CN109251248A - Nano antibody, the Preparation method and use of anti-erabutoxin - Google Patents
Nano antibody, the Preparation method and use of anti-erabutoxin Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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Abstract
The present invention relates to biomedicine technical fields, provide a kind of nano antibody of anti-erabutoxin, preparation method and application, the nano antibody of the anti-erabutoxin is VHH antibody, with amino acid sequence shown in SEQ ID NO.1, the nucleotide sequence of the nano antibody is encoded as shown in SEQ ID NO.2, there is good affinity by affinity analysis nano antibody of the invention, by toy experiments have shown that, the antibody protection group mouse of injection nano antibody of the present invention is after injecting erabutoxin SN36 in advance, there is neurotoxic symptom in no any mouse, one month is observed continuously without toxin lethal cases, illustrate that nano antibody of the invention has the function of excellent anti-lacticotoxin, it bites sea snake with excellent prevention or therapeutic effect, has wide clinic Application prospect.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of nano antibody of anti-erabutoxin, its system
Preparation Method and purposes, especially in the antitoxin preparation of preparation sea snake and the purposes in terms for the treatment of or preventing sea snake and biting.
Background technique
As ocean big country, the critical issue that a strong naval is China's army building is built.However coastal
Poisonous and hazardous marine organisms existing for sea area can there are potential threats to Navy Men, therefore grind to the prevention and treatment of injuries from marine creature
Study carefully work to be of great importance for the health support and curative activity of landing operation, so must set about carrying out anti-sea as early as possible
The scientific application research of foreign biology wound.
Lapemis is a kind of typical toxic marine organisms, belongs to Elapidae, popular name spine sea snake is distributed mainly on eastern print
Du Yangyin Australia sea area, Australia and Philippine is coastal and China's the island of Taiwan and Fujian, Shandong, Hong-Kong, Hainan, wide
The ground such as west.Lapemis event of biting occurs often, constitutes certain threat to the ocean operations and military training in these areas.
Venom is predominantly discharged to the remedy measures that sea snake is bitten at present, reduces venom absorption and antitoxin treatment etc..Although
Injecting the extraordinary antitoxin preparation such as antitoxic serum or antibody drug as early as possible is most effective emergency treatment measure, however directly uses snake
The method of malicious toxin immunity animal has the disadvantages that first come the method for obtaining antitoxic serum or screening more/monoclonal antibody
It is that sea snake captures difficulty, it is difficult to obtain a large amount of natural lacticotoxin and carry out animal immune, separation neurotoxin is remote from poison gland
It is not able to satisfy the needs for preparing antitoxin preparation；Second is that since venom toxin has lethal to animal, it is therefore desirable to be attenuated
Processing, and be attenuated toxin and often lose its antigenicity and epitope, lead to the antitoxin neutralization activity using attenuation toxin preparation
Weaken；Third is that the products such as antitoxic serum of animal origin easily cause the strong allergic reaction of patient, or even dead.Therefore to mesh
Before until, only a few countries are standing in the world the antitoxin preparation of sea snake.
With the development of molecular biology, the technology for preparing venom toxin using genetic engineering is mature, carries out snake venom
The biosynthesis of toxin and the preparation research for carrying out anti-snake venom antibody have become possibility.This team early period, which has recombinated, to be obtained
Three kinds of erabutoxins should screen the specific antibodies medicine for toxin as early as possible.Due to antibody can efficiently, specifically with
Internal and external various antigen proteins combine, so that antibody can not only be applied to adjust function of immune system, can also apply
In various highly sensitive detection methods.
Currently, antibody drug is the most important component part of biotech drug, antibody reagent is also medical diagnosis and life
In object research most commonly used reagent it.Therefore, the relevant biological product of antibody has high application prospect and business valence
Value.Antibody can obtain through a variety of ways, such as: the blood of animal or people, cell culture, the mouse for injecting hybridoma
Ascites etc., but this is required to effective method and is purified, to obtain the antibody product with application value.It is most common
Antibody purification process is to carry out affinity chromatography using the high-affinity between specific proteins and the Fc segment of antibody.Resisted
Affinity chromatography is a wherein the most key step during the industrial production of body product, and is expended in entire production highest
Part.
