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Method for preparing anti-A type botulinus toxin immunoglobulin antibody

A botulinum toxin type A, immunoglobulin technology, applied in the direction of antibacterial immunoglobulins, microorganism-based methods, biochemical equipment and methods, etc. Neutralizing activity and other issues, to achieve high purity, good immune effect, good stability

Active Publication Date: 2009-08-05
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, there are too many non-neutralizing epitopes in the toxoid itself, so although the titer of antibodies produced by immunity can be high, the proportion of neutralizing antibodies in the active ingredient is low, that is, the specific activity is relatively low, and the neutralization of the anti-toxin is relatively low. Activity is affected, and the most important indicator to measure the efficacy of antitoxin is the activity of neutralizing antibody (specific activity)

Method used

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  • Method for preparing anti-A type botulinus toxin immunoglobulin antibody
  • Method for preparing anti-A type botulinus toxin immunoglobulin antibody
  • Method for preparing anti-A type botulinus toxin immunoglobulin antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, the preparation of botulinum toxin type A immunogen

[0034] In the following, AHc denotes a gene, and rAHc denotes a protein encoded by the AHc gene.

[0035] 1. Construction of recombinant prokaryotic expression vector pTIG-Trx-AHc

[0036] Thioredoxin (Trx) is an auxiliary protein involved in the folding of the nascent protein peptide chain. When the recombinant protein rAHc is co-expressed with it (one mRNA, two reading frames, as a result, Trx is first translated, and then the target protein rAHc is translated. Fusion, becoming an independent protein), it can promote the correct folding of the target protein being folded downstream through the isomerization reaction, in addition, it can also enhance the formation of disulfide bonds of intracellular proteins, so that the expression product is not easy to form inclusion bodies, and can be expressed as Soluble form expression, therefore construct the prokaryotic expression vector containing Trx gene and...

Embodiment 2

[0059] Example 2. Recombinant rAHc immunogen immunization horse and horse immune serum ELISA antibody and neutralizing antibody level detection

[0060] The expression and purification scheme of recombinant protein rAHc in Escherichia coli in Example 1 was scaled up, and a large amount of recombinant antigen was obtained by a large amount of purification to be used as an immunogen.

[0061] The recombinant protein rAHc dissolved in PBS was emulsified with an equal volume of incomplete Freund's adjuvant (IFA, Sigma) to immunize horses, 5 ml per horse, a total of 5 horses (horses qualified for biological products after quarantine), subcutaneous and intramuscular More immunity. The first and second recombinant protein doses were 3 mg, the third and fourth recombinant protein doses were 5 mg, and the fifth and sixth recombinant protein doses were 8 mg, for a total of 6 immunizations of horses (four times first, followed by two boosters) ), immunized every two weeks. Before immun...

experiment example 3

[0066] Experimental example 3. Preparation of horse anti-botulinum toxin type A immunoglobulin F(ab')2 antibody

[0067] 1) Collect five and six times of immunized horse anti-botulinum toxin type A plasma in Example 2, inactivate with SD (organic solvent / detergent), and the SD inactivation process is: add Triton X-100 and TNBP solution to The final concentrations were 1% and 0.3%, respectively. After 6 hours at 4 degrees, 0.074% diatomaceous earth was added, and after stirring for 10 minutes, pressure filtration was performed to collect plasma.

[0068] 2) For the plasma inactivated by SD, 1 part of plasma was diluted with 2 parts of water for injection, pepsin was added, toluene was added to make the final concentration 0.2%, the pH was adjusted to 3.2 with hydrochloric acid, and then digested at 30°C for 75 minutes. IgG is F(ab') after pepsin cleavage of the Fc segment 2 .

[0069] 3) Precipitation of ammonium sulfate in plasma after gastric enzyme digestion: add 15% solid...

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Abstract

The invention discloses a method for preparing an anti-type A botulinum neurotoxin immunoglobulin antibody, which comprises the steps of preparing and obtaining the anti-type A botulinum neurotoxin immunoglobulin antibody through taking type A botulinum neurotoxin recombinant protein rAHc as immunogen to immunize animals. The method for preparing the anti-type A botulinum neurotoxin immunoglobulin antibody eliminates a Fc segment in the antibody which can cause side effect and obtains the horse anti-type A botulinum neurotoxin immunoglobulin F(ab')2 antibody; after blood serum obtained by pentalogy hyper-immune and hexalogy hyper-immune are mixed, the content (purity) of F(ab')2 of the antibody can achieve 80.2 percent in a semi-finished product obtained through further purification; the titer of the antibody can achieve 8000 IU / ml; the antibody has good specificity and sensitivity to type A botulinum neurotoxin; and the purity and the specific activity of the antibody are higher than the antitoxin prepared from the traditional toxin immunity horses. Moreover, the invention has the good stability and safety.

Description

technical field [0001] The invention relates to a method for preparing anti-botulinum toxin immunoglobulin antibody. Background technique [0002] Botulinum toxin (BoNT) is currently the most virulent protein known, and 1.0g of botulinum toxin crystals is enough to kill 1 million people. It is produced by Clostridium botulinum in an anaerobic environment. There are 7 serotypes (A-G) of the exotoxins produced. Among them, A and B types of botulinum toxin are the common types that cause human poisoning, E and F types occasionally cause food poisoning epidemics, and C and D types only cause animal and poultry poisoning. Poisoning, there are fewer reports of poisoning caused by G type. Clinical observations have shown that botulinum toxin type A can cause more severe intoxication symptoms and lead to higher mortality than other types. Botulinum toxin poisoning has a small incidence rate in the crowd, but its toxicity is high, the fatality rate is high, and it is extremely harm...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K16/12C12R1/00
Inventor 孙志伟余云舟俞炜源杜韫林建波
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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