CN103087194A - Anti-blood-cycle-venin egg yolk antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-blood circulation type snake venom chicken egg yolk antibody. The antibody is prepared according to the following method: based on the blood circulation type snake venom zinc ion metalloproteinase active domain sequence, the snake venom zinc ion metalloproteinase active domain polypeptide is synthesized; the polypeptide Coupling, conjugating or fusing with the carrier to prepare the antigen peptide complex; injecting the antigen peptide complex into the hen for immunization, and collecting the eggs continuously; extracting the anti-venom chicken egg yolk antibody from the collected eggs to obtain the anti-blood Chicken egg yolk antibody of circulating snake venom; the anti-blood circulating snake venom chicken egg yolk antibody of the present invention has high yield and low cost; the obtained antibody has high safety, good stability, and small allergic adverse reactions; it can be made into oral preparations or Injection; it can be used for the treatment of snake bites with blood circulation toxicity, especially for the treatment of snake bites and pit vipers.

Description

A kind of anti-blood circulation snake venom chicken egg yolk antibody and its application

(1) Technical field

The invention relates to a blood circulation type snake venom chicken egg yolk antibody (Immunoglobulin-yolk, IgY), a preparation method and an application. Specifically, it is a preparation method for preparing an anti-blood circulation type snake venom chicken egg yolk antibody based on the immunization of the blood circulation type snake venom zinc ion metalloprotease active motif polypeptide.

(2) Background technology

For a long time, poisonous snake bites have brought serious harm to people's production and life. According to the figures released by the World Health Organization website: more than 2.5 million people are bitten by snakes in the world every year, resulting in 125,000 deaths. In my country, more than 100,000 patients are bitten by poisonous snakes every year, of which about 73% are young and middle-aged, the mortality rate is 5% to 10%, and the disabled and incapacitated by snakebite account for 25% to 30%. It can be seen that venomous snakes are an important biological safety hazard that endangers human health, and relevant measures must be taken to reduce or eliminate related risks.

Injection of antivenom serum is currently the most effective treatment method recognized in the world, but the composition of snake venom itself is complex, containing hundreds of proteins with different functions. It also contains a large number of antibodies against other non-toxin components of snake venom. This not only greatly increases the clinical dose of antiserum, increases the probability of allergic reactions, but also wastes precious serum resources (Wagstaff S C, LaingG D, Theakston R D, et al. Bioinformatics and multiepitope DNA immunization to design rational snake antibiotic . PLoS Med, 2006, 3:184.). If the main toxic and lethal components in snake venom can be separated and purified to prepare monoclonal antibodies, the ability to neutralize the corresponding snake venom proteins will be greatly improved, the amount of antibodies will be reduced, and adverse reactions will be reduced accordingly, and the production cost of antibodies will also be extremely low. Great reduction, the quality can also be stably controlled. Retrieval of relevant literature has found that many monoclonal antibodies to snake venom proteins have been successfully prepared (Yin Huiqiong. Preparation of Anti-Snake Venom ScFv Antibodies Using Phage Display Library Technology. Yunnan University, 2004 Master Thesis.; Li Lihong et al. Preparation of King Cobra Venom Monoclonal Antibodies . Cellular and Molecular Immunology Journal, 2009,25(3):236-238. etc.), but most of them are limited to the classification and identification of venomous snakes and basic theoretical research; this is most likely due to the diversity of venomous components (Jia Yan et al. Toxic components of snake venom and its application research. Snake Zhi, 2004,16(2):23-32.), so there is almost no monoclonal antibody that can completely neutralize the toxicity of a certain snake venom.

Agkistrodon acutus (Deinagkistrodon acutus) is a common venomous snake in China, belonging to Viperidae Viperidae, also known as five-step snake (Zhejiang), checkerboard snake (Fujian), hundred-step snake (Taiwan), mainly inhabiting northern Vietnam, Laos and southern China, its venom is mainly blood circulation poison (Li QB, Yu QS, Huang GW, et al. Hemostatic disturbances observed in patients with snakebite in south China. Toxicon. 2000, 38(10):1355-1366. Chen CC, Yang CM, Hu FR, et al. Penetrating ocular injury caused by venomous snakebite. Am. J. Ophthalmol. 2005, 140(3):44-546.). After the patient is bitten, the local wound is red and swollen, the pain is severe, the bleeding continues, the swelling is rapid, the toxin spreads to the upper end of the limb, and blisters and ecchymosis often appear. Severe poisoning will also cause blood pressure drop, arrhythmia, oliguria, anuria, and finally death due to circulatory failure. Studies have shown that snake venom metalloproteinases (Wagstaff S C, Laing GD, Theakston R D, et al. Bioinformatics and multiepitope DNA immunization to design rational snake antagonist. PLoS Med, 2006, 3:184) are the main agents that cause symptoms such as bleeding and death. Some of them interfere with the coagulation of wound blood and the formation of thrombus, and some of them degrade the extracellular matrix or basement membrane, which can account for more than 60% of the total snake venom and are the main components of snake venom; they can effectively neutralize these metals Protease, on the other hand, can effectively alleviate the damage of the five-step snake venom to the body.

