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Ribosome display technology-based rabbit monoclonal antibody preparation method

A monoclonal antibody and ribosome display technology, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, DNA preparation, etc., can solve the problems of large difference in antibody quality, unstable performance, and long time consumption

Inactive Publication Date: 2016-10-05
江苏佰润医疗科技有限公司 +1
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Problems solved by technology

However, this technology also has disadvantages: first, the cell fusion technology used is very cumbersome and time-consuming; second, the differences in individual animals lead to large differences in the quality of the prepared antibodies, like mouse monoclonal antibodies, the problem of unstable performance still exist; third, true high-throughput screening cannot be performed

Method used

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specific Embodiment approach

[0004] Specific embodiments: extract total RNA from immunized rabbit spleen, amplify antibody library gene by RT-PCR and construct antibody ribosome display template, perform in vitro transcription and in vitro translation of the constructed library, and target the target antigen , to screen out specific ribosome complexes. By changing the Mg~(2+) concentration, the screened mock antibody-ribosome-mRNA triple complex was dissociated to obtain the mRNA corresponding to the mock antibody. Through several rounds of biopanning, antigen-positive mock antibodies were enriched and highly specific antibodies were obtained. The antibody sequence was highly expressed in Escherichia coli, and the protein was verified as the target antibody by WB blotting. Because the simulated antibody library used is versatile, in principle, any antigen can be used as a target to screen its simulated affinity antibody, which is the specificity of using ribosome display technology to screen multiple ant...

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Abstract

The invention discloses a rabbit monoclonal antibody preparation method. The preparation method is characterized in that a ribosome display technology is adopted to prepare a rabbit monoclonal antibody, and the preparation method comprises the following steps: carrying out PCR amplification on the gene library of the target antibody, adding a promoter, a ribosome binding site and a stem loop, carrying out incubation in a cell-free translation system with couple transcription and translation to make the translation product of the target antibody gene be displayed on the surface of the ribosome and to form an mRNA-protein-ribosome ternary complex, directly screening the target ribosome from the ternary complex through an immobilized target molecule by using a routine immunological detection technology, carrying out next-cycle enrichment and amplification by using RT-PCR amplification, and finally screening the target antibody with high affinity. The preparation method of the rabbit monoclonal antibody has the following advantages: 1, the method is more convenient and fast than the prior art; 2, the screened antibody can be stably reduplicated; and 3, high-flux screening can be realized in order to obtain the antibody with high affinity.

Description

technical field [0001] The invention relates to a method for preparing a rabbit monoclonal antibody, which is characterized in that the rabbit monoclonal antibody is prepared by ribosomal antibody technology. Background technique [0002] In the 1990s, rabbit monoclonal antibodies, as a new generation of monoclonal antibodies, began to be used in scientific research and clinical diagnosis. The principle is to prepare monoclonal antibody by fusion of myeloma-like tumors obtained from transgenic rabbits and immunized rabbit spleen cells. The emergence of rabbit monoclonal antibodies has promoted the advancement of scientific research and clinical diagnostic technology: first, rabbit monoclonal antibodies usually have high affinity and can recognize more types of epitopes than the original mouse monoclonal antibodies; secondly, rabbit monoclonal antibodies Antibodies can recognize many antigens that do not produce immunity in mice; third, due to the larger spleen of rabbits, m...

Claims

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Application Information

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IPC IPC(8): C12N15/13C12N15/10
Inventor 不公告发明人
Owner 江苏佰润医疗科技有限公司
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