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Baculovirus expression system and construction method and application thereof

A baculovirus and expression system technology, applied in the field of protein expression, can solve problems such as time-consuming, labor-intensive, reduced work efficiency, and cumbersome operations

Inactive Publication Date: 2020-10-23
YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

At the same time, this is not conducive to calculating the precise amount of Bacmid DNA added in the baculovirus packaging process, resulting in a non-optimal packaging system
Although purified transposition recombination products can be screened for purified strains by electroporation again, but this process is cumbersome, time-consuming and laborious, which greatly reduces work efficiency

Method used

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  • Baculovirus expression system and construction method and application thereof
  • Baculovirus expression system and construction method and application thereof
  • Baculovirus expression system and construction method and application thereof

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Embodiment 1

[0061] 1. Construction of baculovirus expression vector system

[0062] The common replicons of pFastBac and pHelper were replaced by the temperature-sensitive pSC101ori replicons by the Gibson cloning method, and then the ccdA expression cassette was inserted on the basis of the temperature-sensitive helper plasmid to construct optimized donor vectors pFastBac(ts) and Helper vector pHelper(ts+ccdA).

[0063] Such as figure 1 As shown, the homologous recombinase expression plasmid pRed / ET (tetracycline-resistant Tc) was electrotransferred into the competent DB3.1 containing only the Bacmid vector (Kana-resistant Kan), added to SOC culture and incubated at 30°C for 1 hour .

[0064] The resuscitated culture products were spread on LB plates containing Tc+Kan antibiotics, cultured at 30°C for two days, and positive clones containing both pRed / ET and Bacmid vectors were identified by PCR.

[0065] Pick positive clones and inoculate them into LB liquid medium containing Tc+Kan ...

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Abstract

The invention relates to a baculovirus expression system as well as a construction method and application thereof. The system comprises a baculovirus shuttle vector, an auxiliary vector and a donor vector; the baculovirus shuttle vector contains a Tn7 target site and a toxin protein gene expression cassette; and the auxiliary vector contains an antitoxin protein gene expression cassette, the antitoxin protein can be combined with the toxin protein so as to neutralize the toxicity of the toxin protein, replicons of the auxiliary vector and the donor vector are temperature-sensitive replicons, and the donor vector comprises left and right flanking sequences of a transposon Tn7. By utilizing the baculovirus expression system disclosed by the invention, a high-purity pollution-free recombinantpositive baculovirus vector which can be directly used for downstream baculovirus packaging can be rapidly and directly extracted.

Description

technical field [0001] The invention relates to the technical field of protein expression, in particular to a baculovirus expression system and its construction method and application. Background technique [0002] Autographa californica multiplenuclear polyhedrosis virus (AcMNPV)-based insect baculovirus expression vector system (Baculovirus Expression Vector System, BEVS) is a large-scale expression of foreign recombinant proteins in insect cells. One of the most versatile and powerful eukaryotic expression systems of , has been widely used since the first large-scale production of IL-2 protein was achieved in this system in 1985. Compared with other recombinant protein expression systems, the recombinant baculovirus expression system has obvious advantages: 1) high safety: the host is limited to specific invertebrates, and it is not pathogenic to mammals and plants; 2) easy to expand production Scale: The initially obtained recombinant baculovirus can be used to infect m...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N5/10C12P21/00
CPCC12N15/86C12N2710/14043C12N2820/002C12P21/00
Inventor 施金秀罗燕林映君蒙伟能叶知晟蓝田
Owner YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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