Visual tracking method for rapid expression of NS1 protein by bombyx mori bioreactor

Abstract

The invention relates to a visual tracking method for rapid expression of an NS1 protein by a bombyx mori bioreactor. The method comprises the steps: a donor plasmid carrying an egfp expression cassette and an nsl gene expression cassette is constructed, an scherichia coli DH10B competent cell is converted by the donor plasmid, the egfp expression cassette and the nsl gene expression cassette which are between Tn7R-Tn7L of sequences of the donor plasmid can be specifically transposed onto a Bm-Bacmid vector, an integrated recombinant eukaryotic expression vector for expressing an egfp gene and an nsl gene is constructed, a bombyx mori is infected by recombinant buddedvirus BV particles produced after insect cells are transfected with the integrated recombinant eukaryotic expression vector, and real-time monitoring of a situation of virus multiplication in a bombyx mori body is carried out through visual green fluorescent signals. The method can perform large-scale and real-time monitoring of expression dynamic situation of an exogenous gene in the bombyx mori body, and lays the foundation for revealing characteristics of the NS1 protein and other functional proteins.

Description

technical field [0001] The invention relates to genetic engineering technology, and discloses a construction process and application of a silkworm bioreactor visualization tracking expression system. Specifically, it refers to a preparation method of recombinant budding virion BV, and the use of this virus to rapidly and efficiently express the non-structural protein NS1 of the silkworm bipartite densovirus in the silkworm bioreactor, which is the subsequent function and function of the NS1 protein. This method is also applicable to the expression of other foreign genes and recombinant protein drugs with important functions. Background technique [0002] The baculovirus-insect cell expression system (BEVS) is one of the four major expression systems for genetic engineering today. Most of the baculovirus expression vectors used are the A. californica nuclear polyhedrosis virus (AcNPV) or the silkworm nuclear polyhedrosis virus. body virus (BmNPV). Among many expression syst...

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Problems solved by technology

[0005] However, the genome of Bm-Bacmid is relatively large, and it is replicated in E. coli DH10B cells in a low-copy form. Secondly, the efficiency of liposome-mediated recombinant Bm-Bacmid transfection in BmN cells is relatively low. At the same time, research It shows that there are some proteins with special physical and chemical properties that are often difficult to express or have low expression levels in BEVS. Therefore, it is a good idea to quickly identify whether recombinant virions are produced in cells transfected with recombinant Bm-Bacmid. a key factor in expressing

In the past, in the process of eukaryotic expression, the expressed target protein could not be detected by Western blot, which made us doubt the design and process of the whole experiment, especially the recombinant Bm-Bacmid transfected into BmN cells, in the cell culture supernatant Are infectious recombinant budding virions produced? Because this is not only related to the rapid expression of the target gene in BEVS, but also a bottleneck restricting the research on the function and structure of the target protein

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