Construction method of multigene carrier and its application

A construction method and multi-gene technology, applied in the direction of using vectors to introduce foreign genetic material, DNA preparation, recombinant DNA technology, etc., can solve the problem that there is no effective method for gene assembly

Inactive Publication Date: 2006-07-12
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although it is a well-known technology to use specific recombination system to carry out gene integration, the existing methods can only use this technology to carry out 1-2 rounds of gene integration, and there is no effective method for more than 2 rounds of gene assembly

Method used

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  • Construction method of multigene carrier and its application
  • Construction method of multigene carrier and its application
  • Construction method of multigene carrier and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1: accept the construction of carrier

[0103] Such as image 3 As shown, according to the sequence of transformable artificial chromosome vector pYLTAC7 (Liu et al., 1999), primers P1 and P2 containing XbaI and BamHI sites were synthesized (see image 3 15690bp vector backbone fragment was amplified by PCR method, the cohesive end was cut out with XbaI and BamHI, and then ligated with the synthetic double-stranded DNA fragment MCS-LoxP-I-SceI (sequence 1 in the sequence listing) to form Circular, transform Escherichia coli DH10B to obtain acceptor vector pYLTAC747.

Embodiment 2

[0104] Embodiment 2: Construction of supply vector I and supply vector II

[0105] Such as Figure 4 As indicated, primers P3 and P4 were synthesized according to the sequence of the chloramphenicol resistance gene (see Figure 4Description), from the plasmid pCAMBIA1200 (CAMBIA company) PCR amplified 826bp chloramphenicol resistance gene Cm. Primers P5 and P6 were synthesized according to the sequence of plasmid pBluescript SK (ClonTech Company), and a 1660bp Ori-MCS-LacZ fragment was amplified from plasmid pBluescript SK by PCR. The two fragments were connected into a circle, transformed into Escherichia coli DH10B, and an intermediate product plasmid pYL was obtained. Use the restriction endonuclease BssHII to excise the MCS in the plasmid pYL, connect it with the synthetic double-stranded DNA fragment LoxP-PI-SceI-MCS-I-SceI (sequence 2 in the sequence listing) into a circle, and transform Escherichia coli DH10B , to obtain a donor vector I plasmid, named pYLVS. A rest...

Embodiment 3

[0106] Embodiment 3: the assembly of multigene transformation vector

[0107] (1) Multiple cloning sites are set on the accepting vector pYLTAC747, and a few genes can be directly cloned into the vector by conventional molecular cloning methods. In this example, firstly, the hygrozyme gene HPT was directly subcloned into the NotI site in the cloning site of pYLTAC747. The resulting vector pYLTAC747HPT was as Figure 5 Swimming lane 2 is shown.

[0108] (2) Subcloning the MAR sequence (1.2 kb) into the donor vector I plasmid pYLVS to generate pYLVS-MAR. pYLVS-MAR and pYLTAC747-HPT were co-transformed into E. coli NS3529 competent cells containing Cre recombinase gene, so that the two plasmids were recombined and integrated in E. coli cells. The integrated plasmid was selected with kanamycin and chloramphenicol double antibodies, and then transformed into Escherichia coli DH10B without Cre enzyme gene. Use I-SceI to excise the pYLVS backbone, and use T4DNA ligase to ligate t...

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Abstract

A multi-gene carrier system is composed of a receptor carrier and at least two donor carriers, and is prepared by altermative gene assemblings with the said carriers. It can be used for conversion of multiple genes to obtain different genetically engineered products and multi-gene expression characteristics.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the construction and application of genetic engineering vectors. Background technique [0002] Gene transformation is the basic technology of biological genetic engineering. Existing gene transformation techniques are mainly used to introduce a small number, usually 1-3 genes, into biological cells. In recent years, people have tried to transform more genes, but limited by the existing technology, the construction of multigene vectors and multigene transformation are still very difficult and the efficiency is very low. [0003] Possible approaches to polygenic transformation in current genetic engineering practice are: [0004] (1) Mix multiple vector plasmids containing different genes, and perform co-transformation by gene gun bombardment and other methods (Chen et al., 1998; Ye et al, 2000). [0005] (2) Grouping multiple genes, such as 1-3 genes in each group, subcloning them ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/70C12N15/09C12N15/10C12N15/64C12N15/66
CPCC12N15/10C12N15/66C12N15/64
Inventor 刘耀光
Owner SOUTH CHINA AGRI UNIV
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