阅读说明：本技术 一种制备胡蜂毒素抗毒血清的方法 (Method for preparing bee venom antitoxic serum ) 是由 张鑫 陈则 罗敏 李丹 蒋春英 范铁炯 于 2021-07-19 设计创作，主要内容包括：本发明涉及生物技术领域,具体地说是一种制备胡蜂毒素抗毒血清的方法。一种制备胡蜂毒素抗毒血清的方法,其特征在于：具体方法如下：鉴定并筛选出黑腹虎头蜂；毒素进行甲醛灭活脱毒处理；进行动物免疫；免疫后采血；保存；S/D病毒灭活处理；将收集后的血浆沉淀物进行明矾吸附处理；进行超滤；将超滤后的血浆混合物进行DEAE凝胶柱层析处理；将F(ab’)-(2)免疫球蛋白抗体加入适量的NaCl和间甲酚的溶液中；将胡蜂毒素抗毒血清原液进行除菌过滤,分装。同现有技术相比,制备出来的胡蜂毒素抗毒血清对七里蜂,凹纹胡蜂,黑腹虎头蜂等不同胡蜂种类,云南大理胡蜂,陕西安康胡蜂等不同地域的胡蜂的毒素具有有效的中和效果。(The invention relates to the field of biotechnology and particularly relates to a biological enzyme preparation,in particular to a method for preparing bee venom antitoxic serum. A method for preparing bee venom antitoxic serum is characterized by comprising the following steps: the specific method comprises the following steps: identifying and screening the black abdominal tiger head bees; performing formaldehyde inactivation and detoxification treatment on the toxin; carrying out animal immunization; blood is collected after immunization; storing; S/D virus inactivation treatment; carrying out alum adsorption treatment on the collected plasma precipitate; carrying out ultrafiltration; carrying out DEAE gel column chromatography treatment on the plasma mixture after ultrafiltration; mixing F (ab') 2 Adding the immunoglobulin antibody into a solution of a proper amount of NaCl and m-cresol; sterilizing and filtering the stock solution of the wasp toxin antitoxic serum, and subpackaging. Compared with the prior art, the prepared wasp toxin antitoxic serum has an effective neutralization effect on the toxins of different wasp species such as seven-li wasps, vespid fossilizid, black-belly tiger head wasps and the like, and different regions such as Yunnan Dali wasps, Shaanxi Ankang wasps and the like.)

1. A method for preparing bee venom antitoxic serum is characterized by comprising the following steps: the specific method comprises the following steps:

(1) identifying and screening the black abdominal tiger head bees;

(2) performing formaldehyde inactivation and detoxification treatment on the tenofovir disoproxil;

(3) carrying out animal immunization on the tiger head melittin subjected to formaldehyde inactivation and detoxification treatment;

(4) blood is collected after immunization;

(5) preserving the collected blood plasma in a cold storage at the temperature of 2-8 ℃ for 5 years;

(6) carrying out S/D virus inactivation treatment on the blood plasma;

(7) carrying out plate-and-frame filter pressing treatment on the inactivated plasma mixture, and removing precipitates;

(8) adding 1.5-2 times of distilled water into the plasma mixture subjected to plate-and-frame filter pressing for plasma dilution;

(9) carrying out gastric enzyme digestion treatment on the diluted plasma mixture;

(10) carrying out primary precipitation treatment on the plasma mixture subjected to the gastric enzyme digestion treatment;

(11) carrying out plate-and-frame filter pressing treatment on the plasma mixture subjected to the first precipitation, and discarding the precipitate;

(12) carrying out secondary precipitation treatment on the plasma mixture subjected to plate-and-frame filter pressing treatment;

(13) carrying out plate-and-frame filter pressing treatment on the plasma mixture subjected to the secondary precipitation, collecting precipitates, and discarding supernatant;

(14) carrying out alum adsorption treatment on the collected plasma precipitate;

(15) carrying out plate-frame filter pressing treatment on the plasma mixture subjected to alum adsorption treatment, and discarding precipitates;

(16) adding 3 times of purified water into the plasma mixture subjected to plate-and-frame filter pressing treatment for ultrafiltration;

(17) subjecting the plasma mixture to DEAE gel column chromatography, and filtering with nanometer membrane to obtain F (ab')₂An immunoglobulin antibody;

(18) mixing F (ab')₂Adding the immunoglobulin antibody into a solution of a proper amount of NaCl and m-cresol, and adjusting the pH value to 6-7 to prepare a wasp toxin antitoxic serum stock solution;

(19) sterilizing and filtering the stock solution of the wasp toxin antitoxic serum, and subpackaging.

