ANTI-PHYSALIATOXIN NANOBODY COZO32, AND PREPARATION METHOD AND USE THEREOF

Abstract

The present disclosure relates to the technical field of biomedicine, and provides an anti-physaliatoxin nanobody, and a preparation method and use thereof. The nanobody is a VHH antibody with an amino acid sequence shown in SEQ ID NO. 1. Affinity analysis shows that the nanobody of the present disclosure has prominent affinity. It is proved by small animal experiments that, after mice in an antibody protection group pre-injected with the nanobody of the present disclosure are injected with physaliatoxin, no mice shows toxic symptoms, and during continuous observation for one month, no toxic lethality occurs, indicating that the nanobody of the present disclosure shows excellent anti-physaliatoxin effects, excellent preventive or therapeutic effects on jellyfish stings, and promising clinical application prospects.

Claims

1. An anti-physaliatoxin nanobody COZO32, wherein the nanobody is a single-domain antibody (sdAb) with an amino acid sequence shown in SEQ ID NO. 1.

2. A nucleotide sequence encoding the anti-physaliatoxin nanobody COZO 32 according to claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 2.

3. A method for preparing the anti-physaliatoxin nanobody COZO32 according to claim 1, comprising the following steps: (A) synthesizing an sdAb fragment of the nanobody COZO32 through gene synthesis; (B) using polymerase chain reaction (PCR) to clone the sdAb fragment obtained in step (A), and purifying and recovering a PCR product by agarose gel electrophoresis; and cloning the PCR product into an expression vector, and conducting verification through sequencing to obtain a correct clone; and (C) introducing the expression vector into a host cell for fusion protein expression.

4. The method according to claim 3, 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 ligated with appropriate transcription and translation regulatory sequences; and the host cell is a prokaryotic cell, a mammalian cell, a bacterial cell, an insect cell, a fungal cell, or a yeast cell.

5. A pharmaceutical composition comprising the anti-physaliatoxin nanobody COZO32 according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable drug carrier.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is an injection or a lyophilized formulation; and the pharmaceutically acceptable drug carrier comprises one or a combination of two or more from a group consisting of a surfactant, a solution stabilizer, an isosmotic adjusting agent, and a buffer.

7. The pharmaceutical composition according to claim 6, wherein the injection or the lyophilized formulation is intravenously injected at a dosage of 1 mg/d to 1,800 mg/d.

8. Use of the anti-physaliatoxin nanobody COZO32 according to claim 1 in preparation of an anti-physaliatoxin drug.

Description

DETAILED DESCRIPTION

[0035] The following examples and experimental examples are provided to further illustrate the present disclosure, and shall be construed as a limitation to the present disclosure. The examples do not include detailed descriptions of traditional methods, such as methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art, and are described in many publications, including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

[0036] Example 1. Construction of a Nanobody Library

[0037] (1) 0.5 mg of physaliatoxin CfTX1 [Brinkman D, Burnell J. Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri [J]. Toxicon, 2008, 51 (5): 853-863.] and a freund's adjuvant were mixed in equal volumes to immunize a Xinjiang Bactrian camel once a week, and a total of 6 consecutive immunizations were conducted. During the immunization process, B cells were stimulated to express a specific nanobody.

[0038] (2) After the 6 immunizations were completed, 200 mL of peripheral blood lymphocyte (PBL) was collected from the camel to extract total RNA.

[0039] (3) cDNA was synthesized, and VHH was amplified by nested PCR.

[0040] (4) Restriction endonucleases Pstl and Notl were used to digest 20 μs of a pMECS phage display vector and 10 μg of VHH, and digestion products were ligated.

[0041] (5) A ligation product was electro-transformed into competent cells TG1, a phage display library was constructed, and a storage capacity thereof was determined, which was approximately 2.5×10.sup.8. Moreover, an insertion rate of the library established was tested by colony PCR, which reached more than 95%.

[0042] Example 2. Nanobody Screening Process

[0043] (1) 200 μL of recombinant TG1 cell was inoculated and cultivated in a 2TY medium, and during the cultivation, 50 μL of helper phage VCSM13 was added to infect the TG1 cell and cultivated overnight to expand the phage; and the next day, the phage was precipitated with PEG/NaCl, and collected by centrifugation.

[0044] (2) 150 μg of physaliatoxin dissolved in 150 mmol/L pH 8.2 NaHCO.sub.3 was coupled on an ELISA plate and incubated overnight at 4° C., and a negative control was set at the same time.

[0045] (3) The next day, 100 μL of 5% bovine serum albumin (BSA) was added to block at room temperature for 2 h.

[0046] (4) 2 h later, 100 μL of the phage (1×10.sup.11 tfu phage display gene library of the nanobody of the immunized camel), and a resulting mixture was incubated at room temperature for 1 h.

[0047] (5) The plate was washed five times with phosphate buffered saline (PBS)+0.05% Tween 20 to remove unbound phage.

