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Baculovirus expression vector capable of rapid titer determination and its construction and application

A baculovirus and expression vector technology, applied in the field of bioengineering, can solve the problem of long time-consuming baculovirus titer, and achieve the effect of rapid determination of virus titer by endpoint dilution method and shortened detection period.

Active Publication Date: 2020-03-17
GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] An object of the present invention is to solve the time-consuming problem of determining the titer of baculovirus, and provide a baculovirus expression vector that is more economical, more convenient to operate and does not affect the expression ability of viral proteins

Method used

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  • Baculovirus expression vector capable of rapid titer determination and its construction and application
  • Baculovirus expression vector capable of rapid titer determination and its construction and application
  • Baculovirus expression vector capable of rapid titer determination and its construction and application

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Embodiment 1

[0051] Materials Sf9 cells are a commonly used insect cell, purchased from Invitrogen; SF900Ⅱ serum-free medium is a product of Gibco; Bac-to-Bac system containing donor plasmid pFastBac1 was purchased from Invitrogen LifeScience; Escherichia coli strain (DH5α) The Escherichia coli strain DH10Bac (including parental BacmidbMON14272 and auxiliary plasmid pMON7124) was purchased from Invitrogen Life Science; the DH10B strain (DH10B-pAcWt-gbaA strain) containing bMON14272 plasmid and pBAD-gbaA plasmid was donated by Sun Yat-sen University. The plysS plasmid (containing the chloramphenicol resistance gene) and the pEGFP-C2 plasmid (containing the enhanced green fluorescent protein gene) were preserved by the inventor's laboratory.

[0052] 1) Construction of baculovirus expression vector, see figure 1

[0053] Construction of recombinant linear fragments with enhanced green fluorescent protein, including: downstream fragment L of bacmid p74 gene, fusion fragment E of ie-1 promote...

Embodiment 2

[0076] 1. Construction of recombinant baculovirus containing CD4 gene: use the baculovirus expression vector constructed by this patent to replace the original parent Bacmid bMON14272 of the Bac to Bac system of Invitrogen Company, and the rest of the construction steps are the same as the Bac to Bac operation manual.

[0077] 2. Preparation of cell plates: Sf9 cells were cultured in suspension in shake flasks with SF900II serum-free medium, the speed of the shaker was 100 rpm, and the culture temperature was 27°C. Sf9 cells in the logarithmic growth phase and in suspension culture were diluted with SF900Ⅱ serum-free medium, and the diluted cell solution was diluted with 2×10 4 The / mL insect cell suspension was spread into a 96-well cell culture plate, 100 μl / well, and placed in a 27°C incubator for static culture, so that the Sf9 cells were attached to the bottom of the well, and set aside.

[0078] 3. After gradient dilution of the recombinant baculovirus, take 10 -1 ~10 ...

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Abstract

The invention discloses a baculovirus expression vector capable of quickly determining titer and construction and application thereof. The vector is named BacG, and is obtained by inserting a green fluorescent protein gene regulated by a baculovirus extremely early promoter ie-1 on a nonessential gene position on a genome of a baculovirus expression vector Bacmid bMON14272. The baculovirus expression vector keeps a complete green protein expression ability of the Bacmid bMON14272, meanwhile because of recombinant baculovirus produced by the baculovirus expression vector, the tilter of the baculovirus can be quickly determined by observing the expression of the green fluorescent protein under a fluorescence microscope, and thus the time for determining the tilter of the recombinant baculovirus is shortened to 24 hours.

Description

technical field [0001] The invention relates to the technical field of bioengineering. More specifically, the present invention relates to a baculovirus expression vector capable of rapid titer determination and its construction and application. Background technique [0002] Baculovirus expression system is a widely used eukaryotic expression system, which has many advantages compared with other protein expression systems, such as protein post-translational modification and so on. There are many commercialized baculovirus expression systems. Among them, Life's Bac-to-Bac system is the most commonly used one, and has successfully expressed thousands of proteins. [0003] Compared with the traditional method, using the Bac-to-Bac expression system to obtain recombinant baculovirus can reduce the time by two weeks. However, according to the Bac-to-Bac system operating manual, it will take nearly a week to determine the titer of the recombinant baculovirus produced. Determina...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/866C12N7/01C12N15/66C12Q1/70C12Q1/06C12R1/93
CPCC12N7/00C12N15/66C12N15/86C12N2710/14021C12N2710/14043C12N2710/14051C12Q1/06
Inventor 吴无畏刘钰
Owner GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
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