goal proposing system determined Staphylococcus aureus as targets: since antibiotics like penicillin was applied in 1944, bacteria, especially pathogenic bacteria threatening human life, like Staphylococcus aureus, streptococcus pneumonia, Pseudomonas aeruginosa, mycobacterium tuberculosis, etc., have generated drug-resistance, and human are urgent to develop novel antibiotics against drug-resistant bacteria.
(69) (2) Designing novel antibiotics:
(70) The following designing work was performed according to general formula
(71) ##STR00007##
wherein F is effect region; R is recognition region.
(72) Establishing recognition region molecular structure library: many cells secrete signal transduction polypeptides to the outside of cells; these polypeptides can automatically search for the corresponding receptors on cell membranes of the same species of bacteria, and bind to said receptors to transport information into said bacteria; staphylococcus secretes signal transduction polypeptides to the outside of cells; these polypeptides can automatically search for the corresponding receptors on cell membranes of the same species of bacteria, and bind to said receptors to transport information into said bacteria. These signal transduction polypeptides consist of several to more than 10 amino acids, and are ideal recognition regions against staphylococcus. Pheromone sequences as SEQ ID NOS:22-26 were collected as the members of recognition region library through searching.
(73) Providing effect region: because the preparation goal was lethal novel antibiotics against target of drug-resistant Staphylococcus aureus-Candida albicans, colicin was a competent candidate substance for effect region owing to its characteristics. Thus, colicins Ia, Ib, A, B and N, and ion channel domains thereof were selected as effect regions, and were provided to laboratory system.
(74) The preliminarily designed molecular structure of novel antibiotic was: from amino terminal to carboxyl terminal: colicin or ion channel domain thereof or mutamers thereof +pheromone, and pheromone+colicin or ion channel domain thereof or mutamers thereof.
(75) (3) Laboratory system: recombinant library was obtained by binding the effect region and recognition region; synthetic gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector; recombinant polypeptides were expressed by transforming said recombinant expression vector into engineered bacteria, which realized operable binding between effect region and recognition region.
(76) A batch of recombinants were obtained, such as recombinant polypeptide expressed by recombinant plasmids pBHC-SA1, pBHC-SA2, pBHC-SA3 pBHC-SA4, pBHC-SE and pBHC-PA (as recorded in Patent No. ZL2009/10157564.5). They all have significant killing effects against methicillin-resistant Staphylococcus aureus, penicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Pseudomonas aeruginosa and multi-drug resistant Pseudomonas aeruginosa.
(77) Experiments and data (e.g., vector construction, transformation) related to obtaining each recombinant in this preparation was recorded in examples of Patent No. ZL2009/10157564.5 or based on the record in prior art, person skilled in the art can obtain the experimental methods required for the preparation of this invention by a limited number of experiments, so they are not repeated here.
(78) It took 5 months to prepare a batch of novel antibiotics against drug-resistant bacteria, whose efficiency and success rate are far higher than that of current preparation for a new drug.
Example 6 Preparation of Novel Antibiotics Against Cyanobacteria
(79) (1) Goal proposing system determined cyanobacteria as targets: cyanobacteria proliferation caused by water eutrophication, water pollution are the severest harm threatening water environment in the world, and they result in huge economic loss to human as well as cause unrepaired harm to earth's biosphere. As the accelerated industrialization and urbanization caused by Chinese economic development, ecological environment pollution and degeneration aggravate gradually, and water environment ecological control has been major problem we must face and solve. The current antimicrobial drugs almost have little effects against cyanobacteria; cyanobacteria is prokaryotic cell belonging to cyanobacteria phylum of bacteria kingdom, and only chemicals of heavy metals can control cyanobacteria at present, e.g., cupric sulfate. However, in practical application, owing to limited effects, it is required to use chemical with overdose repeatedly, and other beneficial algae, aquatic plants and aquatic organisms are destroyed when killing cyanobacteria, which results in irreversible permanent damage to environment.
(80) The purpose is preparing novel antibiotics killing or inhibiting cyanobacteria.
(81) (2) Designing novel antibiotics:
(82) The following designing work was performed according to general formula
(83) ##STR00008##
wherein F is effect region; R is recognition region.
(84) Establishing recognition region molecular structure library: a batch of hybridoma cells secreting anti-cyanobacteria monoclonal antibodies were obtained by immunizing mice with cyanobacteria as antigen, and the deposit No. of one strain of said hybridoma cells is CGMCC No. 4783.
(85) Based on amino acid sequences of said monoclonal antibodies obtained by sequencing, a batch of antibody mimetics were designed, which are shown in Table 1, and mutamers of antibody mimetics were obtained through random point mutation on the first 5 and the last 9 amino acids of antibody mimetics; recognition region molecular structure library was built by taking said antibody mimetic molecules and mutamers thereof as members. Wherein, the amino acid sequence of antibody mimetic with a structure of V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 of monoclonal antibody secreted by hybridoma cells (CGMCC No. 4783) is shown as SEQ ID NO:27.
