Homonymous tandem co-expression dual-color fluorescent protein reporter gene vector (independent fusion expression type)

Abstract

The invention relates to a homonymous tandem co-expression dual-color fluorescent protein reporter gene vector pDsRed2-IRES-EGFP substantive fusion (independent fusion expression type). On the basis of pIRES2-EGFP, the homonymous tandem co-expression dual-color fluorescent protein reporter gene vector pDsRed2-IRES-EGFP is prepared by subjecting DsRed2 and IRES to overlap extension PCR and gene subcloning. The target gene is cloned into DsRed2 3' end MCS1 zone or EGFP5' MCS2 zone of the homonymous tandem co-expression dual-color fluorescent protein reporter gene vector, and the homonymous tandem co-expression dual-color fluorescent protein reporter gene vector is used for cell transfection; transcription of mRNA containing DsRed2 or DsRed2 and 3' end fusion target gene, IRES and EGFP or EGFP and 5' end target gene is initiated by pCMV; translation of the section containing DsRed 2 is initiated by 5' cap sturcture; independent translation of the section containing EGFP is mediated by IRES. Fusion DsRed2 or EGFP in maturation proteins can be carried by target gene products so as to realize gene delivery and distribution in cells, and the tracking of the process. The optimized two MCS sequences are suitable for wider range of exogenous gene segment cloning operation; the detection aging ranges of DsRed2 and EGFP are wide, are mutually independent and are mutually characterized; and the imaging effect is clear and bright, so that the experimental operation is more convenient, and efficient.

Description

technical field [0001] The present invention relates to co-expressing two-color fluorescent protein reporter gene carrier (independent fusion expression type) in series in the same direction, which is a carrier plasmid used for expressing target gene fragments and locating their distribution in cells. Background technique [0002] In view of the characteristics of the expression product of fluorescent protein gene in living cells, such as stable biological activity, strong signal specificity, long effective activity maintenance time, convenient detection and little interaction with fusion common exogenous gene, it is often used as a biological screening marker in genetic engineering. , to study the expression of exogenous target genes and the intracellular distribution and localization of their protein products. Usually, the same reporter gene vector contains only one fluorescent protein gene, and the target DNA fragment is cloned into the N-terminal or C-terminal of the flu...

AI Technical Summary

Problems solved by technology

Usually, the same reporter gene vector contains only one fluorescent protein gene, and the target DNA fragment is cloned into the N-terminal or C-terminal of the fluorescent protein gene, and the constructed recombinant plasmid is transfected into the corresponding host cell, and the expression of the fluorescent protein can be detected. Determine whether the target nucleic acid fragment has the corresponding function, but it is difficult to express at the N-terminal or C-terminal of the fluorescent protein gene at the same time, and it is also difficult to simultaneously reflect the transfection efficiency of the recombinant reporter gene plasmid and determine the biological activity of the target gene

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