ST cell lines for stably expressing T7 RNA polyase, constructing process and applications thereof

Abstract

The invention discloses a cell line ST/T7 capable of stably expressing T7RNA polymerase, the microorganism preservation number of which is: CGMCC No.24444. In the invention, a T7RNA polymerase gene is inserted into a eukaryotic expression vector pIRES2-EGFP to get the pIRES2-EGFP-T7 to transfect ST cells to obtain the cell line ST/T7 capable of stably expressing T7RNA polymerase. Through RT-PCR detection, expression plasmid containing a red fluorescent protein gene controlled by T7 promotors can be used to transfect the cell line so as to observe the expression of red fluorescent protein in a transcription product amplified form a ST/T7 cell to the T7RNA polymerase. The cell line ST/T7 can provide reverse T7RNA polymerase for use in reverse genetic manipulation of RNA-virus such as hog cholera virus, etc., and can be used as transcription and expression systems in vitro for research on genic structures and functions.

Description

technical field [0001] The invention relates to an ST cell line, in particular to an ST cell line capable of stably expressing T7 RNA polymerase. The invention also relates to a construction method and application of the cell line, belonging to the field of biotechnology. Background technique [0002] More than ten years ago, a reverse genetic manipulation technology emerged in the field of molecular virology research, that is, the infectious full-length cDNA cloning technology of viruses, which solved the difficult problem of RNA virus genome manipulation in vitro. The reverse genetic system of RNA virus is to use the genome of the virus to rescue the live virus or its derivative virus in cultured cells or susceptible hosts. Genomic clones capable of rescuing viruses are called infectious clones. Generally, a copy of the entire viral genome cDNA is inserted into a bacterial plasmid, so that the cDNA itself or the RNA transcribed from the cDNA in vitro is infectious. There ...

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Problems solved by technology

Moormann et al. (1996) took the lead in using one-step method to transcribe infectious RNA from the full-length cDNA of CSFV rabbitized attenuated vaccine strain (C strain), but its infectivity is low (Moormann RJM, van Gennip HGP, Miedema GKW, et al. al. Infectious RNA from an engineered full-length cDNA template of the genome of apestivirus. J Virol, 1996, 70: 763-770.)

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