阅读说明：本技术 一种二价抗海蛇毒血清的制备方法 (Preparation method of bivalent anti-sea snake venom serum ) 是由 杜宇 林炽贤 高建芳 李佳颀 吕金更 梁丁文 赵祖越 陈忠荫 刘其则 计翔 于 2020-06-11 设计创作，主要内容包括：本发明涉及抗蛇毒血清的制备技术领域,尤其涉及一种二价抗海蛇毒血清的制备方法。本发明提供的二价抗海蛇毒血清的制备方法,以青环海蛇和平颏海蛇的蛇毒为抗原,以羊为免疫对象,通过改进免疫方案与剂量,获得了二价抗海蛇毒血清。该血清能够对海蛇蛇毒起到良好的中和效果。该血清针对我国优势种海蛇蛇毒,有助于提高蛇伤治疗的成功率,降低致残率,减少血清过敏等并发症。该制备方法稳定有效在不同个体之间重现度良好,为完善海蛇咬伤的临床治疗及药物制备提供重要参考。(The invention relates to the technical field of preparation of snake venom resisting serum, in particular to a preparation method of bivalent sea snake venom resisting serum. The invention provides a preparation method of bivalent anti-sea snake venom serum, which takes snake venom of a green-ring sea snake and a flat-chin sea snake as antigens, takes sheep as an immune object, and obtains the bivalent anti-sea snake venom serum by improving an immune scheme and dosage. The serum has good neutralizing effect on snake venom of sea snake. The serum aims at the dominant species of sea snake venom in China, and is beneficial to improving the success rate of snake bite treatment, reducing the disability rate, reducing the complications such as serum allergy and the like. The preparation method is stable and effective, has good reproducibility among different individuals, and provides important reference for perfecting clinical treatment of sea snake bite and medicine preparation.)

1. The preparation method of the bivalent anti-sea snake venom serum is characterized by comprising the following steps:

step 1: respectively dissolving lyophilized snake venom powders of the green-ring sea snake and the flat-chin sea snake in normal saline, mixing the two to prepare a snake venom solution, sterilizing, inactivating at 80-100 ℃, and mixing with an adjuvant to prepare an emulsified antigen;

step 2: immunizing sheep with the emulsified antigen, wherein the dosage of the first immunization is 0.02 mu g/g, and then boosting immunization is respectively carried out at 2 weeks, 3 weeks, 5 weeks, 7 weeks, 9 weeks, 14 weeks and 17 weeks, and the immunization dosages are 0.05 mu g/g, 0.1 mu g/g, 0.2 mu g/g, 0.5 mu g/g, 1 mu g/g, 0.5 mu g/g and 0.5 mu g/g in sequence; collecting sheep serum to obtain bivalent anti-sea snake venom serum; the dosage is calculated by the sum of the mass of snake venom freeze-dried powder of the green-ring sea snake and the flat-chin sea snake.

2. A preparation method according to claim 1, wherein the concentration of the snake venom in the snake venom solution is 20 μ g/μ L to 30 μ g/μ L; the concentration of the sea flatmental venom is 20 mug/mug-30 mug/mug.

3. The method according to claim 1 or 2, wherein the inactivation is performed at 90 ℃ for 30 min.

4. The method according to any one of claims 1 to 3, wherein the emulsified antigen is adjuvanted with Freund's complete adjuvant or Freund's incomplete adjuvant;

the adjuvant in the emulsified antigen of the first immunization is Freund's complete adjuvant;

the adjuvant in the emulsified antigen of the 2 nd-8 th immunization is Freund incomplete adjuvant;

the adjuvant is Freund's complete adjuvant, and the volume ratio of the adjuvant to the snake venom solution is 1: 1;

the adjuvant is Freund's incomplete adjuvant, and the volume ratio of the adjuvant to the snake venom solution is 1: 1.

5. The method of any one of claims 1 to 4, wherein the sheep is a black goat; the part of the immunity is the groin.

6. The method according to any one of claims 1 to 5, wherein the period of collecting the sheep serum is 3 weeks to 17 weeks.

7. A bivalent anti-serpin prepared by the preparation method of any one of claims 1 to 6.

8. Use of the bivalent anti-sea snake venom serum prepared by the preparation method of any one of claims 1 to 6 in preparation of a medicament for treating sea snake bite.

9. Use according to claim 8, wherein the sea snake comprises a green sea snake and/or a flat-chin sea snake.

10. A medicine for treating sea snake bite is characterized in that raw materials of the medicine comprise bivalent anti-sea snake poison serum prepared by the preparation method of any one of claims 1 to 7.