(86) Establishing effect region molecular structure library: because the preparation goal was novel antibiotics killing or inhibiting cyanobacteria, colicin could form lethal ion channel through cell membrane of Escherichia coli of the same species but different strains by itself to cause death of Escherichia coli of the same species but different strains, and it was a competent candidate substance for effect region. Thus, colicins Ia, Ib, A, B and N or mutant sequences thereof were selected as members of effect region molecular structure library.
(87) Preliminarily obtaining the designed molecular structure of novel antibiotics: from amino terminal to carboxyl terminal: colicin or mutamers thereof+anti-cyanobacteria antibody mimetic/mutamers:
(88) (3) Laboratory system: molecular structure of recombinants was obtained by binding the effect region and recognition region; gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector; the recombinants were expressed by transforming said recombinant expression vector into engineered bacteria and isolated.
(89) It is revealed by verification on the inhibition effect of obtained recombinants against cyanobacteria (the design and operation of verification experiments was the same as recorded in Examples 3-5 of ZL2011/10155221.2) that, said recombinants had significant inhibition effects on Microcystis aeruginosa, anabaena but no inhibition on other beneficial algae like chlorella, diatom and scenedesmus. Some experimental results are shown as
(90) TABLE-US-00007 TABLE7 aminoacidsequencesofantibody mimeticsandmutamersagainstsurface antigensofMicrocystisaeruginosa V.sub.HCDR1-V.sub.HFR2-V.sub.LCDR3 NO. andpointmutantsthereof SEQIDNO:27 SYWMQWVKQRPGQGLEWIGQQYWSTPPWT SEQIDNO:28 SYGMHWVKQRPGQGLEWIGQQYWSTPPWT SEQIDNO:29 DHYMHWVKQRPGQGLEWIGQQYWSTPPWT SEQIDNO:30 SYWIEWVKQRPGQGLEWIGQQYWSTPPWT SEQIDNO:31 SYWMQWVKQRPGQGLEWIGQQQFTSSPWT SEQIDNO:32 SYWMQWVKQRPGQGLEWIGQQQSREYPYT SEQIDNO:33 SYWMQWVKQRPGQGLEWIGQLQGTHQPYT
(91) It took less than 11 months for this preparation, and a batch of verified novel antibiotics which had killing effects against targets were obtained successfully.
(92) Experimental methods and materials adopted to obtain recombinants in this preparation were exactly the same as recorded in Patent No. ZL2011/10155221.2, except for the inserted gene of novel antibiotics in vector construction, so they are not repeated here.
Example 7 Experiments of Screening Suitable Biological Expression Systems for the Methods of this Invention
(93) Recombinant plasmids were obtained during novel antibiotics preparation recorded in examples 5-6: pBHC-SA1, pBHC-SA2, pBHC-SA3, pBHC-SA4, pBHC-SE, pBHC-PA and pBHC-PorA1.
(94) Step 1. Transforming Competent Cells
(95) 4040 ?l various engineered bacteria of pET system like BL-21(DE3), B834(DE3), Nova Blue(DE3) and 618 (Novagen) were transfected by 100 ng recombinant mutant plasmids respectively; ice incubate for 5 minutes, thermal shock at 42? C. for 30 seconds, ice bathing for 2 minutes, adding 160 ul SOC medium, 220 rpm, incubated at 37? C. by shaking for 1 hour, and then spread on plate (LB medium with 1% agar and 50 ?g/ml ampicillin) to incubate at 37? C. overnight. Monoclonal colony was selected for proliferation to get strain, and the strain was preserved at low temperature.
(96) Step 2. Strain Recovery
(97) 1. Strain Recovery
(98) Said preserved strain was unfrozen at 4? C.; 1.5 ml strain was added in 10 ml LB medium (containing AMP 50 ?g/ml), 220 rpm, and incubated at 37? C. for 5-8 hours.
(99) 2. Inoculation of Monoclonal Strain
(100) The recovered strain culturing liquid was diluted at 10.sup.4 or 10.sup.5 times; 10 ?l diluted strain culturing liquid was placed onto the prepared LB solid medium (AMP 50 ?g/ml) plate and spread. The plates were placed in moist box for incubation at 37? C. for 10-12 hours till round single colonies were raised on the surface of plate.
(101) Step 3. Selection and Proliferation of Strain
(102) (1) Regular round single colony with smooth edge was selected by the sterilized toothpick or inoculation loop from the incubated plate, and placed into 1.5 ml LB medium for culture by shaking at 220 rpm and 37? C. for 5-8 hours.
(103) (2) 1.5 ml LB medium was added into 100 ml LB medium for culture by shaking at 220 rpm and 37? C. for 5-8 hours.