In view of the above-mentioned problems, it has been found that nano antibody, nano antibody is derived from camellid or selachian
Distinct antibodies.Studies have shown that the antibody in camel body there are a kind of natural deletions light chain containing only heavy chain, referred to as heavy chain antibody.Gram
The available single domain antibody being only made of a heavy chain variable region in the variable region of grand chain antibody, referred to as VHH antibody.VHH antibody
Crystal diameter only have 2.5nm, long 4nm, therefore the nano antibody that is otherwise known as.The size of nano antibody only has tradition IgG type anti-
/ 10th of body, be it is naturally occurring can be with the minimal segment of antigen binding.Nano antibody can be encoded by single-gene, can be very
It is readily produced using microorganism, and there is very high yield.But have not yet to see the nanometer for erabutoxin
The relevant report of antibody.
Summary of the invention
It is an object of the present invention to rely on the studies above background, by taking erabutoxin SN36 as an example, anti-sea snake mind is studied
Nano antibody, preparation method and purposes through toxin provide nano antibody, its preparation side of a kind of anti-erabutoxin
Method and purposes.
The first aspect of the present invention, provides the nano antibody of anti-erabutoxin SN36 a kind of, which is
VHH antibody has amino acid sequence shown in SEQ ID NO.1, encodes the nucleotide sequence such as SEQ ID of the nano antibody
Shown in NO.2.
Nano antibody amino acid sequence (SEQ ID NO.1) is as follows:
DVQLQESGGGLVQAGGSLRLSCAASGMCKVTHCFRAMGWWRQAPGKEREFVAT GKVDITQAWADSVK
GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYA DIMYVNFQDENDYWGQGTQVTVSS。
The nucleotide sequence (SEQ ID NO.2) for encoding the nano antibody is as follows:
GATGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCC TGCGCCTGAGCTGCG
CGGCGAGCGGCATGTGCAAAGTGACCCATTGCTTTCGCGCG ATGGGCTGGTGGCGCCAGGCGCCGGGCAAAGAAC
GCGAATTTGTGGCGACCGGCA AAGTGGATATTACCCAGGCGTGGGCGGATAGCGTGAAAGGCCGCTTTACCATTA
GCC GCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGAT ACCGCGGTGTATTAT
TGCTATGCGGATATTATGTATGTGAACTTTCAGGATGAAAACG ATTATTGGGGCCAGGGCACCCAGGTGACCGTG
AGCAGC。
The acquisition of nano antibody about anti-erabutoxin first constructs the nano antibody phagocytosis of anti-erabutoxin
Body display library, then screens nano antibody, and screens specificity using the enzyme-linked immunoassay method (ELISA) of bacteriophage
Positive colony obtains the VHH nano antibody with above-mentioned amino acid sequence after sequence is analyzed, and the nano antibody is by FR1-
CDR1-FR2-CDR2-FR3-CDR3-FR4 district's groups at.
The second aspect of the present invention, provides the preparation method of the nano antibody of anti-erabutoxin SN36, including with
Lower step:
(A) full genome synthesizes the nano antibody VHH segment of anti-erabutoxin；
(B) the nano antibody VHH segment progress gram using round pcr to the anti-erabutoxin obtained in step (A)
Grand, PCR product is through agarose gel electrophoresis purification and recovery and is cloned into expression vector, and confirmation obtains correct after sequence verification
Clone；
(C) the above-mentioned expression vector is introduced into the expression that fusion protein is carried out in host cell.
Preferably, in step (B), primer sequence used by PCR is as follows:
In the present invention, any suitable carrier is all suitable for, preferably pGEM-T, Pet32a, pcDNA3.1, pEE6.4,
PEE12.4, pDHFR or pDR1 include the fusion dna for being connected with suitable transcription and translation and adjusting sequence in the expression vector
Sequence.
In the present invention, mammal or insect host cell or prokaryotic cell culture systems are used equally for fusion of the invention
The expression of albumen.Available host cell be the prokaryotic cell containing above-mentioned carrier, can for DH5a, Top10, BL21 (DE3),
One of TG1.
Fusion protein of the invention can easily generate in following cell: mammalian cell, such as CHO, NSO,
HEK293, BHK or COS cell；Bacterial cell, such as Escherichia coli, withered grass bud are from bacillus or Pseudomonas fluorescens；Insect is thin
Born of the same parents or fungi or yeast cells, the cell use known technology culture in this field.