Technical Field

The invention relates to the technical field of preparation of snake venom resisting serum, in particular to a preparation method of bivalent sea snake venom resisting serum.

Background

The poisonous snakes in China are widely distributed and are various in variety, the snake injury is serious, and sea snakes are no exception. With increasing frequency of human marine activity, the number of bites by sea snakes (particularly by the bungarus multicinctus and the sea flathead) increases. In China, the hospital mainly treats the sea snake bite by some methods, such as injecting the same anti-bungarus snake venom serum or anti-cobra venom serum, but the two anti-snake venom sera have weak neutralizing capacity to the sea snake venom and generally need to increase the injection dosage. Still other hospitals will treat sea snake bites by combining traditional Chinese medicines with hormone medicines or by directly cutting the trachea. The anti-snake venom serum is considered to be the most effective medicine for treating snake bites, so that the production of the targeted anti-sea snake venom serum is necessary.

The rabbit-derived bivalent anti-trepang and Chin sea snake venom serum is developed by the university of Guangxi medical science of China, plum bin and professor 2011 in cooperation with the Japanese snake society, and is used for clinical emergency treatment of sea snake bite in the northern sea city of Guangxi, and the like, and the treatment effect is obvious, but the rabbit-derived bivalent anti-trepang and Chin sea snake venom serum is not popularized and marketed in the domestic market on a large scale. At present, relevant units of the military develop serum for resisting the poison of the green-ring sea snake, but the application and the treatment effect of the serum are not reported. The second university of military medicine is also conducting research on the preparation of serum against sea snake venom. Although some countries like Australia have developed monovalent anti-purulent serums against local sea snake venom, the cost is high and the therapeutic effect on sea snake bite in China is unclear. Therefore, the development of the serum for resisting sea snake venom aiming at the dominant species of sea snakes in China has higher application value. The anti-snake venom serum which is currently marketed is usually produced by taking horses as immune animals, but the breeding cost of the horses is high, and the blood collection is difficult.

Generally, snake venom can be neutralized with either a heterologous or a homologous anti-snake venom serum, but the neutralizing effect of a heterologous anti-snake venom serum is generally reduced. When the snake bite cannot be identified, it is often difficult to determine which anti-snake venom serum is used, and the multivalent anti-snake venom serum for the snake in a certain region, particularly sea snakes, can be directly used without identifying the snake type. Therefore, the development of the multivalent serums for resisting the sea snake venom has higher application value.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing bivalent anti-sea snake venom serum, which uses sheep as an immune animal, not only reduces the cost, but also can quickly meet the titer requirement.

The preparation method of the bivalent anti-sea snake venom serum provided by the invention comprises the following steps:

step 1: respectively dissolving lyophilized snake venom powders of the green-ring sea snake and the flat-chin sea snake in normal saline, mixing the two to prepare a snake venom solution, sterilizing, inactivating at 80-100 ℃, and mixing with an adjuvant to prepare an emulsified antigen;

step 2: immunizing sheep with the emulsified antigen, wherein the dosage of the first immunization is 0.02 mu g/g, and then boosting immunization is respectively carried out at 2 weeks, 3 weeks, 5 weeks, 7 weeks, 9 weeks, 14 weeks and 17 weeks, and the immunization dosages are 0.05 mu g/g, 0.1 mu g/g, 0.2 mu g/g, 0.5 mu g/g, 1 mu g/g, 0.5 mu g/g and 0.5 mu g/g in sequence; collecting sheep serum to obtain bivalent anti-sea snake venom serum; the dosage is calculated by the sum of the mass of snake venom freeze-dried powder of the green-ring sea snake and the flat-chin sea snake.

In the invention, the concentration of the green-ring sea snake venom in the snake venom solution is 20-30 mug/mug; the concentration of the sea flatmental venom is 20 mug/mug-30 mug/mug. In some embodiments, the snake venom solution has a concentration of bungarus multicinctus venom of 25 μ g/μ L; the concentration of the sea flathead venom is 25 μ g/μ L.

In the scheme of the invention, the snake venom is inactivated by adopting a high-temperature water bath method, so that the use of formaldehyde in the traditional method is avoided. In some embodiments, the inactivation conditions are 90 ℃ for 30 min.

In the invention, in the emulsified antigen, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant;

the adjuvant in the emulsified antigen of the first immunization is Freund's complete adjuvant;

the adjuvant in the emulsified antigen of the 2 nd-8 th immunization is Freund incomplete adjuvant;

the adjuvant is Freund's complete adjuvant, and the volume ratio of the adjuvant to the snake venom solution is 1: 1;

the adjuvant is Freund's incomplete adjuvant, and the volume ratio of the adjuvant to the snake venom solution is 1: 1.