2. The method of preparing melittin antitoxin serum as set forth in claim 1, wherein: the method for identifying the black-belly tiger head bees in the step (1) is as follows:

(1a) dissolving 0.2g unknown wasp venom with 2ml DDH2O, centrifuging 3000g for 5min, and removing supernatant;

(1b) adding 200ul of LB2, mixing, suspending and precipitating;

(1c) adding 20ul RNase A to the sample of step (1 b) and incubating at room temperature for 2 min;

(1d) adding 20ul PK, mixing by vortex, and incubating at room temperature for 2 min;

(1e) adding 500ul BB2, immediately vortexing for 5s, and incubating at room temperature for 10 min;

(1f) adding all the solution into a centrifugal column, centrifuging at 12000rpm for 30s, and removing effluent liquid;

(1g) adding 500ul of CB2, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

(1h) repeating the step (1 g) once;

(1i) adding 500ul WB2, checking the presence of anhydrous ethanol before use, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

(1j) repeating step (1 i) once;

(1k) centrifuging at 12000rpm for 2min to completely remove the residual WB 2;

(1 l) placing a centrifugal column in a clean centrifugal tube, adding 100ul of preheated EB or deionized water into the center of the column, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, eluting DNA, and storing the eluted DNA at-20 ℃;

(1 m) adopting a universal primer and a specific primer to perform PCR amplification on a wasp DNA nucleic acid sequence to obtain a fragment of the COI sequence, then performing second-generation sequencing to obtain a sequence of the COI fragment, and comparing with a wasp species through an NCBI website.

3. The method of preparing melittin antitoxin serum as set forth in claim 2, wherein: the DDH2O is deionized water; LB2 is lysate; RNase A is RNase A; PK is proteinase K; BB2 is a DNA binding solution; CB2 is a solution formed by combining 0.1 mL of deionized water, 0.1 mL of 5 mol.L < -1 > NaCl and 0.8 mL of precooled 95% ethanol absolute ethyl alcohol; WB2 was 75% absolute ethanol; EB is DNA eluate.

4. The method for preparing the melittin antitoxin serum as set forth in claim 2 or 3, wherein: the pH value of the deionized water is more than 7.0, and the preheating temperature of EB is 65 ℃.

5. The method of preparing melittin antitoxin serum as set forth in claim 2, wherein: the PCR amplification is to identify the wasp species from wasp DNA extracted from wasp toxin, design a universal primer and a specific primer through Oligo7, and amplify a fragment of the COI sequence by using the DNA extracted from the wasp toxin as a template.

6. The method of preparing melittin antitoxin serum as set forth in claim 1, wherein: the specific process of S/D virus inactivation treatment in the step (5) is as follows:

(5a) adding 1% Triton X-100 and 0.3% TNBP into plasma, and standing at 25 deg.C for 6 hr;

(5b) to the plasma mixture after standing for 6 hours, 0.074% of diatomaceous earth was added.

7. The method of preparing melittin antitoxin serum as set forth in claim 1, wherein: and (4) standing the filter liquor subjected to filter pressing by the plate frame in the step (6) at the temperature of 4 ℃ overnight for treatment.

8. The method of preparing melittin antitoxin serum as set forth in claim 1, wherein: the specific process of the gastric enzyme digestion treatment in the step (8) is as follows:

(8a) adding gastric enzyme into the plasma mixture, and digesting for 45-90 minutes in an environment with the temperature of 30 ℃ and the pH value of 3.2;

(8b) adding 150g/L ammonium sulfate into the digested plasma mixture, and stirring for dissolving;

(8c) adjusting the pH value to 5.2-5.4, and heating to 56-58 ℃ for 30 minutes.

9. The method of preparing melittin antitoxin serum as set forth in claim 1, wherein: the specific process of the first precipitation treatment in the step (9) is as follows: cooling to below 45 ℃, and adding 5-10 g/L of diatomite.

10. The method of preparing melittin antitoxin serum as set forth in claim 1, wherein: the specific process of the second precipitation treatment in the step (11) is as follows:

(11a) adding 200g/L ammonium sulfate, adjusting the pH value to 7.0-7.4, and stirring for dissolving;

(11b) and adding 5-10 g/L of diatomite, and standing for 30-60 minutes.

11. The method of preparing melittin antitoxin serum as set forth in claim 1, wherein: the specific process of alum adsorption treatment in the step (13) is as follows:

(13a) dissolving the plasma precipitate in purified water at 35 ℃ by weight which is 2-4 times of the amount of the plasma precipitate;

(13b) adding 0.8% of alum, adjusting the pH value to 7.7-7.9, and stirring for 60 minutes.