Studies have shown that snake venom metalloproteases are the same as mammalian matrix metalloproteases, they are both metallozinc proteins with a zinc-binding domain HEXXHXXGXXH. Since 1950, more than 150 kinds of proteases (including more than 100 metalloproteases) have been purified from snake venom, and the complete amino acid sequences of more than 40 kinds (more than 20 metalloproteases) have been determined. According to the reported cDNA sequences and protein structures of snake venom metalloproteases, snake venom metalloproteases are divided into 4 categories: PI category only contains metalloproteinase domain (metalloproteinase domain), molecular weight 20-30kDa; PII category contains metalloproteinase domain and disintegrin disintegrin domain, molecular weight 30-50kDa; PIII class contains metalloprotease domain, disintegrin domain and cysteine-rich domain (cysteine-rich domain), molecular weight 50-80kDa; PIV class has two more Sulfur bonded C-type lectin domain (Cynthia Tallant, Aniebrys Marrero, F.Xavier Gomis-Rüth. Matrix metalloproteinases: Fold and function of their catalytic domains. Biochimica et Biophysica Acta, 2010, 1803 :20-28).

The metalloprotease domain is a common part of the four classes of snake venom metalloenzymes. The results of comparative analysis of the metalloprotease domains of snake venoms showed that the HEXXHXXGXXH sequence is a highly conserved sequence in the active center of the enzyme. For this reason, in theory, immunizing animals with this conserved sequence as an antigenic peptide can obtain specific antiserum against this type of antigenic site, and the antiserum is likely to be able to cross-react with the above-mentioned various snake venom metalloproteins, To neutralize the toxicity of snake venom. Due to the singleness of the antigenic peptide used to immunize animals, the antiserum antibody prepared by it is relatively single, which can reduce the amount of antiserum and adverse reactions; at the same time, based on this antigenic peptide, effective monoclonal antibody preparations can be further screened and established.

(3) Contents of the invention

The purpose of the present invention is to provide a method for preparing anti-blood circulation type snake venom chicken egg yolk antibody based on the active domain sequence of blood circulation type snake venom zinc ion metalloprotease as an antigen peptide; the method has high antibody yield and low cost; The obtained antibody has high safety, good stability, and small allergic adverse reactions; it can be made into oral preparations and injections; it can be used for the treatment of snake venom bites with blood circulation toxicity, especially for snake bites and pit viper bites treat.

The technical scheme adopted in the present invention is:

The present invention provides an anti-blood circulation snake venom chicken egg yolk antibody, and the antibody is prepared as follows:

(1) Synthesize a snake venom zinc ion metalloprotease active domain polypeptide based on the active domain sequence of blood circulation type snake venom zinc ion metalloprotease; the amino acid sequence of the polypeptide is CX _n HEXXHXXGXXHX _n or X _n HEXXHXXGXXHX _n C, where X is except Any amino acid other than cysteine, n is any positive integer including zero;

(2) Coupling, conjugating or fusing the polypeptide in step (1) with a carrier to prepare an antigen peptide complex;

(3) Hen immunization: inject the antigen peptide complex in step (2) into the hen for immunization, and collect eggs continuously;

(4) Extracting the anti-venomous chicken yolk antibody from the eggs collected in step (3), that is, obtaining the anti-circulating type snake venomous chicken yolk antibody.

Further, preferably, the n is any positive integer between 0 and 20.

Further, the carrier includes protein or lipid. More preferably, the carrier is: Keyhole Limpet Hemocyanin, Human Serum Albumin, Bovine Serum Albumin, Bovine Thyroglobulin, Chicken Ovalbumin ( Ovalbumin) and other protein carriers such as gamma globulin; other recombinant carriers can be Poly-L-Lysine, Dipalmitoyl-Lysine, Polyglutamic acid (Poly -γ-Glutamic acid) and other poly-mixed amino acids, etc.

