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

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...

AI Technical Summary

Problems solved by technology

However, because the antibody library constructed by this method only expands the library capacity through the exchange of light and heavy chains, and has insufficient diversity in the CDR region, it has certain defects.

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