12. The method of preparing melittin antitoxin serum as set forth in claim 1, wherein: the anti-toxin serum stock solution of the wasp toxin has a neutralizing effect on the toxins of black abdomen tiger head bees, Yunnan big reason wasps, vespid fossa venomosa and Shaanxi Ankang wasps.

13. The method of claim 11, wherein the anti-toxin serum is prepared by: the neutralization process is that after the prepared wasp toxin antitoxic serum stock solution and the wasp toxin are neutralized, the wasp toxin antitoxic serum stock solution and the wasp toxin are injected into a mouse in an abdominal cavity, the death rate is observed, and the neutralization titer obtained according to a formula is used for evaluating the efficacy of antiserum.

Technical Field

The invention relates to the technical field of biology, in particular to a method for preparing bee venom antitoxic serum.

Background

The black tiger head bees are also named as killer bees, have black abdomen and villi all over the world, one of the most toxic poisonous bees in the world has super-strong aggressivity and super-strong toxicity, and because the black tiger head bees are living organisms and one honeycomb contains thousands of bees, so that the black tiger head bees often sting and hibernating people.

However, no special medicine for treating stinging of black-belly tiger head bees exists in China so far, only general measures can be taken for the patient suffering from the stinging of the wasps, the general measures have certain effect on mild symptoms, but the toxins in the body are not fundamentally eliminated, and the toxins in the body continue to damage the body, so that serious consequences are finally caused.

The antitoxic serum is the most effective first-aid medicament for treating bite or sting of toxic animals all over the world at present, such as antitoxin serum and the like; if a patient can inject anti-virus serum at the first time in time, the possibility of survival of the patient is greatly increased, and valuable time is won for subsequent rescue and treatment. However, at present, no wasp toxin antitoxic serum exists in China, and the preparation research of the effective wasp toxin antitoxic serum is blank; moreover, due to the difference of the species of the wasps, the toxin compositions of the wasps are greatly different, the laboratory identification of the black-belly tiger head wasps has great value, and the preparation of the wasp toxin antitoxic serum, particularly the antitoxic serum with high titer and good purity, has great difficulty and challenge.

Disclosure of Invention

The prepared wasp toxin antitoxic serum has a neutralizing effect on wasp toxins of black-belly tiger-head bees and has an effective neutralizing effect on various wasp toxins of Yunnan big-reason wasps, vespids, Shaanxi Ankang wasps and the like.

In order to achieve the purpose, the method for preparing the bee venom antitoxic serum is designed, and is characterized in that: the specific method comprises the following steps:

(1) performing molecular identification and screening the black abdominal tiger head bees;

(2) performing formaldehyde inactivation and detoxification treatment on the wasp toxin of the black-belly tiger head wasp;

(3) performing animal immunization on the wasp toxin of the black-belly tiger head wasp after the formaldehyde inactivation and detoxification treatment;

(4) blood is collected after immunization;

(5) preserving the collected blood plasma in a cold storage at the temperature of 2-8 ℃ for 5 years;

(6) carrying out S/D virus inactivation treatment on the blood plasma;

(7) carrying out plate-and-frame filter pressing treatment on the inactivated plasma mixture, and removing precipitates;

(8) adding 1.5-2 times of distilled water into the plasma mixture subjected to plate-and-frame filter pressing for plasma dilution;

(9) carrying out gastric enzyme digestion treatment on the diluted plasma mixture;

(10) carrying out primary precipitation treatment on the plasma mixture subjected to the gastric enzyme digestion treatment;

(11) carrying out plate-and-frame filter pressing treatment on the plasma mixture subjected to the first precipitation, and discarding the precipitate;

(12) carrying out secondary precipitation treatment on the plasma mixture subjected to plate-and-frame filter pressing treatment;

(13) carrying out plate-and-frame filter pressing treatment on the plasma mixture subjected to the secondary precipitation, collecting precipitates, and discarding supernatant;

(14) carrying out alum adsorption treatment on the collected plasma precipitate;

(15) carrying out plate-frame filter pressing treatment on the plasma mixture subjected to alum adsorption treatment, and discarding precipitates;

(16) adding 3 times of purified water into the plasma mixture subjected to plate-and-frame filter pressing treatment for ultrafiltration;

(17) subjecting the plasma mixture to DEAE gel column chromatography, and filtering with nanometer membrane to obtain F (ab')₂An immunoglobulin antibody;

(18) mixing F (ab')₂Adding the immunoglobulin antibody into a solution of a proper amount of NaCl and m-cresol, and adjusting the pH value to 6-7 to prepare a wasp toxin antitoxic serum stock solution;

(19) sterilizing and filtering the stock solution of the wasp toxin antitoxic serum, and subpackaging.