(104) (3) 1.sup.st grade amplification culture: 100 ml strain culturing liquid obtained from the last step was added into 700 ml improved FB-M9 complex medium for culture by shaking at 220 rpm and 37? C. for 5-8 hours.
(105) (4) 2.sup.nd grade amplification culture: 700 ml strain culturing liquid obtained from the last step was added into 6?700 ml improved FB-M9 complex medium for culture by shaking at 220 rpm and 37? C. for 5-8 hours.
(106) (5) 3.sup.rd grade amplification culture: 6?700 ml strain culturing liquid obtained from the last step was added into 20 L improved FB-M9 complex medium for culture in fermenter with shaking speed at 220 rpm and maximum oxygen content, at 37? C. for 3-5 hours.
(107) (6) Engineered bacteria fermentation and induction of protein expression: 20 L strain culturing liquid obtained from the last step was added into 200 L improved FB-M9 complex medium for culture and protein expression in fermenter, with shaking speed at 220 rpm and maximum oxygen content, at 30? C. for 2-4 hours; then at 42? C. for 0.5 hours; finally at 37? C. for 1-2 hours. Note: IPTG with final concentration of 0.5 mM was added when reaching 42? C.
(108) Step 4. Strain Collection by Centrifugation
(109) Strain culturing liquid was centrifuged at 6000 g, 4? C. for 20 min. The precipitate was collected after centrifugation, and resuspended in 50 mM borate buffer (pH9.0). Note: 2 mM PMSF (benzyl sulfuryl fluoride serine proteases inhibitor) was added into borate buffer, and the operation after thalli resuspending must be conducted at 4? C.
(110) Step 5. Thalli Fragmentation
(111) After thalli were suspended in pH9.0 borate buffer totally, thalli were fragmentated by high pressure homogenizer at 500-600 bar; fragmentation was repeated for 7 times, and there was 3-5 minutes interval between two fragmentations.
(112) Step 6. Precipitation of Thalli DNA
(113) Fragmentated strain culturing liquid was centrifuged at 55000 g, 4? C. for 40 min. The supernatant was isolated, added with streptomycin sulfate (16 bottles of streptomycin sulfate with 1 million units were added into every 200 ml liquid), and stirred on magnetic stirrer for 1 h.
(114) Step 7. Dialysis
(115) Strain culturing liquid obtained by the last step was centrifuged at 55000 g, 4? C. for 20 min. The supernatant was isolated, placed into dialysis bag, placed in borate buffer for dialysis for 8-12 hours, and the dialysate was changed once every 4 hours.
(116) Step 8. Antibacterial Engineered Polypeptide Medicine Obtained by Protein Purification
(117) Strain culturing liquid after dialysis was centrifuged at 55000 g, 4? C. for 20 min. The supernatant was placed into beaker to conduct protein purification by ion exchange method. The supernatant was loaded in CM ion exchange column, and protein concentration was detected to count protein content per unit volume; the ratio of loading volume and CM ion gel particles was fixed according to operation manual. After rinsing thoroughly, novel antibacterial engineered polypeptide was obtained through elution by 50 mM borate buffer containing 0.2M NaCl.
(118) It is described by results shown in Table 8 that, the expression rate of PMC-SA in E. coli B834 (DE3) was the highest.
(119) TABLE-US-00008 TABLE 8 comparison of expression rates in different strains Engineered strains TG1 BL-21 618 NavaBlue B834 the average yield per unit (mg/L) 0.8 10 5.8 8.1 24.4 (the average yield per unit = total production of extracted PMC-SA1/the volume of strain culturing liquid)
(120) The same operation and comparison were conducted on other 6 kinds of recombinant mutant plasmids, and the results all show the same tendency as shown in Table, that is, compared with other engineered bacteria, the expression rates of 7 kinds of recombinant mutant plasmids in E. coli B834(DE3) are all the highest.
(121) On the basis of this screening experiment, it is preferable but not limited to select E. coli B834(DE3) as expression system in the method of this invention, in order to efficiently express and prepare to obtain novel antibiotics.
(122) In summary, novel antibiotic preparation method and platform system of this invention are capable of providing novel antibiotics with recognition region and effect region specifically against most pathogenic microorganisms and targets. For most targets, the currently-known colicin is competent to playing the role of effect region, so the cycle time of preparing a novel antibiotic depends on the time to design molecular structure, the time to provide recognition region molecular information, as well as the time to prepare and verify recombinants. Because recognition region depends not only on the selected nature substances but mainly depends on artificial-prepared antibody mimetic as recognition region, in view of the current biotechnological level, the time to prepare a novel antibiotic can be basically controlled in a very short period. Platform for operating said preparation method of this invention fully optimizes, utilizes and integrates human resources and technology resources, to ensure efficient conduct of development process and make novel antibiotic development operating as flow-line production.