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High-titer anti-cobra neurotoxin serum as well as preparation method and application thereof

A technology of neurotoxin and cobra venom, which is applied in the field of immunity, can solve the problems of poor therapeutic effect and low antiserum titer, and achieve the effect of improving therapeutic effect, reducing production cost and long residence time

Inactive Publication Date: 2017-06-09
WEST ANHUI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the strong stability of cobra venom neurotoxins, it is difficult to change its toxicity by general attenuation methods. In the process of preparing neurotoxin antiserum, only a small amount of injected antigen can survive, and the small amount of antigen leads to low titer of antiserum. , poor treatment effect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Step 1: Obtaining Cobra Neurotoxin

[0020] Dissolve 0.1 g of cobra venom in 1.0 mL of normal saline, heat in a boiling water bath for 10 minutes, centrifuge (13,000 rpm) to remove non-neurotoxin precipitates, then add 50 mg of SDS and 2 uL of β-mercaptoethanol to fully mix to reduce its neurotoxicity ;

[0021] Step 2: Entrapment of Neurotoxin

[0022] Add the above neurotoxin solution to 1.4mL 30% acrylamide, 50uL 10% ammonium persulfate, 2uL tetramethylethylenediamine (TEMED), mix thoroughly, slowly add 0.5mL isopropanol along the wall of the test tube, and after 1 hour Solidify, remove the isopropanol, take out the colloid and divide it into 4 equal portions with a scalpel, each portion is about 1 g, and store it in a freezer;

[0023] Step 3: Inject immunized rabbits to obtain antiserum

[0024] Take 1 portion of the above colloid, add 2.0 mL of normal saline, fully grind it in a mortar, inhale it into a 5 mL disposable syringe, the total volume is about 3.5 mL,...

Embodiment 2

[0036] Step 1: Obtaining Cobra Neurotoxin

[0037] Except using 0.09g cobra venom, other steps are the same as the first step of "Example 1".

[0038] Step 2: Entrapment of Neurotoxin

[0039] Add 1.3mL of 30% acrylamide, 50uL of 10% ammonium persulfate, 2uLTEMED to the neurotoxin solution obtained in the first step, and mix well, and the other steps are the same as the second step of "Example 1";

[0040] Step 3: Inject immunized rabbits to obtain antiserum

[0041] It is the same as the third step of "Example 1".

[0042] Step 4: Determination of antiserum titer

[0043] The determination process is the same as the fourth step of "Example 1".

[0044] After calculation, the titer of the antiserum prepared in "Example 2" reached 544, while the titer of the antiserum prepared by emulsifying with conventional snake venom plus adjuvant was only 80, so the titer of the antiserum prepared by the present invention was increased by about 6.8 times.

Embodiment 3

[0046] Step 1: Obtaining Cobra Neurotoxin

[0047] Except using 0.11g cobra venom, other steps are the same as the first step of "Example 1".

[0048] Step 2: Encapsulation of snake venom protein

[0049] Add 1.5mL of 30% acrylamide, 50uL of 10% ammonium persulfate, 2uLTEMED to the neurotoxin solution obtained in the first step, and mix well, and the other steps are the same as the second step of "Example 1";

[0050] Step 3: Inject immunized rabbits to obtain antiserum

[0051] It is the same as the third step of "Example 1".

[0052] Step 4: Determination of antiserum titer

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PUM

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Abstract

The invention discloses a high-titer anti-cobra neurotoxin serum and a preparation method thereof. The preparation method comprises the following steps: firstly, reducing neurotoxicity of cobra venom; then, embedding the cobra venom in polyacrylamide gel; and finally, grinding neurotoxin-containing gel, performing immune injection on rabbits to obtain antiserum, and performing cryopreservation. In the invention, different doses of cobra venom are adopted; the neurotoxicity is reduced by heating, SDS and beta-mercaptoethanol treatment; the emulsification is replaced with polyacrylamide gel embedding, so as to ensure healthy survival of immunized animals, reduce production cost of he antiserum, and prepare high-titer antiserum.

Description

technical field [0001] The invention relates to a preparation method of high titer anti-cobra neurotoxin serum, which belongs to the technical field of immunization. Background technique [0002] cobra( Naja naja atra Cantor) belongs to the Cobra family and the genus Cobra, and is widely distributed in my country. It is a neurovenomous snake, and its main toxin is neurotoxin. Neurotoxins are divided into two categories: pre-synaptic neurotoxins and post-synaptic neurotoxins. The former inhibit the release of acetylcholine in nerve conduction, and the latter can bind to motor endplate acetylcholine receptors. Both lead to blockade of nerve conduction. Class II neurotoxins are all basic proteins, do not contain non-protein components, have small molecular weight, contain 4-5 disulfide bonds, and have strong heat resistance. [0003] Due to the strong stability of cobra venom neurotoxins, it is difficult to change its toxicity by general attenuation methods. In the process o...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06A61K39/395A61P39/02
CPCA61K39/00C07K16/06C07K16/18C07K2317/35
Inventor 韦传宝
Owner WEST ANHUI UNIV
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