CN109251248B

CN109251248B - Nano antibody for resisting sea snake neurotoxin, preparation method and application

Info

Publication number: CN109251248B
Application number: CN201810966599.2A
Authority: CN
Inventors: 胡适; 刘烁吾; 雷长海; 李天�
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2018-08-23
Filing date: 2018-08-23
Publication date: 2021-07-20
Anticipated expiration: 2038-08-23
Also published as: CN109251248A

Abstract

The invention relates to the technical field of biological medicines, and provides a nano antibody for resisting sea snake neurotoxin, a preparation method and application thereof, wherein the nano antibody for resisting sea snake neurotoxin is a VHH antibody, has an amino acid sequence shown in SEQ ID NO.1, and a nucleotide sequence for coding the nano antibody is shown in SEQ ID NO. 2.

Description

Nano antibody for resisting sea snake neurotoxin, preparation method and application

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a nano antibody for resisting sea snake neurotoxin, a preparation method and application thereof, in particular to application in preparing a sea snake antitoxic preparation and treating or preventing sea snake bite.

Background

As a large ocean country, the construction of a powerful navy is a key problem for the construction of military troops in China. However, toxic and harmful marine organisms existing in coastal sea areas have potential threats to navy fighters, so that the prevention and treatment research work on marine organism injuries has great significance to the medical service support and medical work in the sea-crossing landing operation, and scientific application research on marine organism injury resistance must be started as soon as possible.

The sea snake with flat chin is a typical toxic marine organism, belongs to the family of elapidae, commonly known as sea snake with spine, and is mainly distributed in east India ocean, Australia, coastal areas of Philippines and Taiwan islands of China, Fujian, Shandong, hong Kong, Hainan, Guangxi, and the like. The flat-chin sea snake bite often happens, and certain threats are formed to marine operation and military training in the areas.

At present, the treatment measures for sea snake bite mainly comprise the steps of discharging venom, reducing venom absorption, resisting poison, and the like. Although early injection of anti-virus agents such as specific anti-virus serum or antibody drugs is the most effective first aid treatment, the method of directly immunizing animals with snake venom toxin to obtain anti-virus serum or screening multi/monoclonal antibodies has the following disadvantages: firstly, sea snakes are difficult to capture, a large amount of natural sea snake toxins are difficult to obtain for animal immunization, and separation of neurotoxin from venom glands can not meet the requirement of preparation of antitoxic preparations; secondly, because the snake venom toxin is lethal to animals, the attenuation treatment is needed, and the attenuated toxin usually loses the antigenicity and epitope thereof, so that the neutralizing activity of the antitoxin prepared by the attenuated toxin is weakened; thirdly, products such as antitoxic serum derived from animals and the like easily cause strong anaphylactic reaction of patients and even death. Thus, only a few countries in the world have sea snake antitoxic formulations available to date.

With the development of molecular biology, the technology for preparing snake venom toxin by adopting genetic engineering is mature, and the biosynthesis of snake venom toxin and the preparation and research of anti-snake venom antibody become possible. Three sea snake neurotoxins have been obtained by recombination in earlier groups, and specific antibody drugs aiming at the toxins should be screened as soon as possible. The antibody can be combined with various in-vivo and in-vitro antigen proteins efficiently and specifically, so that the antibody can be applied to the regulation of immune system functions and various high-sensitivity detection methods.

Currently, antibody drugs are the most important components of biotechnology drugs, and antibody reagents are also among the most commonly used reagents in medical diagnostics and biological research. Therefore, the antibody-related biological product has extremely high application prospect and commercial value. Antibodies can be obtained in a variety of ways, for example: animal or human blood, cell culture, ascites from mice injected with hybridoma cells, etc., but all require purification by efficient methods to obtain antibody products of practical value. The most commonly used method of antibody purification at present is affinity chromatography using high affinity between a particular protein and the Fc fragment of the antibody. Affinity chromatography is the most critical step in the industrial production of antibody products and is the most costly part of the overall production.