The preparation method of fusion protein disclosed in the present invention is to cultivate above-mentioned host cell under expression condition, from
And it expresses, separate, purifying the fusion protein.It can be substantially uniform substance by antibody purification using the above method, such as
It is single band on SDS-PAGE electrophoresis.
The method that can use affinity chromatography isolates and purifies fusion protein disclosed by the invention, according to what is utilized
The characteristic of affinity column can be used conventional method such as high-salt buffer, change the methods of PH elution of bound on affinity column
Fusion protein polypeptide.
Various method for purifying proteins can be used, and such method is known in this field and is described in for example
(Wilchek and Bayer,1990,Methods in enzymology)(Scopes,2013,Protein
purification: principles and practice)。
Analyzed by Biacore, nano antibody of the invention have good affinity, by toy it is demonstrated experimentally that
After injecting erabutoxin SN36, no any mouse the antibody protection group mouse of injection nano antibody of the present invention occurs in advance
Neurotoxic symptom is observed continuously one month without toxin lethal cases.Illustrate that nano antibody of the invention has excellent anti-sea
The effect of ophiotoxin.
Therefore, the third aspect of the present invention provides a kind of medicine group of nano antibody containing anti-erabutoxin
Close object.The pharmaceutical composition further includes that pharmaceutically acceptable drug carries in addition to the nano antibody including anti-erabutoxin
Body.
The nano antibody of anti-erabutoxin of the invention and pharmaceutically acceptable auxiliary material forms drug system together
Agent composition, to more stably play curative effect, these preparations can guarantee receiving for anti-erabutoxin disclosed by the invention
The conformation integrality of rice antibody amino acid core sequence, while also wanting the polyfunctional group of protected protein matter, prevent its degradation (including
But it is not limited to cohesion, deamination or oxidation).
Under normal conditions, liquid preparation can save under the conditions of 2 DEG C -8 DEG C and at least stablize 1 year, and lyophilized preparation is at 30 DEG C
Holding at least six months is stablized.The preparations, preferably water needle or freeze-drying such as preparation can commonly be suspended for pharmaceutical field, water needle, freeze-drying
Preparation.
For the water needle or lyophilized preparation of the nano antibody of above-mentioned anti-erabutoxin disclosed by the invention, pharmaceutically may be used
Auxiliary material with receiving includes one or a combination set of surfactant, solution stabilizer, isotonic regulator and buffer.Wherein, table
Face activating agent includes nonionic surface active agent such as Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80)；poloxamer
(such as poloxamer 188)；Triton；Lauryl sodium sulfate (SDS)；Sodium Laurylsulfate；Myristyl, sub- oil base or 18
Alkylsarcosines；Pluronics；MONAQUATTM etc., additional amount should make the granulating of difunctional bispecific antibody albumen
Trend is minimum；Solution stabilizer can be carbohydrate, including reducing sugar and nonreducing sugar, amino acids include monosodium glutamate
Or histidine, alcohols include one or a combination set of trihydroxylic alcohol, advanced sugar alcohol, propylene glycol, polyethylene glycol, the addition of solution stabilizer
Amount should make the preparation those skilled in the art eventually formed think to reach stable time interior holding stable state；It is isotonic
Regulator can be one of sodium chloride, mannitol；Buffer can be one of TRIS, histidine buffering liquid, phosphate buffer.
Above-mentioned preparation is the composition of the nano antibody comprising anti-erabutoxin, is given to animal including people
After medicine, anti-snake venom effect is obvious.Specifically, the prevention and/or treatment bitten to sea snake effectively, can be used as anti-venom drug
It uses.
Nano antibody of anti-erabutoxin and combinations thereof is when to animal administration including people in the present invention,
Age and weight of the dosage because of patient, disease traits and seriousness and administration route and it is different, zoopery can be referred to
Result and various situations, total dosage is no more than a certain range.The dosage being specifically injected intravenously is 1~1800mg/
It.
The fourth aspect of the present invention provides a kind of purposes of the nano antibody of anti-erabutoxin, is specifically making
Purposes in the standby antitoxin preparation medicine of sea snake.
