Exhibiting method of preparing antibody by antigen utilizing bacteriophage exhibiting technique

A phage display and phage technology, applied in the field of genetic engineering, can solve the problems of small molecule peptide immunogenicity, small molecular weight, short half-life, etc.

Inactive Publication Date: 2004-03-31
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for some rare antigens with small molecular weight and short half-life, such as certain hormones and their metabolites, certain tumor markers, etc., it brings difficulties to antigen purification and antibody preparation
Genetic engineering can effectively solve this problem. For example, the general expression system can solve the production and preparation

Method used

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  • Exhibiting method of preparing antibody by antigen utilizing bacteriophage exhibiting technique
  • Exhibiting method of preparing antibody by antigen utilizing bacteriophage exhibiting technique
  • Exhibiting method of preparing antibody by antigen utilizing bacteriophage exhibiting technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0012] 1. Construction of phage display vector

[0013] wxya 3 Excision of the second cloning site

[0014] wxya 3 Digested with NheI+XbaI, a 272bp band can be seen. After excising the above fragments, recover the remaining large fragments, transform and extract the plasmids after self-ligation, and check with NheI, XbaI, SpeI, XhoI and XhoI+SpeI respectively. The results showed that XbaI and NheI had no enzyme digestion, SpeI and XhoI could see a single enzyme digestion band, and the enzyme digestion site was correct.

[0015] Fragment insertion and identification

[0016] pET-28(a) + (stored in the Biotechnology Research Center of China Pharmaceutical University) After PCR amplification as a template, a 550bp band can be seen. After the fragment is recovered, it is connected to the same Pa carrier with the same enzyme digestion. After transformation, the positive plasmid is screened, and the 1.5% agarose electrophoresis band appears in After Pa. 10 single colonies were...

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Abstract

The invention comprises, fusing exogenous antigen genes and filobactivirus III gene proteins and expressed onto the tail of filobactivirus, fusing antigen epitopes and filobactivirus III gene proteins and expressed onto the surface of filobactivirus, preparing antibodies taking filobactivirus with exogenous antigen and antigen epitopes as the immunogen. The invention claims:1)a method of preparing monoantibodies and polyantibodies based on filobactivirus showing technology;2) a method of preparing cTnI monoantibody and polyantibody based on filobactivirus showing technology showing antigen and antigen epitopes of human troponin; 3) a PIII filobactivirus showing carrier pFD5; 4) a filobactivirus showing fusion expressing carrier pFD5-cTnI;5) a PIII filobactivirus showing fusion expressing carrier PC89-cTnI[95??08];6)taking filobactivirus particles showing cTnI and cTnI[95í½108] as immunogens, immunizing new zealand white rabbit to get cTnI antibody.

Description

1. Technical field [0001] The invention belongs to the technical field of genetic engineering. This technology uses the principle of phage display technology to display the fusion of exogenous antigen gene and filamentous phage III gene protein on the tail of phage particle, and display the fusion of antigen epitope and filamentous phage VIII gene protein on the surface of filamentous phage particle. Antibodies are prepared by using phage particles displaying foreign antigens and antigenic epitopes as immunogens. 2. Background technology [0002] In the past 20 years, antibodies have been widely developed as in vitro diagnostic drugs in the fields of medicine and biology. In the past half century, detection technologies based on the principles of enzymatic reaction kinetics, chemiluminescence, ligand-receptor combination, etc., especially based on the three major protein labeling technologies (fluorescence, nuclides, enzymes) And the establishment of specific and sensitive...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K16/18C12N15/63C12P21/00C12P21/08
Inventor 沈子龙吴国球
Owner CHINA PHARM UNIV
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