The method for identifying the wasps in the black-belly tiger head in the step (1) comprises the following steps:

(1a) dissolving 0.2g unknown wasp venom with 2ml DDH2O, centrifuging 3000g for 5min, and removing supernatant;

(1b) adding 200ul of LB2, mixing, suspending and precipitating;

(1c) adding 20ul RNase A to the sample of step (1 b) and incubating at room temperature for 2 min;

(1d) adding 20ul PK, mixing by vortex, and incubating at room temperature for 2 min;

(1e) adding 500ul BB2, immediately vortexing for 5s, and incubating at room temperature for 10 min;

(1f) adding all the solution into a centrifugal column, centrifuging at 12000rpm for 30s, and removing effluent liquid;

(1g) adding 500ul of CB2, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

(1h) repeating the step (1 g) once;

(1i) adding 500ul WB2, checking the presence of anhydrous ethanol before use, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

(1j) repeating step (1 i) once;

(1k) centrifuging at 12000rpm for 2min to completely remove the residual WB 2;

(1 l) placing a centrifugal column in a clean centrifugal tube, adding 100ul of preheated EB or deionized water into the center of the column, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, eluting DNA, and storing the eluted DNA at-20 ℃;

(1 m) adopting a universal primer and a specific primer to perform PCR amplification on a fragment of a mitochondrial COI sequence from a wasp DNA nucleic acid sequence, measuring the sequence of the mitochondrial COI fragment through second-generation sequencing, and comparing the wasp species through an NCBI website.

The DDH2O is deionized water; LB2 is lysate; RNase A is RNase A; PK is proteinase K; BB2 is a DNA binding solution; CB2 is a solution formed by combining 0.1 mL of deionized water, 0.1 mL of 5 mol.L < -1 > NaCl and 0.8 mL of precooled 95% ethanol absolute ethyl alcohol; WB2 was 75% absolute ethanol; EB is DNA eluate.

The pH value of the deionized water is more than 7.0, and the preheating temperature of EB is 65 ℃.

The PCR amplification is to identify the wasp species from wasp DNA extracted from unknown wasp toxin, design a universal primer and a specific primer through Oligo7, and amplify the fragment of the mitochondrial COI sequence by using the DNA extracted from the wasp toxin as a template.

The specific process of S/D virus inactivation treatment in the step (5) is as follows:

(5a) adding 1% Triton X-100 and 0.3% TNBP into plasma, and standing at 25 deg.C for 6 hr;

(5b) to the plasma mixture after standing for 6 hours, 0.074% of diatomaceous earth was added.

And (4) standing the filter liquor subjected to filter pressing by the plate frame in the step (6) at the temperature of 4 ℃ overnight for treatment.

The specific process of the gastric enzyme digestion treatment in the step (8) is as follows:

(8a) adding gastric enzyme into the plasma mixture, and digesting for 45-90 minutes in an environment with the temperature of 30 ℃ and the pH value of 3.2;

(8b) adding 150g/L ammonium sulfate into the digested plasma mixture, and stirring for dissolving;

(8c) adjusting the pH value to 5.2-5.4, and heating to 56-58 ℃ for 30 minutes.

The specific process of the first precipitation treatment in the step (9) is as follows: cooling to below 45 ℃, and adding 5-10 g/L of diatomite.

The specific process of the second precipitation treatment in the step (11) is as follows:

(11a) adding 200g/L ammonium sulfate, adjusting the pH value to 7.0-7.4, and stirring for dissolving;

(11b) and adding 5-10 g/L of diatomite, and standing for 30-60 minutes.

The specific process of alum adsorption treatment in the step (13) is as follows:

(13a) dissolving the plasma precipitate in purified water at 35 ℃ by weight which is 2-4 times of the amount of the plasma precipitate;

(13b) adding 0.8% of alum, adjusting the pH value to 7.7-7.9, and stirring for 60 minutes.

The stock solution of the anti-toxin serum of the wasp toxin has a neutralizing effect on the toxins of wasps such as the Yunan Dali wasps, the Vespid with depressed veins, the Shanxi Ankang wasps and the like.

The neutralization process is that after the prepared wasp toxin anti-virus serum stock solution and the wasp toxin are neutralized, the wasp toxin anti-virus serum stock solution and the wasp toxin are injected into a mouse in an abdominal cavity, the death rate is observed, and the neutralization titer obtained by calculation according to a Reed-muench formula is used for evaluating the efficacy of the antiserum.

