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Method for building polypeptide library by phagemid display system

A display system and phagemid technology, which is applied in the fields of biotechnology and biopharmaceuticals, can solve problems such as difficult operation and small fragments, and achieve the effects of convenient construction, increased display efficiency and stability

Pending Publication Date: 2019-07-26
艾柏森(江苏)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems of small fragments and difficult operation in the current phage display polypeptide technology, the present invention develops a polypeptide library construction method that is not limited by the size of the displayed fragments

Method used

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  • Method for building polypeptide library by phagemid display system
  • Method for building polypeptide library by phagemid display system
  • Method for building polypeptide library by phagemid display system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Example 1: Taking the construction of a dodecapeptide library as an implementation case, the construction method is described in combination with specific steps and data, and the construction method of a phagemid display random dodecapeptide library:

[0022] 1) Gene synthesis of random dodecapeptides:

[0023] Twelve random peptides adopt (NNB)12, (NNM)12, (NNV)12 and other coding methods (N represents the four bases of A, T, G, and C, B represents the three bases of G, T, and C, and M Indicates two bases of A and C, and V indicates three bases of G, A, and C), and some bases of the sfiI restriction site are added to the upstream of the twelve random peptide gene sequence as the linker of the upstream primer for the synthesis of the target gene fragment (adaptor), the base sequence of the upstream part of the linker (linker) is added downstream of the twelve random peptide gene sequence as the adapter (base complementary pairing sequence) connected with the linker-s...

Embodiment 2

[0040] 2. Example 2: Taking the construction of a random heptapeptide library as an implementation case, the construction method is described in combination with specific steps and data. The construction method of the phagemid display random heptapeptide library:

[0041] 1), gene synthesis of random heptapeptide:

[0042] The heptapeptide random peptide adopts (NNB)7, (NNM)7, (NNV)7 and other encoding methods, and some bases of the sfiI restriction site are added to the upstream of the heptapeptide gene sequence as the adapter for the upstream primer of the synthetic target gene fragment, and the heptapeptide The base sequence of the upstream part of the linker is added downstream of the gene sequence as an adapter connected downstream with the linker-sumo, and the final sequence form is 5'-CCAAGCGGCC-(NNB)7\(NNM)7\(NNV)7-GGCGGCGGTAGCG.

[0043] Step 2)-step 9) refer to step 2)-step 9) in embodiment 1.

Embodiment 3

[0044] 3. Example 3: Taking the construction of a random nonapeptide library as an implementation case, the construction method is described in conjunction with specific steps and data. The construction method of the phagemid display random nonapeptide library:

[0045] 1), gene synthesis of random nonapeptide:

[0046] The nonapeptide random peptide adopts (NNB)9, (NNM)9, (NNV)9 and other coding methods, and some bases of the sfiI restriction site are added to the upstream of the nonapeptide gene sequence as the adapter for the upstream primer of the synthetic target gene fragment, nonapeptide The base sequence of the upstream part of the linker is added downstream of the gene sequence as an adapter connected downstream with the linker-sumo, and the final sequence form is 5'-CCAAGCGGCC-(NNB)9\(NNM)9\(NNV)9-GGCGGCGGTAGCG.

[0047] Step 2)-step 9) refer to step 2)-step 9) in embodiment 1.

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Abstract

The invention discloses a Method for building a polypeptide library by a phagemid display system. According to the method, polypeptide is connected with a fixed sequence, restriction enzyme cutting sites are inserted at two ends and connected with a carrier, fragment recovery rate and display efficiency are greatly improved, a flexible linker is added between the polypeptide and the fixed sequence, displayed polypeptide keeps better flexibility, steric hindrance is reduced, and functions cannot be affected. A method for building the polypeptide library cannot be limited by the size of the polypeptide, the fixed sequence is increased, the length of a subject sequence is indirectly increased, and displayed random peptide cannot be affected by the fixed sequence under the action of the flexible connection linker. Several to dozens of polypeptide libraries can be built and can apply to screening of drug targets, epitope analysis and the like.

Description

technical field [0001] The invention relates to the fields of biotechnology and biopharmaceuticals, and relates to a method for preparing a polypeptide library through a phage display system. Background technique [0002] Random peptide libraries play an increasingly important role in the study of protein ligand / receptor interactions, the analysis of enzyme substrates, the search for mimetic peptides of proteins with biological functions, and the screening of new drugs. There are two main categories of methods for constructing peptide libraries. The first type of method is in vitro chemical synthesis, and the second type of method is to use synthetic random oligonucleotides to express soluble fusion proteins in vivo, but the above two types of methods have their limitations. Although the design of artificially synthesized peptides in vitro is relatively simple, expensive instruments and complicated methods are used, which is time-consuming, laborious, and costly, and the sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/10
CPCC40B50/06C40B40/10
Inventor 贺建望谢朋辉
Owner 艾柏森(江苏)生物科技有限公司