Beneficial guarantee of the invention and effect:
The present invention provides nano antibody, the preparation method and its usage of a kind of anti-erabutoxin, the anti-sea snake minds
Nano antibody through toxin is VHH antibody, has amino acid sequence shown in SEQ ID NO.1, and size only has tradition IgG type anti-
/ 10th of body are naturally occurring can be encoded by single-gene, microorganism easy to use with the minimal segment of antigen binding
It is produced, it is simple to construct and express process, and there is very high yield, be advantageously implemented industrialized production.
In addition, there is good affinity by affinity analysis nano antibody of the invention, is tested and demonstrate,proved by toy
Bright, the antibody protection group mouse of injection nano antibody of the present invention is after injecting erabutoxin SN36 in advance, without any mouse
There is neurotoxic symptom, is observed continuously one month without toxin lethal cases, it is excellent to illustrate that nano antibody of the invention has
The effect of anti-lacticotoxin bites with excellent prevention or therapeutic effect to sea snake, has wide potential applicability in clinical practice.
Detailed description of the invention
Fig. 1 is the nano antibody ELISA the selection result of anti-erabutoxin.
Specific embodiment
The present invention is further detailed in following embodiment, experimental example, should not be construed as limiting the invention.It is real
Applying example does not include detailed descriptions of conventional methods, such as the method that those draw for carrier construction and matter, will encode the base of albumen
Plasmid is introduced method as the method for host cell for this field by the method drawn by carrier as being inserted into and matter
In be well-known with the personnel of ordinary skill, and be all described in many publications, including Sambrook,
J., Fritsch, E.F.and Maniais, T. (1989) Molecular Cloning:A Laboratory Manual,
2ndEdition, Cold spring Harbor Laboratory Press.
Building of the embodiment 1. for the nano antibody library of erabutoxin SN36
(1) by 0.5mg erabutoxin SN36 (Hu Shi etc., the anti-full source of people monoclonal of Lapemis short-chain neurotoxin
Screening, preparation and the bioactivity research liberation army medical journal 42.7 (2017) of antibody: 612-616.) antigen and Freund help
Agent mixes in equal volume, and an Xinjiang two-humped camel is immunized, once a week, co-continuous 6 times immune, and B cell table is irritated in immunologic process
Up to the nano antibody of specificity；
After (2) 6 times immune, extract camel peripheral blood lymphocytes 200mL and extract total serum IgE；
(3) it synthesizes cDNA and expands VHH using sleeve type PCR, primer sequence employed in the step is as shown in table 1:
1 PCR primer sequence of table
(4) simultaneously using 20 μ gpMECS Vector for Phage Display of restriction enzyme Pstl and NotI digestion and 10 μ g VHH
Connect two kinds of segments；
(5) connection product is converted to electricity and is turned in competent cell TGl, building erabutoxin SN36 nano antibody is bitten
Phage-displayed peptide libraries simultaneously measure storage capacity, and the size of storage capacity is about 2.5 × 108.At the same time, built text is detected by bacterium colony PCR
The insertion rate in library reaches 95% or more.
Embodiment 2. is directed to the nano antibody screening process of erabutoxin SN36
(1) it takes 200uL recombination TGl cell to cultivate into 2TY culture medium, 50 μ L helper phage VCSM13 is during which added
TGl cell is infected, and overnight incubation, to expand bacteriophage, next day utilizes PEG/NaCl precipitating phage, amplification is collected by centrifugation and bites
Thallus；
(2) it is dissolved in 8.2 NaHCO of 150mmol/L pH3In erabutoxin SN36150ug be coupled at enzyme mark
On plate, 4 DEG C stand overnight, while setting up negative control；
The 5%BSA of 100 μ L is added within (3) second days, room temperature closes 2h；
(4) after 2h, 100 μ L is added and expand bacteriophage (1 × 1011Camel nano antibody phage display gene is immunized in tfu
Library), room temperature acts on 1 hour；
(5) it is washed 5 times with PBS+0.05%Tween 20, to wash off the bacteriophage of combination；
(6) bacteriophage that will be specifically bound in erabutoxin SN36 with the membrane proteolytic enzyme of final concentration of 25mg/mL
Under dissociation, and the Escherichia coli TGl cell for being in logarithmic growth phase is infected, 37 DEG C of culture lh are generated and collected bacteriophage and are used for
The screening of lower whorl, identical screening process repeat 3 wheels, are gradually enriched with.
Embodiment 3. is cloned with enzyme-linked immunoassay method (ELISA) the screening specific positive of bacteriophage
(1) after above-mentioned 3 wheel screening in tissue culture plate, 200 single colonies is selected and are inoculated in respectively containing 100 μ g/mL ammonia
In 96 deep-well plates of parasiticin TB culture medium, and blank control is set, after 37 DEG C of culture to logarithmic phases, is added final concentration of 1
The IPTG of mmol/L, 28 DEG C of overnight incubations；
(2) method acquisition is burst using infiltration and slightly mention antibody, and antibody is transferred on the elisa plate through antigen coat, room
Temperature places lh；
(3) unbonded antibody is washed away with PBST, and Mouse anti-HA tag of the 100 μ L after 1:2000 dilutes is added
Antibody (the anti-HA antibody of mouse is purchased from Ke Wensi), is being placed at room temperature for lh；
(4) unbonded antibody is washed away with PBST, and Anti-mouse of the 100 μ L after 1:2000 dilutes is added
Alkaline phosphatase conjugate (goat-anti-mouse alkaline phosphatase enzyme mark antibody is purchased from Sigma),
It is placed at room temperature for 1h；
(5) it washes away unbonded antibody with PBST, alkaline phosphatase developing solution is added, react after 10min in microplate reader
At 405 wavelength, absorption value is read；
(6) when sample well OD value be greater than 6 times of control wells or more when, be determined as positive colony hole, as a result as shown in Figure 1,
The OD value in the hole SN36 is significantly greater than control wells group；
(7) bacterium in positive colony hole is turned to shake in the LB culture medium containing 100 μ g/ μ L ampicillins to extract matter
Grain is simultaneously sequenced.The gene order that each clone strain is analyzed according to sequence alignment program VectorNTI, FRl, FR2, FR3,
The identical strain of FR4, CDR1, CDR2, CDR3 sequence is considered as same clone strain, and the different strain of sequence is considered as different clone strains, most
1 plant of anti-erabutoxin SN36 specific nano antibody is obtained eventually, and the amino acid sequence of antibody is SEQ ID NO.1, is compiled
The nucleotide sequence of code antibody is as shown in SEQID NO.2.
Expression and purifying of the 4. erabutoxin SN36 specific nano antibody of embodiment in host strain Escherichia coli
(1) it by above-mentioned sequencing analysis Cloning Transformation obtained into Escherichia coli WK6, and is coated on containing ammonia benzyl
On the culture plate of penicillin and glucose, 37 DEG C of overnight incubations；
(2) it selects single bacterium colony and is seeded in 5mL and contain in the LB culture solution of ammonia Wei penicillin, 37 DEG C of shaking table cultures are stayed overnight；
(3) be inoculated with 1 mL is incubated overnight strain into 330mL TB culture solution, and OD is arrived in 37 DEG C of shaking table cultures, culture
When 600nm value reaches 0.6-0.9,1M IPTG is added, 28 DEG C of shaking table cultures are stayed overnight；
(4) it is centrifuged, collects Escherichia coli, burst method using infiltration, obtain antibody crude extract；
(5) antibody is purified by affinity chromatography method, obtains the nano antibody of high-purity, and enrichment method.
Embodiment 5.Biacore analysis
Anti- polyhistidine antibody (abcam) is coated in CM5M5 chip (GE company), is captured after being detected antibody,
The affinity of each fusion protein is detected with Biacore T100 (GE Healthcare), the specific affinity numerical value that detects is shown in Table 2.
Table 2.Biacore analyzes result
The experiment of 6. toy of embodiment
Kunming mice 30 of weight (20 ± 2) g are taken, fasting 12h (can't help water) before testing.Mouse is grouped at random, often
Group 10, mouse is divided into using half lethal dose SN36 group by half male and half female, and injection nano antibody 10mg/kg medicine in advance
Object protection group.Other 1 group is blank control group (physiological saline), and mouse peritoneal drug administration by injection allusion quotation occurs after mouse administration in 1h
There is neurotoxic symptom without any mouse in the neurotoxic symptom of type, antibody protection group, are observed continuously 1 month without endotoxin lethality
Situation, concrete outcome are shown in Table 3.
3 antibody antitoxin of table acts on testing result
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Sequence table
<110>Second Military Medical University, PLA
<120>nano antibody of anti-erabutoxin, Preparation method and use
<130>claims specification
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 124
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 1
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Met Cys Lys Val Thr His
20 25 30
Cys Phe Arg Ala Met Gly Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg
35 40 45
Glu Phe Val Ala Thr Gly Lys Val Asp Ile Thr Gln Ala Trp Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Tyr Ala Asp Ile Met Tyr Val Asn Phe Gln Asp Glu Asn Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 2
<211> 372
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
gatgtgcagc tgcaggaaag cggcggcggc ctggtgcagg cgggcggcag cctgcgcctg 60
agctgcgcgg cgagcggcat gtgcaaagtg acccattgct ttcgcgcgat gggctggtgg 120
cgccaggcgc cgggcaaaga acgcgaattt gtggcgaccg gcaaagtgga tattacccag 180
gcgtgggcgg atagcgtgaa aggccgcttt accattagcc gcgataacgc gaaaaacacc 240
gtgtatctgc agatgaacag cctgaaaccg gaagataccg cggtgtatta ttgctatgcg 300
gatattatgt atgtgaactt tcaggatgaa aacgattatt ggggccaggg cacccaggtg 360
accgtgagca gc 372
<210> 3
<211> 15
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
gatgtgcagc tgcag 15
<210> 4
<211> 16
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
gctgctcacg gtcacc 16
Claims (9)
1. a kind of nano antibody of anti-erabutoxin, which is characterized in that the nano antibody is VHH antibody, has SEQ
Amino acid sequence shown in ID NO.1.
2. encoding the nucleotide of the nano antibody of anti-erabutoxin described in claim 1, which is characterized in that the nucleosides
Acid sequence is as shown in SEQ ID NO.2.
3. the preparation method of the nano antibody of anti-erabutoxin described in claim 1, which is characterized in that including following step
It is rapid:
(A) full genome synthesizes the nano antibody VHH segment of anti-erabutoxin；
(B) it is cloned using nano antibody VHH segment of the round pcr to the anti-erabutoxin obtained in step (A),
PCR product is through agarose gel electrophoresis purification and recovery and is cloned into expression vector, and confirmation obtains correct gram after sequence verification
It is grand；
(C) the above-mentioned expression vector is introduced into the expression that fusion protein is carried out in host cell.
4. the preparation method of the nano antibody of anti-erabutoxin according to claim 3, it is characterised in that:
Wherein, in step (B), primer sequence used by PCR is as shown in SEQ ID NO.3 and SEQ ID NO.4.
5. the preparation method of the nano antibody of anti-erabutoxin 6 according to claim 3, it is characterised in that:
Wherein, the expression vector is pGEM-T, Pet32a, and pcDNA3.1, pEE6.4, pEE12.4, pDHFR or pDR1 are described
It include the fusion dna sequence for being connected with suitable transcription and translation and adjusting sequence in expression vector,
The host cell is mammalian cell, bacterial cell, insect cell, fungal cell or yeast cells.
6. the pharmaceutical composition of the nano antibody containing the described in any item anti-erabutoxins of Claims 1 to 5, feature
It is, further includes pharmaceutically acceptable pharmaceutical carrier.
7. the pharmaceutical composition of the nano antibody of anti-erabutoxin according to claim 6, it is characterised in that:
Wherein, described pharmaceutical composition be water needle or lyophilized preparation,
The pharmaceutically acceptable pharmaceutical carrier include surfactant, solution stabilizer, isotonic regulator and buffer it
One or combination.
8. the pharmaceutical composition of the nano antibody of anti-erabutoxin according to claim 7, it is characterised in that:
Wherein, the water needle or the dosage of lyophilized preparation intravenous injection are 1~1800mg/ days.
9. the nano antibody of the described in any item anti-erabutoxins of Claims 1 to 5 is in the preparation antitoxin preparation of sea snake
Purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810966599.2A CN109251248B (en) | 2018-08-23 | 2018-08-23 | Nano antibody for resisting sea snake neurotoxin, preparation method and application |
Applications Claiming Priority (1)
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| CN113214392A (en) * | 2020-12-30 | 2021-08-06 | 中国人民解放军海军军医大学 | Anti-aquatoxin nano antibody KY031, preparation method and application |
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