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 inserti

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