Method for connecting DNA molecule fragment with carrier and application thereof in construction of high-capacity antibody library

A DNA molecule and fragment technology, applied in the field of DNA molecule fragment and carrier connection, can solve the problem of insufficient diversity of CDR regions, and achieve the effects of high-efficiency and high-quality connection, simple components, and high-efficiency electroshock transformation.

Inactive Publication Date: 2010-01-06
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Abstract
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Problems solved by technology

However, because the antibody library constructed by this method only expands the library capacity through the

Method used

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  • Method for connecting DNA molecule fragment with carrier and application thereof in construction of high-capacity antibody library
  • Method for connecting DNA molecule fragment with carrier and application thereof in construction of high-capacity antibody library
  • Method for connecting DNA molecule fragment with carrier and application thereof in construction of high-capacity antibody library

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Embodiment Construction

[0025] The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0026] The present invention takes the construction of a large-capacity fully synthetic phage single-chain antibody library as a specific example, and aims to provide an efficient two-step enzyme digestion and ligation method, which can be used to construct various large-capacity antibody libraries and other protein libraries.

[0027] 1. Construction of the connection carrier PHB-pIX-EcoR used in the present invention

[0028] PHB-pIX display vector (such as figure 1 ) is transformed on the basis of the phage coat protein pIX protein display vector PHB1HCSFV preserved in the laboratory, which is derived from the phage display vector pAK600. First, the pIX protein coding...

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Abstract

The invention discloses a method for connecting a DNA molecule fragment with a carrier. The carrier comprises at least three different restriction enzyme cutting sites E1, E2 and E3, wherein the E2 is located between the E1 and E3; the DNA molecule fragment to be connected at least comprises the restriction enzyme cutting sites E1 and E3. The method comprises the following steps: firstly carrying out enzyme cutting and dephosphorylation on the restriction enzyme cutting sites E1 and E2 of the carrier; and then carrying out a first step of the enzyme cutting and connection on the restriction enzyme cutting site E1 of the DNA molecule fragment to obtain a linear connection product of the DNA molecule fragment and the carrier, and then recycling the connection product with a correct size; finally carrying out a second step of the enzyme cutting and connection on the restriction enzyme cutting site E3 of the connection product to obtain a self-connected annular connection product. The invention can realize the effective connection between the DNA molecule fragment and the carrier, so that the generation of incorrect connected polymers with different forms is reduced, the proportion of the correct connected product accounts for over 30% in the connection system so as to realize an efficient electro-transformation.

Description

technical field [0001] The invention relates to a method for linking DNA molecular fragments to a carrier and its application in the construction of a large-capacity antibody library. Background technique [0002] DNA molecular ligation has always been a key link in the construction of recombinant plasmids. Due to the limitation of ligation efficiency, it is difficult to obtain some recombinant clones. Especially in the process of constructing various DNA molecular libraries, ligation efficiency is particularly important. Therefore, improving the connection efficiency is of great significance for increasing the number of transposons, especially in the process of constructing libraries including antibodies, polypeptides and various other proteins by means of genetic engineering. [0003] In recent years, the use of phage antibody library technology to prepare various diagnostic and therapeutic antibodies has become an important technical means in the field of genetic engineer...

Claims

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Application Information

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IPC IPC(8): C12N15/66C40B40/08
Inventor 孙志伟王双俞炜源杜威世余云舟
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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