Further, step (3) is carried out as follows: mix and emulsify the antigen-peptide complex prepared in step (2) with an equal volume of Freund's complete adjuvant (Freund's complete adjuvant) at room temperature, and then inject it subcutaneously in hens at multiple points For the first immunization, the dosage of the antigen peptide complex for the first immunization is 0.1-3 mg/monkey (preferably 1 mg/bird), and the first booster immunization is carried out 1-2 weeks after the first immunization, and the dosage of the antigen-peptide complex is 0.1-3 mg/monkey (preferably 0.5mg/bird), the second booster immunization is carried out 2~4 weeks after the first immunization, the dosage of antigen peptide complex is 0.1~3 mg/bird (preferably 0.5mg/bird), and the subsequent interval is 3 Immunize once every ~4 weeks, the dose of antigen peptide complex is 0.1~3 mg/bird (preferably 0.5mg/bird), and start to collect eggs continuously 3 weeks after the initial immunization. For each booster immunization or immunization after the first immunization, the antigen-peptide complex prepared in step (2) and an equal volume of Freund's incomplete adjuvant were mixed and emulsified at room temperature, and then subcutaneously injected into hens at multiple points.

The method for extracting chicken yolk antibody from collected eggs described in step (4) of the present invention and the separation and purification method are well-known techniques in the art, and ammonium sulfate precipitation method, polyethylene glycol precipitation method, column chromatography and related products can be used. Chemical kits and other purification methods.

The present invention also relates to an application of the chicken yolk antibody against blood circulation type snake venom in the preparation of anti blood circulation type snake venom medicine.

The chicken egg yolk antibody against blood-circulating snake venom can treat blood-circulating snake bites, and can be administered orally or by injection, preferably by injection. The dosage of egg yolk antibody against blood-circulating snake venom chicken is preferably 4-10 mg/kg body weight.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the anti-blood-circulating snake venom chicken egg yolk antibody of the present invention has high yield and low cost; the obtained antibody has high safety, good stability and no allergic adverse reactions. Small; it can be made into oral preparations or injections; it can be used for the treatment of snake venom bites with blood circulation toxicity, especially for the treatment of snake bites by quinqueras and hakistrodon.

(4) Description of drawings

Figure 1: HPLC chart of M15 polypeptide;

Figure 2: M15 peptide mass spectrum;

Figure 3: SDS-PAGE electrophoresis of IgY samples;

Figure 4: The results of the subcutaneous hemorrhage toxicity test in mice: A is the result of the subcutaneous hemorrhage toxicity test of the five-step snake venom; B is the result of the subcutaneous hemorrhage test of the viper venom; 0 in the figure refers to the IgY 50 μg injection point before the venom plus immunization, and D1 is Refers to the IgY 50μg injection point after snake venom plus immunization, D2 refers to the IgY 25μg injection point after snake venom plus immunization, D3 refers to the IgY 10μg injection point after snake venom plus immunization, D4 refers to the IgY 5μg injection point after snake venom plus immunization, D5 Refers to the injection point of snake venom plus 2.5 μg of IgY after immunization.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

If not specified, it can be based on "Making and Using Antibodies: A Practical Handbook" (CRC Press, Florida, 2006) and "Antibody Preparation and Use Experimental Guide" (Science Press, Beijing, 2010) familiar to those skilled in the art. 2010) and other manuals, as well as the methods listed in the references cited herein. In addition, unless otherwise specified, the materials used in the examples can be purchased from the market through commercial channels.

Example 1: Preparation of novel anti-circulatory snake venom chicken egg yolk antibody based on snake venom zinc ion metalloprotease activity motif

1. Synthesis and coupling of peptides

Through comparative analysis of the amino acid sequences of the catalytic domains of various snake venom metalloproteases (such as Acutolysin A, Acutolysin F, Acutolysin E, Aculysin 2, Metalloprotease H1, Metalloprotease H2, Metalloprotease H3, Metalloprotease H4 and Metalloprotease H5), the following peptides were determined to be synthesized Sequence (M15): CAHEMAHNLGVRHDE, and submitted to Hangzhou Jucheng Biotechnology Co., Ltd. to synthesize the peptide. The results of HPLC and MS identification are shown in Figures 1 and 2. Then the polypeptide was coupled to keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH, purchased from sigma company) by carbodiimide method to form antigen peptide complex KLH-M15.

