Site-specific recombination-based tetrahymena thermophile expression carrier and construction and application thereof

Abstract

The invention discloses a site-specific recombination-based tetrahymena thermophile expression carrier and construction and application thereof. The carrier is constructed by comprising the following steps: with plasmid pNeo4 as a skeleton, and combined serially connected HA tag sequence, restriction enzyme digestion site and tetrahymena thermophile ACTIN1 gene transcription termination sequence as modules, digesting and inserting module synthesis and digestion sites into 5'-terminal polyclonal digestion site of pNeo4, so as to obtain an expression carrier pNeo4-3HA. In use, the carrier is amplified to obtain recombinant homologous arms, namely target gene coding region C-terminal sequence and 3' flanking sequence thereof, which are subjected to enzyme-cut and connection into the 5' upstream and 3' downstream polyclonal digestion site of pNeo4-3HA. The obtained construction body is subjected to transformation of tetrahymena thermophile and resistance screening to obtain an expression mutant cell line. The carrier realizes the purpose that the C-terminal expresses the endogenous gene of a fusion tag under the regulation of self starters, the operation is simple and rapid, the immunofluorescence localization sensitivity can be enhanced through 3 HA tags, and the site-specific recombination-based tetrahymena thermophile expression carrier can also be used for protein affinity purification and protein interaction studies.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a site-specific recombinant expression vector pNeo4-3HA containing three HA tags in series, and a construction method and application thereof. Background technique [0002] The expression vector of the target protein fusion tag is widely used in the study of protein localization, interaction and function. In the model organism - ciliated thermophilic Tetrahymena, the fusion tag can be efficiently recombined into the endogenous gene locus through homologous recombination, so the protein expression vector of the fusion tag is an extremely useful method in Tetrahymena. molecular tools (Cassidy-Hanley et al, 1997). However, there are very few such construction vectors that can be used at present. Currently, there are two methods for constructing target gene fusion tag constructs: one method is to construct an expression construct of target gene fusion tag under the regula...

AI Technical Summary

Problems solved by technology

These methods are complex in the construction of recombinant plasmids, often need to use multiple pairs of primers and long overlapping PCR primers, resulting in increased economic costs and therefore cannot increase the number of tags, which may be due to many target genes under the regulation of their own promoters The level of expression is not high and the number of tags is small, so that the antibody affinity is low, and finally a clear localization signal cannot be obtained; another method is to construct an overexpression construct of the target gene, which can make the target gene In the metal ion Cd 2+ Under the induction, it is overexpressed under the regulation of the MTT1 promoter, so as to observe the localization pattern of the target gene in Tetrahymena cells (Shang et al, 2002; Zweifel et al, 2009). The construction method is simple and easy to operate. Overexpression of the target gene can improve the clarity of the localization signal of the low-expression target gene and find localization details, but it may also cause cell development to be blocked or non-specific localization due to overexpression of the gene

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