In response to the above problems, nanobodies, which are specific antibodies derived from camelids or cartilaginous fish, have been discovered. The study shows that an antibody which naturally lacks a light chain and only contains a heavy chain exists in the camel body, and is called a heavy chain antibody. Cloning of the variable region of a heavy chain antibody results in a single domain antibody consisting of only one heavy chain variable region, referred to as a VHH antibody. The crystal diameter of VHH antibodies is only 2.5nm and the length is 4nm, and are therefore also referred to as nanobodies. Nanobodies are only one tenth the size of traditional IgG-type antibodies, the smallest fragment that naturally occurs and can bind to an antigen. The nano antibody can be coded by a single gene, can be easily produced by microorganisms and has high yield. However, no report related to the nano antibody against the sea snake neurotoxin is found at present.

Disclosure of Invention

The invention aims to research the nano antibody for resisting sea snake neurotoxin, a preparation method and application by taking sea snake neurotoxin SN36 as an example based on the research background, namely the nano antibody for resisting sea snake neurotoxin, the preparation method and the application are provided.

In a first aspect of the invention, a nano antibody for resisting sea snake neurotoxin SN36 is provided, wherein the nano antibody is a VHH antibody, has an amino acid sequence shown in SEQ ID NO.1, and a nucleotide sequence for coding the nano antibody is shown in SEQ ID NO. 2.

The amino acid sequence of the nano antibody (SEQ ID NO.1) is as follows:

DVQLQESGGGLVQAGGSLRLSCAASGMCKVTHCFRAMGWWRQAPGKEREFVAT GKVDITQAWADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYA DIMYVNFQDENDYWGQGTQVTVSS。

the nucleotide sequence (SEQ ID NO.2) for coding the nano antibody is as follows:

GATGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCC TGCGCCTGAGCTGCGCGGCGAGCGGCATGTGCAAAGTGACCCATTGCTTTCGCGCG ATGGGCTGGTGGCGCCAGGCGCCGGGCAAAGAACGCGAATTTGTGGCGACCGGCA AAGTGGATATTACCCAGGCGTGGGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCC GCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGAT ACCGCGGTGTATTATTGCTATGCGGATATTATGTATGTGAACTTTCAGGATGAAAACG ATTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC。

the nano antibody for resisting sea snake neurotoxin is obtained by constructing a nano antibody phage display library for resisting sea snake neurotoxin, screening the nano antibody, screening specific positive clones by adopting a phage enzyme-linked immunosorbent assay (ELISA), and obtaining the VHH nano antibody with the amino acid sequence after sequence analysis, wherein the nano antibody consists of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 regions.

In a second aspect of the invention, a preparation method of a nano antibody against sea snake neurotoxin SN36 is provided, which comprises the following steps:

(A) synthesizing a nano antibody VHH fragment of the sea snake neurotoxin in a whole gene manner;

(B) cloning the nano antibody VHH fragment of the anti-sea snake neurotoxin obtained in the step (A) by adopting a PCR technology, purifying and recovering a PCR product through agarose gel electrophoresis, cloning the PCR product into an expression vector, and confirming to obtain correct clone after sequencing verification;

(C) the expression vector is introduced into host cells for expression of the fusion protein.

Preferably, in step (B), the primer sequences used for PCR are as follows:

in the present invention, any suitable vector is suitable, preferably pGEM-T, Pet32a, pcDNA3.1, pEE6.4, pEE12.4, pDAFR or pDR1, including a fusion DNA sequence linked to appropriate transcriptional and translational regulatory sequences.

In the present invention, mammalian or insect host cells or prokaryotic cell culture systems can be used for the expression of the fusion proteins of the present invention. The host cell to be used is a prokaryotic cell containing the above-mentioned vector, and may be one of DH5a, Top10, BL21(DE3) and TG 1.

The fusion protein of the present invention can be easily produced in the following cells: mammalian cells such as CHO, NSO, HEK293, BHK or COS cells; bacterial cells such as E.coli, Bacillus subtilis or Pseudomonas fluorescens; insect cells, or fungal or yeast cells, which cells are cultured using techniques known in the art.

The preparation method of the fusion protein disclosed by the invention is to culture the host cell under the expression condition so as to express, separate and purify the fusion protein. Using the above method, the antibody can be purified to a substantially homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis.

The fusion protein disclosed in the present invention can be isolated and purified by affinity chromatography, and the fusion protein polypeptide bound to the affinity column can be eluted by a conventional method such as high salt buffer, PH change, etc., depending on the characteristics of the affinity column used.