2. Hens are immunized

Mix and emulsify the above antigen peptide complex KLH-M15 with an equal volume of Freund's complete adjuvant (purchased from MP Biomedicals) at room temperature, take 22-week-old Laihang hens, and inject the above-mentioned Freund's complete adjuvant into the hens subcutaneously. The antigen peptide complex KLH-M15 emulsified with adjuvant was used for initial immunization, and the amount of KLH-M15 for the initial immunization was 1.0 mg/monkey; after 2 weeks, the antigen peptide complex KLH- M15 booster immunization once, the dosage of KLH-M15 was 0.5mg/monkey; the second booster immunization (antigenic peptide complex KLH-M15 emulsified in Freund's incomplete adjuvant) was carried out 4 weeks later, and the dosage of KLH-M15 was 0.5mg/ After that, they were immunized once every 4 weeks (antigen-peptide complex KLH-M15 emulsified in Freund's incomplete adjuvant), and the amount of KLH-M15 was 0.5 mg/mouse each time; and they were collected continuously 3 weeks after the first immunization Eggs were stored at 4°C for later use; at the same time, eggs before immunization were kept as negative controls.

3. Antibody extraction

Extract and purify anti-venom chicken yolk antibody (IgY) from eggs collected in step 2 by polyethylene glycol precipitation (Pauly D, Chacana PA, Calzado EG, Brembs B, Schade R. IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation.J Vis Exp. 2011(51). doi:pii:3084.10.3791/3084.): take egg yolk, add 2 times the volume of egg yolk with PBS buffer at pH7.4, mix well and add The final concentration is 3.5% PEG6000 (mass concentration), shake and dissolve in ice bath for 10 minutes, centrifuge at 12 000g, 4°C for 20 minutes, and take the supernatant; according to the volume of supernatant, add 8.5% (mass concentration) PEG6000, shake and dissolve in ice bath 15min, 12 000g, centrifuge at 4°C for 20min, remove the supernatant; dissolve the precipitate with 10ml of PBS pH7.4, add 12% (mass concentration) PEG6000, shake and dissolve in ice bath for 15min, centrifuge at 12 000g, 4°C for 20min, remove The precipitate was dissolved in 1ml of PBS at pH 7.4, and then dialyzed overnight in PBS at pH 7.4 in an ice bath with a 14 000Da cut-off dialysis bag, and the medium was changed 3 times. The absorbance value of the dialyzed sample was measured at 280nm, and the calculated antibody IgY content reached 56.7mg/ml (calculation method reference: Pauly D, Chacana PA, Calzado EG, Brembs B, Schade R. IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation.J Vis Exp. 2011(51).doi:pii:3084.10.3791/3084.); samples were stored at -20°C for later use.

4. SDS-PAGE detection

SDS-PAGE method (see application number: 200610035761.6) was used to detect the extracted and purified IgY sample in step 3; there were two obvious bands at 65kDa (heavy chain) and 25kDa (light chain) in the sample (as shown in Figure 3) , consistent with relevant literature reports (Pauly D, Chacana PA, Calzado EG, Brembs B, Schade R. IgY technology: extraction of chicken antibodies from egg yolk bypolyethylene glycol (PEG) precipitation. J Vis Exp. 2011(51). doi: pii:3084.10.3791/3084.) (see Figure 3).

5. ELISA titer detection

Referring to the method of application number: 200610035761.6, the potency of the antibody sample and antigen peptide complex KLH-M15 prepared in step 3 and the cross-immunity between the sample and the venom of the five-step snake and the venom of the Agkistrodon halys were measured. The results showed that the titer of the sample and KLH-M15 could reach 320,000-fold dilution, but its cross-reactivity with the venoms of pentapods and Agkistrodon vipers was poor, and the dilutions were only about 5,000-fold and 2,000-fold, respectively.

6. Subcutaneous bleeding test in mice

Healthy male mice of the ICR line were selected and divided into random groups, 10 in each group. Experimental group 1: Mix 10 μL of 1.0 mg/ml five step snake venom with different dilutions of IgY samples dissolved in PBS (pH7.4) in a sterile EP tube (see Table 1), and place in a constant temperature water bath at 25°C Incubate for 60 min, and then inject subcutaneously on the back of depilated mice. Experimental group 2: Mix 10 μL of Agkistrodon halys venom at 0.5 mg/ml with 10 μL of different dilutions of IgY samples dissolved in PBS (pH7.4) in a sterile EP tube (see Table 1), and place in a constant temperature water bath at 25°C Incubate for 60 min, and then inject subcutaneously on the back of depilated mice. Eighteen hours later, they were sacrificed by cervical dislocation, dissected, and photographed to record the length and diameter of the bleeding plaque. IPP software was used to calculate the bleeding area and the bleeding inhibition rate of each antiserum. The results are shown in Table 1 and Figure 4.

