Cloning vector pGreen-S based on enhanced green fluorescent protein gene deletion as screen marker and construction method thereof

Abstract

The invention discloses a cloning vector pGreen-S based on enhanced green fluorescent protein, (EGFP) gene deletion as a selection marker, and a construction method thereof. The pGreen-S vector inserts an EGFP gene into an Xba I restriction enzyme site of a pUC18 vector, and sequentially comprises main components as follows: a replication origin sequence, an EGFP gene upstream polyclone site, an EGFP gene sequence, an EGFP gene downstream polyclone site, a lacZ gene sequence and a resistance selection gene sequence. The pGreen-S vector can be used for screening gene recombinants, is more reliable in screening results compared with a lac blue / white spot screening method, and has the characteristics of convenience, economic property, time conservation and high efficiency.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a cloning vector pGreen-S based on enhanced green fluorescent protein gene deletion as a screening marker and a construction method thereof. Background technique [0002] The β-galactosidase gene (lacZ) is the most commonly used reporter gene in molecular biology, such as the pUC series is the lacZ insertion inactivation vector. The screening principle is: the vector contains the DNA segment of the lacZ operon and the gene segment (α peptide segment) encoding the N-terminal 146 amino acids of β-galactosidase, and a multiple cloning site is formed in the coding region at the same time, and does not affect the reading. Coding boxes and gene function. The chemical isopropyl-β-D-thiogalactoside (IPTG) induces the synthesis of this fragment. Select α-peptide-deficient host bacteria (which can synthesize the rest of the peptides at the C-terminal by itself), a...

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Problems solved by technology

[0003] However, in practical applications, the problem with this method is that when the inserted DNA fragment (smaller) cannot destroy the reading frame of the N-terminal fragment of β-galactosidase, the recombinant colony may show light blue or even blue Color, false negative clones appear, the misselection rate is about 30%

Acta Bot, 1999, 41 (5): 487-489], there are obvious deficiencies in the carrier formed based on the GFP expression strategy: first, the foreign gene insertion sites are selected upstream of the GFP gene, and the screening strategy is based on whether the inserted gene Resulting in a frame shift of the GFP gene reading frame and affecting the expression of GFP to screen recombinants, the inserted gene sequence is strictly required, and once the requirements cannot be met, recombinants cannot be screened. , fewer restriction endonuclease sites, reducing the flexibility of gene recombination; thus, its utility is significantly limited

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