An anti-ricin immunoglobulin f(ab') 2 and its preparation method

A technology of immunoglobulin and ricin, applied in the direction of anti-plant immunoglobulin, immunoglobulin, immunoglobulin from serum, etc., can solve problems that are easy to cause social harm and difficult to supervise

Active Publication Date: 2021-08-31
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The source of ricin is easy, easy to extract, easy to obtain, difficult to supervise, and easy to cause social harm. There have been many terrorist incidents using ricin in history

Method used

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  • An anti-ricin immunoglobulin f(ab') <sub>2</sub> and its preparation method
  • An anti-ricin immunoglobulin f(ab') <sub>2</sub> and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, anti-ricin immunoglobulin F (ab') 2 Antibody preparation

[0053] 1. Preparation and identification of ricin immunogen

[0054] (1) Obtaining of recombinant ricin protein mt RTA

[0055] The amino acid sequence of the recombinant ricin protein mt RTA is sequence 2, and the nucleotide sequence of its coding gene is sequence 1.

[0056] mt RTA is a low-toxic protein obtained by modifying the A chain of ricin (patent publication number: 201010552949.4)

[0057] The preparation process is as follows:

[0058] 1. Preparation of engineering bacteria expressing recombinant ricin protein

[0059] The engineering bacterium mt RTA / pET-His / BL21(DE3) expressing the recombinant ricin protein is a strain obtained by introducing the plasmid mt RTA / pET-His into Escherichia coli BL21(DE3).

[0060] Plasmid mt RTA / pET-His is the insertion of mt RTA (sequence 1) gene into pET-His (Sun Simei, Wang Jinglin, etc. Soluble expression, purification and antigenicity analysis o...

Embodiment 2

[0124] Embodiment 2, refining anti-ricin immunoglobulin F (ab') 2 Evaluation of therapeutic and preventive effects on BALB / c mice

[0125] 1. Anti-ricin immunoglobulin F (ab') obtained from different immunogens 2 Comparison of immunogenicity of

[0126] Use neutralizing antibody titer detection, as follows:

[0127] The purified ricin (natural ricin) PBS in (three) of Example 1 is diluted to 2LD50 / 0.2ml, and the recombinant ricin protein mt RTA prepared in Example 1 is used as the horse anti-ricin prepared by immunogen Immunoglobulin F (ab') 2 Injection-type finished product, recombinant ricin protein rRTA as immunogen prepared as immunogen for horse anti-ricin immunoglobulin F (ab') 2 Equine anti-ricin immunoglobulin F (ab') prepared by injectable finished product and detoxified purified ricin as immunogen 2 The injection-type finished products were diluted 1000, 2000, 4000, and 6000 times respectively, and 0.2ml and 2LD50 / 0.2ml of natural ricin were placed in 37°C for 1...

Embodiment 3

[0149] Example 3. Evaluation of the therapeutic effect of refined anti-ricin immunoglobulin in rhesus monkeys

[0150] In the examples, horse anti-ricin immunoglobulin F (ab') injection was used to prepare detoxified and purified ricin as the immunogen.

[0151] 1. Infected animals

[0152] Use 0.01MPBS (PH 7.2) to be formulated into the purified ricin (natural ricin) prepared in 72 μg / ml (three) of Example 1, and intraperitoneally inject rhesus monkeys (body weight 5.5-6.3kg, male and female half and half) 15 animals were obtained as animal models of infection.

[0153] 2. Immunity

[0154] At the 4th hour after the injection of ricin, 5 rhesus monkeys in the animal model of infection were given 3000U (7.5 mg) of equine anti-ricin immunoglobulin F (ab') diluted in 0.01M PBS ) / ml, 5 rhesus monkeys were given horse anti-ricin immunoglobulin F (ab') injection 5000U (12.5mg) / ml, and the other 5 rhesus monkeys were intraperitoneally injected with the same volume of 0.01MPBS (PH...

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Abstract

The invention discloses an anti-ricin immunoglobulin F(ab')2 and a preparation method thereof. The invention provides a method for preparing anti-ricin immunoglobulin F(ab')2, comprising the steps of: immunizing animals with an immunogen, preparing ricin IgG antibodies, and then removing the ricin IgG antibodies The Fc segment obtains the anti-ricin immunoglobulin F (ab') 2; the antigen of the immunogen is as follows: 1), low toxicity or attenuated ricin mutant or its recombinant protein; 2) encoding the A DNA molecule of a low-toxic or attenuated ricin mutant or its recombinant protein, or a vector, an expression cassette, a recombinant bacterium or a recombinant virus expressing the DNA molecule; 3) detoxified natural ricin. The invention utilizes the non-toxic or low-toxic ricin antigen to immunize healthy animals to produce high titer neutralizing ricin antiserum, which has a specific protective effect on the treatment and prevention of the ricin.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and relates to an anti-ricin immunoglobulin F (ab') 2 and its preparation method. Background technique [0002] Ricin (ricin) is a highly toxic glycoprotein derived from plant castor seeds. The intraperitoneal median lethal dose (LD50) of ricin to mice is about 3-8 μg / kg, and the lethal dose to adults is about 150μg, its toxicity is equivalent to about 6000 times that of cyanide. Ricin is a highly toxic plant protein with two peptide chains, which can easily damage liver, kidney and other solid organs, causing bleeding, degeneration and necrosis. It can agglutinate and dissolve red blood cells, inhibit and paralyze the cardiovascular and respiratory centers, and is one of the main causes of death. The source of ricin is easy, easy to extract, easy to obtain, difficult to supervise, and easy to cause social harm. There have been many terrorist incidents using ricin in history. The 1993 Chemica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/16C07K16/06A61K39/395A61P39/02
CPCA61K2039/505A61K2039/552A61P39/02C07K16/065C07K16/16C07K2317/35C07K2317/54C07K2317/94
Inventor 杨鹏辉王希良段跃强赵忠鹏谷宏婧杨晓岚
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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