Various Protein purification Methods can be employed, and such Methods are known in the art and described, for example (Wilchek and Bayer,1990, Methods in enzymology) (Scopes,2013, Protein purification: principles and practice).

According to Biacore analysis, the nano antibody has good affinity, and small animal experiments prove that no mouse has any neurotoxicity symptom after the antibody protection group mouse pre-injected with the nano antibody is injected with sea snake neurotoxin SN36, and the condition of no toxin death in one month is continuously observed. The nano antibody of the invention has excellent anti-sea snake toxin effect.

Accordingly, in a third aspect of the present invention, there is provided a pharmaceutical composition comprising a nanobody against sea snake neurotoxin. The pharmaceutical composition comprises the nano antibody for resisting the sea snake neurotoxin and a pharmaceutically acceptable pharmaceutical carrier.

The nano antibody for resisting sea snake neurotoxin and pharmaceutically acceptable auxiliary materials form a medicinal preparation composition together, so that the curative effect is exerted more stably, the preparations can ensure the conformation integrity of the amino acid core sequence of the nano antibody for resisting sea snake neurotoxin, and simultaneously protect the polyfunctional group of protein to prevent the protein from degrading (including but not limited to agglomeration, deamination or oxidation).

In general, liquid formulations can be stable for at least one year at 2 ℃ to 8 ℃ and lyophilized formulations can be stable for at least six months at 30 ℃. The preparation can be suspension, injection, or lyophilized preparation, preferably injection or lyophilized preparation.

For the hydro-acupuncture or freeze-dried preparation of the nano antibody for resisting sea snake neurotoxin, pharmaceutically acceptable auxiliary materials comprise one or the combination of a surfactant, a solution stabilizer, an isotonic regulator and a buffer solution. Wherein the surfactant comprises nonionic surfactant such as polyoxyethylene sorbitol fatty acid ester (Tween-20 or Tween-80); poloxamer (such as poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; tetradecyl, oleyl, or octadecyl sarcosine; pluronics; monaquatm, etc., in an amount that minimizes the tendency of the bifunctional bispecific antibody protein to granulate; the solution stabilizer can be saccharides including reducing saccharides and non-reducing saccharides, amino acids including monosodium glutamate or histidine, alcohols including one of trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol or combinations thereof, and should be added in an amount such that the final formulation remains stable for a period of time deemed stable by one skilled in the art; the isotonic regulator can be one of sodium chloride and mannitol; the buffer may be one of TRIS, histidine buffer, and phosphate buffer.

The preparation is a composition containing nano antibody for resisting sea snake neurotoxin, and has obvious effect of resisting snake venom after being administrated to animals including human beings. Specifically, it is effective for preventing and/or treating sea snake bite and can be used as an anti-snake poison drug.

When the nano antibody for resisting sea snake neurotoxin and the composition thereof are administrated to animals including human, the administration dosage varies with the age and the weight of a patient, the disease property and the severity and the administration route, the result of animal experiments and various conditions can be referred, and the total administration dose cannot exceed a certain range. In particular, the dosage of intravenous injection is 1-1800 mg/day.

The fourth aspect of the invention provides an application of a nano antibody for resisting sea snake neurotoxin, in particular an application in preparing sea snake antitoxic preparation medicaments.

The invention has the following beneficial guarantee and effects:

the invention provides a nano antibody for resisting sea snake neurotoxin, a preparation method and application thereof, wherein the nano antibody for resisting sea snake neurotoxin is a VHH antibody, has an amino acid sequence shown in SEQ ID NO.1, has one tenth of the size of the traditional IgG type antibody, is a naturally-occurring minimum fragment capable of being combined with an antigen, can be encoded by a single gene, is easy to produce by using microorganisms, is simple in construction and expression processes, has high yield, and is beneficial to realizing industrial production.

In addition, the nano antibody has good affinity through affinity analysis, and small animal experiments prove that after the antibody protection group mice pre-injected with the nano antibody has been injected with sea snake neurotoxin SN36, no neurotoxicity symptom appears in any mice, and no toxin lethal condition is continuously observed for one month, so that the nano antibody has excellent sea snake toxin resisting effect, has excellent prevention or treatment effect on sea snake bite, and has wide clinical application prospect.

Drawings

FIG. 1 shows the result of ELISA screening for nano antibody against sea snake neurotoxin.