Compared with the prior art, the invention provides a method for preparing the wasp toxin antitoxic serum, and the prepared wasp toxin antitoxic serum has effective neutralization effects on the toxins of different regions such as seven-miles wasps, vespid dimorphism wasps, black-belly tiger-head wasps and the like, Yunnan big reason wasps, Shaanxi Ankang wasps and the like.

Drawings

FIG. 1 shows PCR products of the universal primers for the genome of Vespa mandarina in Yunnan.

FIG. 2 shows PCR products of genome universal primers for Ankang wasps, Shanxi.

FIG. 3 shows PCR products of the universal primers for the female wasp, positive control wasp genome.

FIG. 4 shows PCR products of genome-specific primers for Ankang wasps in Shaanxi, Dali wasps in Yunnan, positive control wasps, and Vespa velutina aurantiaca.

FIG. 5 shows the immunological double-diffusion potency of the wasp anti-toxin serum Y2102 against Yunnan Dali wasp venom, Shaanxi Ankang wasp venom and the wasp anti-toxin serum Y2103 against Yunnan Dali wasp venom and Queen wasp venom.

FIG. 6 shows the immunological double-diffusion potency of Wasp antiserum Y2101 to Rou bee venom in Yunnan, Shanxi Ankang Wau bee venom, Vespa velutina virescens venom and Wasp antiserum Y2102 to Vespa velutina virescens venom.

FIG. 7 shows the immunological double-diffusion potency of the wasp anti-virus serum Y2103 against Shaanxi Ankang wasp venom and the cross-protection ability of the wasp anti-virus serum Y2101, Y2102 and Y2103 against Yunan Dali wasp venom, Shaanxi Ankang wasp venom and Queen wasp venom.

Detailed Description

The invention is further illustrated below with reference to the accompanying drawings.

A method for preparing bee venom antitoxic serum comprises the following steps:

(1) identifying and screening the black abdominal tiger head bees;

(2) performing formaldehyde inactivation and detoxification treatment on the tenofovir disoproxil;

(3) carrying out animal immunization on the tiger head melittin subjected to formaldehyde inactivation and detoxification treatment;

(4) blood is collected after immunization;

(5) preserving the collected blood plasma in a cold storage at the temperature of 2-8 ℃ for 5 years;

(6) carrying out S/D virus inactivation treatment on the blood plasma;

(7) carrying out plate-and-frame filter pressing treatment on the inactivated plasma mixture, and removing precipitates;

(8) adding 1.5-2 times of distilled water into the plasma mixture subjected to plate-and-frame filter pressing for plasma dilution;

(9) carrying out gastric enzyme digestion treatment on the diluted plasma mixture;

(10) carrying out primary precipitation treatment on the plasma mixture subjected to the gastric enzyme digestion treatment;

(11) carrying out plate-and-frame filter pressing treatment on the plasma mixture subjected to the first precipitation, and discarding the precipitate;

(12) carrying out secondary precipitation treatment on the plasma mixture subjected to plate-and-frame filter pressing treatment;

(13) carrying out plate-and-frame filter pressing treatment on the plasma mixture subjected to the secondary precipitation, collecting precipitates, and discarding supernatant;

(14) carrying out alum adsorption treatment on the collected plasma precipitate;

(15) carrying out plate-frame filter pressing treatment on the plasma mixture subjected to alum adsorption treatment, and discarding precipitates;

(16) adding 3 times of purified water into the plasma mixture subjected to plate-and-frame filter pressing treatment, and performing ultrafiltration until the content of ammonium sulfate is less than 0.1%;

(17) subjecting the plasma mixture to gel column chromatography to obtain F (ab')₂An immunoglobulin antibody;

(18) mixing F (ab')₂Adding the immunoglobulin antibody into 7.5-9.5 g/L NaCl solution with the m-cresol content of less than 0.25%, and adjusting the pH value to 6-7 to prepare a wasp toxin antitoxic serum stock solution;

(19) sterilizing and filtering the stock solution of the wasp toxin antitoxic serum, and subpackaging.

The specific process of S/D virus inactivation treatment in the step (5) is as follows:

(5a) adding 1% Triton X-100 and 0.3% TNBP into plasma, and standing at 25 deg.C for 6 hr;

(5b) to the plasma mixture after standing for 6 hours, 0.074% of diatomaceous earth was added.

And (4) standing the filter liquor subjected to filter pressing by the plate frame in the step (6) at the temperature of 4 ℃ overnight for treatment.

