Polygene vector system and application thereof

Abstract

The invention belongs to the field of molecular biology, and discloses a polygene vector system and application thereof. The multi-gene vector system comprises a receiving vector p32RR, a linker donor vector pP1dor and a gene donor vector pGemor, BP or LR clonase recombinase is utilized to catalyze gene recombination exchange reaction between the gene donor vector and the receiving vector, and a target gene is exchanged to the receiving vector; and performing enzyme digestion and connection on the joint donor vector and the accepting vector containing the target gene by using restriction enzyme digestion and ligase, forming paired joints recognized by BP or LR clonase recombinase by the connected accepting vector containing the target gene, and performing a second round of recombination exchange reaction on the accepting vector containing the target gene and the gene donor vector. The steps are repeated in a circulating manner, so that any multiple target genes can be connected to the receiving carrier in series.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a multigene carrier system and its application. Background technique [0002] Gene carrier is one of the important tools of genetic engineering. Traditional vector construction methods include restriction endonuclease and DNA ligase enzyme-cut ligation vector construction method; recombinase-catalyzed homologous recombination exchange construction method. [0003] The enzyme-cut ligation vector construction method relies on the multiple cloning sites on the vector. Only when the multiple cloning sites on the vector and the target gene match, can the target gene and the vector be ligated after enzyme digestion. However, the number of multiple cloning sites on the vector is limited, so the enzyme-cut ligation method can only meet the vector assembly of a single or a few genes. Homologous recombination methods include λ-att system and Cre-LoxP system. The att site in ...

AI Technical Summary

Problems solved by technology

However, this system still has some disadvantages, such as the low efficiency of Cre / LoxP reaction, many side reactions, and the reaction cannot be carried out in vitro. It is necessary to use NS3529 competent cells that produce Cre recombinase; the recombination reaction occurs in NS3529 cells, and the subsequent positive The screening of clones is relatively complicated. It is necessary to use a special homing endonuclease (homing ednonucleas) to digest the transformation vector and then carry out the blue and white spot screening for the second transformation. Re-identification of colony PCR for white spots is required

Therefore, two conversions are required to use this system, and the whole operation process requires high experimental experience of the experimenter, and the technical threshold is high

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