Detailed Description

The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cellsAnd are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2^ndedition， Cold spring Harbor Laboratory Press。

Example 1 construction of Nanobody library against sea Snake neurotoxin SN36

(1) 0.5mg of sea snake neurotoxin SN36 (Hu fit, and the like, screening, preparation and biological activity research of a full-human monoclonal antibody against short-chain neurotoxin of sea snake, namely, sea snake, Jiejun Jun J42.7 (2017):612 and 616.) antigen is mixed with Freund's adjuvant in equal volume, a bactrian camel in Xinjiang is immunized once a week for 6 times continuously, and B cells are stimulated to express specific nano antibodies in the immunization process;

(2) after 6 times of immunization, extracting 200mL of camel peripheral blood lymphocytes and extracting total RNA;

(3) cDNA was synthesized and VHH was amplified using nested PCR, the primer sequences used in this step are shown in table 1:

TABLE 1 PCR primer sequences

(4) Digesting 20 mu g of pMECS phage display vector and 10 mu g of VHH by using restriction enzymes Pstl and NotI and connecting the two fragments;

(5) the ligation product is transformed into electrotransformation competent cell TGl, and sea snake neurotoxin SN36 nanometer antibody phage display library is constructed and the library capacity is determined, the size of the library capacity is about 2.5X 10⁸. Meanwhile, the insertion rate of the constructed library is more than 95% through colony PCR detection.

Example 2 Nanobody screening procedure against sea Snake neurotoxin SN36

(1) Culturing 200uL recombinant TGl cells in a 2TY culture medium, adding 50 uL helper phage VCSM13 to infect TGl cells, culturing overnight to amplify the phage, precipitating the phage by using PEG/NaCl on the next day, and centrifuging to collect the amplified phage;

(2) dissolving in 150mmol/LpH 8.2 NaHCO₃Coupling the sea snake neurotoxin SN36150ug on an enzyme label plate, standing overnight at 4 ℃, and simultaneously setting a negative control;

(3) adding 100 μ L of 5% BSA the next day, blocking for 2h at room temperature;

(4) after 2h, 100. mu.L of amplified phage (1X 10) was added¹¹tfu immune camel nanometer antibody phage display gene library), acting for 1 hour at room temperature;

(5) wash 5 times with PBS + 0.05% Tween 20 to wash away bound phage;

(6) dissociating phage specifically bound with sea snake neurotoxin SN36 with membrane protease with final concentration of 25mg/mL, infecting Escherichia coli TGl cells in logarithmic growth phase, culturing at 37 deg.C for lh, generating and collecting phage for next round of screening, repeating the same screening process for 3 rounds, and gradually enriching.

Example 3 screening of specific Positive clones by enzyme-linked immunosorbent assay (ELISA) with phages

(1) Selecting 200 single colonies from the cell culture plates after the 3 rounds of screening, respectively inoculating the single colonies into a 96-deep-well plate containing 100 mu g/mL ampicillin TB culture medium, setting a blank control, culturing at 37 ℃ until the logarithmic phase, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1 mmol/L, and culturing at 28 ℃ overnight;

(2) obtaining a crude antibody by using a permeation bursting method, transferring the antibody to an ELISA plate coated by an antigen, and standing for lh at room temperature;

(3) unbound antibody was washed away with PBST, 100. mu.L of Mouse anti-HA tag antibody (murine anti-HA antibody, available from Covins) diluted 1:2000 was added, and left at room temperature for lh;

(4) unbound antibody was washed away with PBST, 100. mu.L of Anti-mouse alkaline phosphatase conjugate (goat Anti-mouse alkaline phosphatase-labeled antibody, ex Sigma) diluted at 1:2000 was added, and the mixture was allowed to stand at room temperature for 1 h;

(5) washing away unbound antibodies by PBST, adding alkaline phosphatase developing solution, reacting for 10min, and reading absorption value at 405 wavelengths on an enzyme labeling instrument;

(6) when the OD value of the sample well is more than 6 times that of the control well, the positive clone well is judged, and the result is shown in figure 1, wherein the OD value of the SN36 well is obviously more than that of the control well group;

(7) the bacteria of the positive cloning wells were shaken in LB medium containing 100. mu.g/. mu.L ampicillin to extract plasmids and to sequence. Analyzing the gene sequence of each clone strain according to sequence comparison software VectorNTI, regarding the strains with the same sequence of FRl, FR2, FR3, FR4, CDR1, CDR2 and CDR3 as the same clone strain, and regarding the strains with different sequences as different clone strains, finally obtaining 1 strain of anti-sea snake neurotoxin SN36 specific nano antibody, wherein the amino acid sequence of the antibody is SEQ ID NO.1, and the nucleotide sequence of the coded antibody is shown as SEQ ID NO. 2.