The specific process of the gastric enzyme digestion treatment in the step (8) is as follows:

(8a) adding gastric enzyme into the plasma mixture, and digesting for 45-90 minutes in an environment with the temperature of 30 ℃ and the pH value of 3.2;

(8b) adding 150g/L ammonium sulfate into the digested plasma mixture, and stirring for dissolving;

(8c) adjusting the pH value to 5.2-5.4, and heating to 56-58 ℃ for 30 minutes.

The specific process of the first precipitation treatment in the step (9) is as follows: cooling to below 45 ℃, and adding 5-10 g/L of diatomite.

The specific process of the second precipitation treatment in the step (11) is as follows:

(11a) adding 200g/L ammonium sulfate, adjusting the pH value to 7.0-7.4, and stirring for dissolving;

(11b) and adding 5-10 g/L of diatomite, and standing for 30-60 minutes.

The specific process of alum adsorption treatment in the step (13) is as follows:

(13a) dissolving the plasma precipitate in purified water at 35 ℃ by weight which is 2-4 times of the amount of the plasma precipitate;

(13b) adding 0.8% of alum, adjusting the pH value to 7.7-7.9, and stirring for 60 minutes.

The obtained product has neutralizing effect on toxin in Yunnan QILI Wasp, black abdomen tiger head bee, Yunnan Dali Wasp, Vespa velutina aurantiaca, and Wasp in Shaanxi.

The prepared anti-toxin serum stock solution of the wasp toxin has a neutralization effect on the toxins of different types of wasps in different regions of Yunnan Dali, Shaanxi Ankang, Qili wasps, black abdomen tiger head wasps, vespid bees and the like, so that how to identify the types of the wasps has a great significance on clinical guidance, and the anti-toxin serum stock solution not only can help doctors to judge the types of stinging so as to help follow-up correct medication. And the toxin of the wasp can be identified, and the toxin of the most toxic black tiger head wasp is used for preparing the antitoxic serum, so that the antitoxic serum with high neutralization effect and high titer is obtained for clinical treatment.

Secondly, identifying the species of the wasps, which comprises the following specific steps:

1. the wasp total DNA extraction method comprises the following steps:

(1) dissolving 0.2g unknown wasp venom with 2ml DDH2O, centrifuging 3000g for 5min, and removing supernatant;

(2) adding 200ul of LB2, mixing, suspending and precipitating;

(3) adding 20ul of RNase A to the sample of step (2) and incubating at room temperature for 2 min;

(4) adding 20ul PK, mixing by vortex, and incubating at room temperature for 2 min;

(5) adding 500ul BB2, immediately vortexing for 5s, and incubating at room temperature for 10 min;

(6) adding all the solution into a centrifugal column, centrifuging at 12000rpm for 30s, and removing effluent liquid;

(7) adding 500ul of CB2, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

(8) repeating the step (7) once;

(9) adding 500ul WB2, checking the presence of anhydrous ethanol before use, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

(10) repeating the step (9) once;

(11) centrifuging at 12000rpm for 2min to completely remove the residual WB 2;

(12) placing the centrifugal column in a clean centrifugal tube, adding 100ul preheated EB or deionized water into the center of the column, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, eluting DNA, and storing the eluted DNA at-20 ℃.

DDH2O was deionized water; LB2 is lysate; RNase A is RNase A; PK is proteinase K; BB2 is a DNA binding solution; CB2 is a solution formed by combining 0.1 mL of deionized water, 0.1 mL of 5 mol.L < -1 > NaCl and 0.8 mL of precooled 95% ethanol absolute ethyl alcohol; WB2 was 75% absolute ethanol; EB is DNA eluate.

The pH of the deionized water was greater than 7.0 and the EB had a preheat temperature of 65 ℃.

2. Wasp DNA positive standard:

DNA of the wasp cadaver of the black-belly tiger head is extracted as a positive standard substance, and the extraction steps are the same as 1.

3. The wasp DNA molecule identification method comprises the following steps:

the general primer and the specific primer are adopted to amplify the segment of the mitochondrial COI sequence from the wasp DNA nucleic acid sequence by PCR, the sequence of the mitochondrial COI segment is measured by second-generation sequencing, and the wasp species is obtained by NCBI website blast.

(1) And (3) PCR amplification:

identifying the species of wasps by using wasp DNA extracted from the wasp toxins, designing a universal primer and a specific primer by using Oligo7, using the DNA extracted from the wasp toxins as a template, and using the DNA extracted from the black-belly tiger head wasp corpses as a positive control to amplify a fragment of a mitochondrial COI sequence.

