Preparation of soluble human selenium-containing single-chain abzyme

A single-chain antibody and soluble technology, which is applied in the field of preparing glutathione-derived selenium-containing single-chain antibody enzymes, can solve the problems of long antibody enzyme cycle, slow recovery activity, and decreased yield of antibody enzymes

Inactive Publication Date: 2008-10-08
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method for the human source selenium-containing single-chain antibody enzyme prepared by this method has the following disadvantages: (1) the inclusion body protein expressed in the preparation process is a kind of inactive protein molecule without normal spatial conformation and in a precipitated state. Denaturation - renaturation to be active
The renaturation of inclusion body protein is a difficult and complicated process. Only in the renaturation step will a large amount of s

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Synthesize glutathione dibutyl ester with existing technology (see Example 1 of patent 99104234.4), take 50 μg of glutathione dibutyl ester, dissolve it with dimethyl sulfoxide (DMSO), and coat it on an immunoplate Above, the remaining sites were blocked with BSA. Using the coated glutathione dibutyl ester as the target antigen, the phage-displayed human single-chain antibody library was screened for 5 rounds by standard immunoaffinity screening method, and finally the positive clones were further screened by ELISA method to obtain the affinity for GSH. Higher specificity single chain antibody B3, sequenced to determine the entire gene and amino acid sequence.

[0046] The phage DNA was extracted, and the phage DNA containing the single-chain antibody B3 gene was used as a template, and the public primers of the antibody library (5'-CAGGAAACAGCTATGAC-3' and 5'-GAATTTTCTGTATGAGG-3') were used for PCR amplification of the single-chain antibody B3 gene. The PCR product wa...

Embodiment 2

[0050] Synthesize hapten-glutathione dibutyl ester according to the method of patent 99104234.4, take 50 μg of glutathione dibutyl ester, dissolve it with dimethyl sulfoxide (DMSO), coat it on an immunoplate, and use BSA blocked. Using the coated glutathione dibutyl ester as the target antigen, the phage-displayed human single-chain antibody library was screened for 5 rounds by standard immunoaffinity screening method, and finally the positive clones were further screened by ELISA method to obtain the affinity for GSH. Higher specificity single chain antibody B3, sequenced to determine the entire gene and amino acid sequence.

[0051] Under the premise of keeping the amino acid sequence unchanged, primers (5'-CGGAATTCATGGCCCGGGT-3' and 5'-GTGCGGCCGCACCTAGGA-3') were designed according to the gene sequence of the single-chain antibody B3, and a start codon (ATG ) and EcoRI restriction site, the 3' end primer added a NotI restriction site downstream of the stop codon. The phag...

Embodiment 3

[0056] Synthesize glutathione dibutyl ester with existing technology (see Example 1 of patent 99104234.4), take 50 μg of glutathione dibutyl ester, dissolve it with dimethyl sulfoxide (DMSO), and coat it on an immunoplate Above, the remaining sites were blocked with BSA. Using the coated glutathione dibutyl ester as the target antigen, the phage-displayed human single-chain antibody library was screened for 5 rounds by standard immunoaffinity screening method, and finally the positive clones were further screened by ELISA method to obtain the affinity for GSH. Higher specificity single chain antibody B3, sequenced to determine the entire gene and amino acid sequence.

[0057] The phage DNA was extracted, and the phage DNA containing the single-chain antibody B3 gene was used as a template, and the public primers of the antibody library (5'-CAGGAAACAGCTATGAC-3' and 5'-GAATTTTCTGTATGAGG-3') were used for PCR amplification of the single-chain antibody B3 gene. The PCR products w...

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Abstract

The invention discloses a method to prepare soluble human selenium-containing catalytic single-chain antibodies, belonging to biotechnology field. The method includes the following steps: with glutathione derivatives as target antigen, filtrating the recombinant phage display human single-chain antibody library through immune affinity selection method to obtain single-chain antibodies B3 and D8; assembling the single-chain antibodies to secretory procaryon or eucaryon expression vector; translating colon bacillus or yeast cell; expressing and purifying the single-chain antibody proteins in procaryon or eucaryon; introducing GPX catalytic group SeCys at the substrate combining sites of the antibodies through chemical mutation method or directly expressing the soluble single-chain antibody proteins which contain GPX catalytic groups at the substrate combining sites with auxotrophic colon bacillus through genetic mutation techniques to endue the antibodies with GPX catalytic activity. The method of the invention is simple and the human selenium-containing catalytic single-chain antibodies prepared with the method are of high catalytic activity; the proteins expressed by the antibodies are soluble; therefore the method is good for large-scale production and is of broad application prospect in biological pharmacy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing glutathione human source selenium-containing single-chain antibody enzyme by applying the technology of combining genetic engineering and chemical mutation. Background technique [0002] The substrate of selenium-containing glutathione peroxidase (GPX) is glutathione (GSH), and the catalytic group is selenocysteine ​​(SeCys). In organisms, GPX together with superoxide dismutase (SOD) and catalase (CAT) constitute the body's antioxidant defense system. GPX plays an important role in this system. It uses GSH as a reducing agent to decompose hydrogen peroxide and various hydroperoxides in the body, so it can remove reactive oxygen species (ROS) in the body and prevent lipid peroxidation. Various diseases caused by active oxygen, such as ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataracts, tumors, etc. Different from other an...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K1/107C12N15/13C12N15/70C12N15/81
Inventor 魏景艳徐俊杰霍锐李杰帅刘慧娟罗际巡罗贵民阎岗林李唯
Owner JILIN UNIV
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