Example 4 expression and purification of sea snake neurotoxin SN36 specific nano antibody in host bacterium Escherichia coli

(1) The clone obtained by the above sequencing analysis was transformed into E.coli WK6, spread on a culture plate containing ampicillin and glucose, and cultured overnight at 37 ℃;

(2) selecting a single colony to be inoculated in 5mL LB culture solution containing ampicillin, and carrying out shake culture at 37 ℃ overnight;

(3) inoculating 1 mL of overnight cultured strain into 330mL of TB culture solution, performing shake culture at 37 ℃, adding 1M IPTG when OD 600nm value reaches 0.6-0.9, and performing shake culture at 28 ℃ overnight;

(4) centrifuging, collecting Escherichia coli, and obtaining crude antibody extractive solution by use of osmotic bursting method;

(5) purifying the antibody by a nickel column affinity chromatography to obtain a high-purity nano antibody, and concentrating and enriching the nano antibody.

Example 5 Biacore analysis

Anti-polyhistidine antibodies (abcam) were coated on a CM5M5 chip (GE corporation), and after capturing the antibodies to be detected, the affinity of each fusion protein was measured using Biacore T100(GE Healthcare), and the specific values of the measured affinities are shown in table 2.

TABLE 2 Biacore analysis results

Example 6 Small animal experiments

30 Kunming mice with the weight of 20 +/-2 g are taken and fasted for 12h (without water prohibition) before the experiment. The mice are randomly divided into 10 groups with each group having half male and female, and divided into groups using half lethal dose SN36 and groups pre-injected with nano antibody 10mg/kg drug protection. In addition, 1 group is a blank control group (normal saline), mice are administrated by intraperitoneal injection, typical neurotoxicity symptoms appear in 1h after the mice are administrated, no neurotoxicity symptoms appear in any mice in an antibody protection group, no toxin death condition exists in 1 month of continuous observation, and specific results are shown in table 3.

TABLE 3 results of the detection of the antitoxic Effect of the antibodies

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Sequence listing

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

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Claims

1. A nano antibody for resisting sea snake neurotoxin is characterized in that the nano antibody is a VHH antibody and has an amino acid sequence shown in SEQ ID NO. 1.

2. Nucleic acid encoding the nano-antibody against sea snake neurotoxin of claim 1, characterized in that the nucleotide sequence of said nucleic acid is shown in SEQ ID No. 2.

3. The method for preparing nano antibody against sea snake neurotoxin of claim 1, which comprises the following steps:

4. The method for preparing nano antibody against sea snake neurotoxin according to claim 3, characterized in that:

wherein, in the step (B), the primer sequences adopted by the PCR are shown as SEQ ID NO.3 and SEQ ID NO. 4.

5. The method for preparing nano antibody against sea snake neurotoxin according to claim 3, characterized in that:

wherein the expression vector is pGEM-T, Pet32a, pcDNA3.1, pEE6.4, pEE12.4, pDHFR or pDR1, the expression vector comprises a fusion DNA sequence connected with a proper transcription and translation regulatory sequence,

the host cell is a mammalian cell, a bacterial cell, an insect cell, or a fungal cell.

6. A pharmaceutical composition comprising the nano antibody against sea snake neurotoxin of any one of claims 1 to 5, characterized in that it further comprises a pharmaceutically acceptable pharmaceutical carrier.

7. The pharmaceutical composition of nanobodies against sea snake neurotoxins of claim 6, characterized in that:

wherein the pharmaceutical composition is a hydro-acupuncture or freeze-drying preparation,

the pharmaceutically acceptable drug carrier comprises one or a combination of a surfactant, a solution stabilizer, an isotonic regulator and a buffer.

8. Use of the nano antibody against sea snake neurotoxin according to any one of claims 1 to 5 in the preparation of sea snake antitoxic formulations.