Using PCR amplification primers:

the COI sequence universal upstream primer in the wasp mitochondrial genome is H-F: GGTCAACAAATCATAAAGATATTGG, respectively; the general downstream primer is H-R: TAAACTTCAGGGTGACCAAAAAATCA, respectively; the COI sequence specific upstream primer in the wasp mitochondrial genome is HT-F: GTATTAAAGTTTCGATCGGTA, respectively; the specific downstream primer is HT-R: GATATAGCTTTTCCTCGAAT are provided.

An amplification system of wasp COI sequence: the amplification system (20. mu.L) is shown in Table 1.

Table 1.

The amplification procedure is shown in Table 2.

Table 2.

(2) Agarose gel electrophoresis:

the PCR product was subjected to 2% agarose gel electrophoresis at a constant pressure of 120V for 40 min.

(3) Second-generation sequencing:

and (3) carrying out bidirectional sequencing and sequence splicing on the fragments amplified by the PCR to obtain an amplified sequence, wherein the sequence is as follows.

Sequence 1:

sequencing sequence of Yunnan Qili wasp:

CCTTTTTATTTTATTTTTGCTTTATGATCAGGATCTTTAGGAGCCTCTATAAGTTTAATTATTCGTATAGAACTTAGATCCCCAGGAAGATTAATTAACAACGATCAAATCTATAATTCTATTATTACCGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTTTTATAATTGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGAATTCCAGATATAGCTTTTCCTCGAATAAATAATATAAGATTCTGACTATTACCTCCCTCTTTATTTCTATTAATTATAAGAAATTTTATTGGAGGAGGAGTAGGAACTGGATGAACTCTTTACCCACCTCTATCATCAATTACTGGTCATAATTCTCCAGCTGTTGATCTTAGAATCTTTTCATTACATATTGCAGGAATTTCATCAATTATAGGAGCCATTAATTTCATTGTTACAATTTTAAACATACACATTAAAACTCACTCACTAAGATTCTTACCTTTATTTTCATGATCAGTTTTAATTACAGCATTTTTACTATTATTATCCTTACCTGTTCTAGCAGGAGCAATTACAATACTTCTTACCGATCGAAACTTTAATACATCATTTTTTGATCCCACAGGAGGAGGAGACCCTATTTTATATCAACATTTATTTTGATTTTTTGGTCACCTGAAAGTTTAAAAA。

sequence 2:

sequencing sequence of Wasp of Shanxi:

TTAAACTTCCACAGGTGACCAAAAAATCAAAATAAATGTTGATATAAAATAGGGTCTCCTCCTCCTGTGGGATCAAAAAATGATGTATTAAAGTTTCGATCGGTAAGAAGTATTGTAATTGCTCCTGCTAGAACAGGTAAGGATAATAATAGTAAAAATGCTGTAATTAAAACTGATCATGAAAATAAAGGTAAGAATCTTAGTGAGTGAGTTTTAATGTGTATGTTTAAAATTGTAACAATAAAATTAATGGCTCCCTATAATTGATGAAATTCCTGCAATATGTAATGAAAAGATTCTAAGATCAACAGCTGGAGAATTATGACCAGTAATTGATGATAGAGGTGGGTAAAGAGTTCATCCAGTTCCTACTCCTCTTCCAATAAAATTTCTTATAATTAATAGAAATAAAGAGGGAGGTAATAGTCAGAATCTTATATTATTTATTCGAGGAAAAGCTATATCTGGAATTCCTAATATTAAAGGAATTAATCAATTTCCAAATCCTCCAATTTATAAAAGGTATAACTATAAAAAAAATTATAATAAAAGCATGAGCGGTAATAATAGAATTATAGATTTGATCGTTGTTAATTAATCTTCCTGGGGATCTAAGTTCTATACGAATAATTAAACTTATAGAGGCTCCTAAAGATCCTGATCATAAAGCAAAAATAAAATAAAGTATTCCAATATCTTTATGTTTGTTTTGACACAAGAAA。

and (3) sequence:

sequencing sequence of positive control wasp:

TTAAACTTCCACAGGTGACCAAAAAATCAAAATAAATGTTGATATAAAATAGGGTCTCCTCCTCCTGTGGGATCAAAAAATGATGTATTAAAGTTTCGATCGGTAAGAAGTATTGTAATTGCTCCTGCTAGAACAGGTAAGGATAATAATAGTAAAAATGCTGTAATTAAAACTGATCATGAAAATAAAGGTAAGAATCTTAGTGAGTGAGTTTTAATGTGTATGTTTAAAATTGTAACAATAAAATTAATGGCTCCCTATAATTGATGAAATTCCTGCAATATGTAATGAAAAGATTCTAAGATCAACAGCTGGAGAATTATGACCAGTAATTGATGATAGAGGTGGGTAAAGAGTTCATCCAGTTCCTACTCCTCTTCCAATAAAATTTCTTATAATTAATAGAAATAAAGAGGGAGGTAATAGTCAGAATCTTATATTATTTATTCGAGGAAAAGCTATATCTGGAATTCCTAATATTAAAGGAATTAATCAATTTCCAAATCCTCCAATTTATAAAAGGTATAACTATAAAAAAAATTATAATAAAAGCATGAGCGGTAATAATAGAATTATAGATTTGATCGTTGTTAATTAATCTTCCTGGGGATCTAAGTTCTATACGAATAATTAAACTTATAGAGGCTCCTAAAGATCCTGATCATAAAGCAAAAATAAAATAAAGTATTCCAATATCTTTATGTTTGTTTTGACACAAGAAA。

(4) and (3) detecting results and analyzing data:

by using a general primer, a specific primer and a DNA sample of the Roughage Yunnanensis, Shaanxi Ankang and Vespa velutina virens extracted from the Wasp toxin, performing amplification for 40 cycles by adopting the reaction system and the program, performing electrophoresis by using 2% agarose gel to obtain a band of a COI sequence in a mitochondrial genome, wherein the specific primer can only P out of a band of the COI sequence in the Roughage tiger bee mitochondrial genome, and if the Roughage nigra virens are not, the specific primer does not generate a positive band by PCR (polymerase chain reaction), and as a result, the Roughage Yunnanensis, Shaanxi Ankang Vespa velutina virens and the positive control Vespa velutina DNA generate a positive band, and the Roughage virens do not have the Roughage virens, further sequencing analysis, sequencing of three PCR products of the general primer, splicing the sequencing result, and blast sequence homology of more than 98% can be determined to be a species. Through identification, the Yunnan Dali wasp and the Shaanxi Ankang wasps belong to black abdomen tiger head wasps, so that the Shaanxi Ankang wasp toxin with strong toxicity is selected for preparing the wasp antitoxic serum.

Thirdly, determining the toxin neutralization effect of the antitoxic serum:

and finally, according to the verification of the neutralization effect of the prepared wasp toxin antitoxic serum, in vitro, after the wasp toxin antitoxic serum stock solution and the wasp toxin are neutralized, injecting a mouse into the abdominal cavity, observing the death rate, and obtaining the neutralization titer according to a Reed-muench formula to evaluate the efficacy of the antiserum.

Three batches of the anti-virus serum stock solution of the wasp toxin, Shanxi Ankang wasp toxin, Yunnan Dali wasp toxin and Queen wasp toxin have immune double-amplification potency, as shown in Table 3.

Table 3.

Original solution of anti-toxin serum of wasp toxin	Toxin of Wasp of Dali Yunan	Shaanxi Ankang wasp poison	Venom of Vespa velutina
				Y2101	1:8	1:8	1:8
Y2102	1:8	1:8	1:8
				Y2103	1:8	1:8	1:8

As shown in FIGS. 5 to 7, the three batches of the anti-virus serum stock solutions of the wasp toxins have precipitation lines with the Shanxi Ankang wasp toxins, Yunan Dali wasp toxins and Queen wasp toxins, and show good cross protection for different wasp toxins.

The neutralization titers of the wasp toxin antitoxic serum stock solution Y2102 and Shaanxi Ankang wasp venom, Yunan Dali wasp venom and Vespa wasp venom animals are 266U/ml, 296U/ml and 403U/ml respectively, and the wasp toxin antitoxic serum stock solution F (ab') 2 has a certain protection effect on different wasp toxins and shows good cross protection.

The result of immune double diffusion shows that the anti-virus serum of the wasp toxin not only combines with the Royal wasp toxin in Yunnan, but also combines with Shaanxi Ankang wasp toxin and Queen wasp toxin, and has cross reaction.

Animal experiment results show that the anti-virus serum of the wasp toxin has protective effects on wasp toxins in Shanxi Ankang, Yuanli wasp toxins in Yunnan, and also has protective effects on the wasp toxins in intaglio, so that the anti-virus serum of the wasp toxins has protective effects on neutralizing antibodies.

Since the wasp toxin antiserum has a broad cross-protective effect on the wasp venom of the family Vespidae, a broad-spectrum antiserum against the family Vespidae can be developed on the basis of the wasp toxin antiserum.