The results show that the sample IgY has a good neutralizing effect on the hemorrhagic toxicity of pentapod venom and Agkistrodon venom in vivo, and the neutralization activity against pentapod venom is better, 50 μg IgY sample can completely neutralize 10 μg pentapod venom, while The same dose can only neutralize about 70% of Agkistrodon halys venom (see Table 1 and Figure 4). The significant difference between sample in vitro ELISA and in vivo hemorrhagic toxicity may be related to snake venom metalloproteinase conformation (Tallant C, Marrero A, Gomis-Rüth FX.Matrix metalloproteinases: fold and function of theircatalytic domains.Biochim Biophys Acta. 2010,1803(1 ):20-28.).

Each sample of table 1 is to the mouse subcutaneous hemorrhage inhibition rate (%) that snake venom causes

7. Snake venom neutralization test

Healthy male mice of the ICR line were selected and divided into random groups, 10 in each group. Experimental group 1: Mix KLH-M15 immunized sample IgY (10 mg/kg mouse body weight) with five-step snake venom at twice the half-lethal dose LD ₅₀ in sterile EP tubes, and incubate in a constant temperature water bath at 25°C for 60 minutes , and then the mice were intraperitoneally injected with 0.2ml, and the survival rate of the mice within 24 hours after administration was recorded. Experimental group 2: Mix KLH-M15 immunized sample IgY (20 mg/kg mouse body weight) with Agkistrodon halys venom twice the half lethal dose LD ₅₀ in a sterile EP tube, and incubate in a constant temperature water bath at 25°C for 60 minutes , and then the mice were intraperitoneally injected with 0.2ml, and the survival rate of the mice within 24 hours after administration was recorded. IgY samples before immunization were used as controls.

The results showed that no matter whether it was the venom of the five-step snake or the venom of the Agkistrodon halys, all the mice given the IgY samples after KLH-M15 immunization survived, while all the mice given the IgY samples before immunization died.

Claims (5)

1. an anti-blood circulation type snake venom chicken egg yolk antibody is characterized in that said antibody is prepared as follows: (1) Synthesize a snake venom zinc ion metalloprotease active domain polypeptide based on the active domain sequence of blood circulation type snake venom zinc ion metalloprotease; the amino acid sequence of the polypeptide is CX _n HEXXHXXGXXHX _n or X _n HEXXHXXGXXHX _n C, where X is except Any amino acid other than cysteine, n is any positive integer including zero; (2) Coupling, conjugating or fusing the polypeptide in step (1) with a carrier to prepare an antigen peptide complex; (3) Hen immunization: inject the antigen peptide complex in step (2) into the hen for immunization, and collect eggs continuously; (4) Extracting the anti-venomous chicken yolk antibody from the eggs collected in step (3), that is, obtaining the anti-circulating type snake venomous chicken yolk antibody. 2. The anti-blood circulating snake venom chicken egg yolk antibody according to claim 1, wherein said n is any positive integer between 0 and 20. 3. The anti-circulating snake venom chicken egg yolk antibody according to claim 1, characterized in that the carrier comprises protein or lipid. 4. Anti-blood-circulating snake venom chicken yolk antibody as claimed in claim 1, characterized in that step (3) is carried out as follows: the antigen-peptide complex prepared in step (2) is mixed with an equal volume of complete Freund's adjuvant in Mix and emulsify at room temperature, and then inject multi-point subcutaneous injections for the first immunization. The dosage of the antigen peptide complex for the first immunization is 0.1-3 mg/hen. The first booster immunization is carried out 1-2 weeks after the first immunization, and the antigen peptide compound The dosage of the drug was 0.1-3 mg/bird, and the second booster immunization was carried out 2-4 weeks after the first immunization. The dosage of the peptide complex was 0.1-3 mg/egg, and eggs were collected continuously 3 weeks after the initial immunization. 5. The application of the chicken yolk antibody against blood circulation type snake venom according to claim 1 in the preparation of anti blood circulation type snake venom medicine.

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