[0048] (6) Specifically-bound phages were dissociated with trypsin at a final concentration of 25 mg/mL, used to infect E. coli TG1 cells in logarithmic growth phase, and cultivated at 37° C. for 1 h; and resulting phages were collected for the next round of screening; and the same screening process was repeated 3 times to gradually achieve enrichment.

[0049] Example 3. Screening of Specific Positive Clones by Phage ELISA

[0050] (1) 200 single colonies were picked from a cell culture plate obtained after the 3 rounds of screening and inoculated in a 96 deep-well plate with a 100 μg/mL ampicillinum-containing TB medium, and a blank control was set; and after the colonies were cultivated at 37° C. to a logarithmic phase, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 1 mmol/L was added, and then the colonies were cultivated overnight at 28° C.

[0051] (2) A crude antibody was extracted by the osmotic burst method, transferred to an antigen-coated ELISA plate, and incubated at room temperature for 1 h.

[0052] (3) The unbound antibody was washed off with phosphate-buffered saline with Tween 20 (PBST), then 100 μL of Mouse anti-HA tag antibody (purchased from Covance) diluted at 1:2000 was added, and a resulting mixture was incubated at room temperature for 1 h.

[0053] (4) The unbound antibody was washed off with PBST, then 100 μL of Anti-mouse alkaline phosphatase conjugate (goat anti-mouse alkaline phosphatase-labeled antibody, purchased from Sigma) diluted at 1:2000 was added, and a resulting mixture was incubated at room temperature for 1 h.

[0054] (5) The unbound antibody was washed off with PBST, an alkaline phosphatase chromogenic solution was added to allow a reaction for 10 min, and an absorbance value was read at 405 nm on a microplate reader.

[0055] (6) When an OD value of a sample well was more than 6 times greater than that of a control well, the sample well was determined as a positive clone well. Results were shown in FIG. 1. An OD value of the SN160 well was significantly greater than that of the control well group.

[0056] (7) Bacteria in the positive clone well were transferred to a 100 μg/μL ampicillinum-containing LB medium and cultivated under shaking, and then the plasmid was extracted and sequenced. Gene sequences of each cloned strain were analyzed according to the sequence alignment software Vector NTI. Strains with the same FR1, FR2, FR3, FR4, CDR1, CDR2, and CDR3 sequences were regarded as the same cloned strain, and strains with different sequences were regarded as different cloned strains. Finally, a specific nanobody was obtained, with an amino acid sequence shown in SEQ ID NO. 1, and a nucleotide sequence encoding the antibody was shown in SEQ ID NO. 2.

[0057] Example 4. Expression and Purification of the Nanobody in Host E. coli

[0058] (1) A clone obtained after the above sequencing analysis was transformed into E. coli WK6, and then the E. coli was coated on a culture plate with ampicillinum and glucose, and then cultivated overnight at 37° C.

[0059] (2) Single colonies were picked and inoculated into 5 mL of an ampicillinum-containing LB medium, and cultivated overnight at 37° C. on a shaker.

[0060] (3) 1 mL of a strain obtained after the overnight cultivation was inoculated into 330 mL of a TB medium and cultivated at 37° C. on a shaker until an OD.sub.600 nm value reached 0.6 to 0.9, then 1 M IPTG was added, and then the strain was further cultivated overnight at 28° C. on a shaker.

[0061] (4) E. coli was collected by centrifugation, and the osmotic burst method was used to obtain a crude antibody extract.

[0062] (5) Purification was conducted by nickel column affinity chromatography to obtain a high-purity nanobody, which was concentrated and enriched.

[0063] Example 5. Biacore analysis

[0064] An anti-polyhistidine antibody (abcam) was coated on a CM5M5 chip (GE), and after the antibody to be tested was captured, the affinity of each fusion protein was detected by Biacore T100 (GE Healthcare). Specific detected affinity values were shown in Table 1.

TABLE-US-00003 TABLE 1 Biacore analysis results Parameter Unit Value Binding affinity/kinetics KD (nM) 109.34

[0065] Example 6. Small Animal Experiment

[0066] 32 C57 mice with a body weight of (20±2) g were selected and fasted for 12 h before the experiment (without water deprivation). The mice were randomly divided into three groups (half female and half male for each group): half-lethal dosage physaliatoxin group: 12 mice; drug protection group: 10 mice, which were pre-injected with the nanobody at 10 mg/kg; and blank control group: 10 mice, which were administered with normal saline. The mice were administered intraperitoneally. Within 1 h after the administration, the mice in the blank control group all showed typical neurotoxic symptoms. None of the mice in the antibody protection group showed neurotoxic symptoms, and during continuous observation for one month, no toxic lethality occurred. Specific results were shown in Table 2.

TABLE-US-00004 TABLE 2 Test results of the anti-physaliatoxin effect of the antibody Number of survivors Number of survivors after Total on the day one month Nanobody 10 10 10 Control group 10 0 0

[0067] The preferred examples of the present disclosure have been described in detail above, but the present disclosure is not limited to these examples. Those skilled in the art can make various equivalent variations or substitutions without departing from the spirit of the present disclosure, and these equivalent variations or substitutions are all included in the scope defined by the claims of this application.