In the invention, the immune cycle and the immune dose are set aiming at the sheep, the sheep are adapted when the low dose is adopted in the initial immunity, the dose is gradually increased in the adaptation range of the sheep, and the immune interval time is prolonged. During the course of immunization, Elisa tests showed that the absorbance after the seventh and ninth weeks of immunization differed little, indicating that the immunization dose was not increased until the immunization dose reached 0.5. mu.g/g, followed by a booster dose of 0.5. mu.g/g. After the sixth immunization, the absorbance of the serum decreases, and the interval period of the immunization can be judged to be 3-4 weeks, so that after the fourteenth immunization, the immunization is performed once again in the seventeenth week. Attempts of this immunization protocol in a large number of subjects showed that the reproducibility was good and that the immune responses generated between multiple individuals should be highly similar.

In the invention, the sheep is a black goat; the part of the immunity is the groin. In the examples, the sheep used were 6-month-old female black goats; the female black goat weighs 15 kg.

In the art, an anti-snake venom serum absorbance greater than three times that of negative serum absorbance may be considered effective, while the titer of anti-snake venom serum is greater than 1×10⁵The anti-snake venom serum is considered to be successfully prepared. In the present example, all of the anti-snake venom sera at week 1 were negative sera, i.e., sera from sheep themselves when not immunized. And (3) immunizing after taking blood, wherein the serum in week 2 is the serum after the first immunization for one week, the absorbance of the serum is not much different from that of the negative serum, and the serum in week 2 does not show a positive result, so that the serum is continuously immunized, and the serum after week 3 is more than three times that of the negative serum, and is considered as effective serum. Therefore, in the invention, the time for collecting the sheep serum is 3 weeks to 17 weeks.

The divalent sea snake poison resisting serum prepared by the preparation method is provided by the invention.

The divalent sea snake poison resisting serum prepared by the preparation method is applied to preparing the medicine for treating sea snake bite.

The method provided by the invention obtains the crude product of bivalent anti-sea snake venom serum, and can be applied to clinical treatment of sea snake bite after separating and purifying specific antibodies in the crude product and evaluating safety. In the method provided by the invention, the snake venom of the blue-ring sea snake and the flat-chin sea snake is taken as an antigen to prepare the anti-snake venom serum, but in the previous research, the components of snake venom proteins of the same family and the same genus are similar, so that clinically, when the serum which is specific to a certain snake venom does not exist, the snake venom of the same family or even the same family can be generally used for treatment. Therefore, the divalent anti-sea snake poison blood removing agent provided by the invention is applied to the bite of the green-ring sea snake and the flat-chin sea snake, and can also be applied to the bite of other sea snakes. The sea snakes comprise green sea snakes and/or flat-chin sea snakes.

The invention also provides a medicine for treating sea snake bite, and the raw materials of the medicine comprise the divalent sea snake poison resisting serum prepared by the preparation method.

The invention also provides a method for treating sea snake bite, which is to administer the medicament.

The invention provides a preparation method of bivalent anti-sea snake venom serum, which takes snake venom of green-ring sea snake and flat-chin sea snake as antigen; the sheep are taken as an immune object, and the bivalent anti-sea snake venom serum is obtained by improving the immune scheme and the dosage. The serum has good neutralizing effect on snake venom of sea snake. The serum aims at the dominant species of sea snake venom in China, and is beneficial to improving the success rate of snake bite treatment, reducing the disability rate, reducing the complications such as serum allergy and the like. The preparation method is stable and effective, has good reproducibility among different individuals, and provides important reference for perfecting clinical treatment of sea snake bite and medicine preparation.

Drawings

FIG. 1 illustrates the immune cycle and dose in the examples;

FIG. 2 shows the example of the immunoreaction of bivalent anti-sea snake venom serum with respectively green-ring sea snake venom (Hcy) and Chignan sea snake venom (Hcu): a shows the immune response in sheep No. 1, B shows the immune response in sheep No. 2;

FIG. 3 shows polyacrylamide gel electrophoresis of the venom of the Rohdea viridis (Hcy) and Genchin sea snake (Hcu);

FIG. 4 shows a schematic representation of Western blot detection of monovalent and bivalent anti-sea snake venom sera in the examples: A. b is a Western blot detection chart of bivalent anti-sea snake venom serum of two sheep and two snake venom respectively; c is a Western blot assay of monovalent anti-Serpentis venom serum and two snake venoms; d is a Western immunoblot assay of monovalent anti-bunyah venom serum with both snake venoms.

Detailed Description

The invention provides a preparation method of bivalent anti-sea snake venom serum, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the preparation method. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples: