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Baculovirus expression vector capable of quickly determining titer and construction and application thereof

A technology of baculovirus and expression vector, which is applied in the field of bioengineering, can solve the problems of time-consuming baculovirus titer, etc., and achieve the effect of fast determination of virus titer by terminal dilution method and shortening the detection cycle

Active Publication Date: 2017-03-15
GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 quickly determining titer and construction and application thereof
  • Baculovirus expression vector capable of quickly determining titer and construction and application thereof
  • Baculovirus expression vector capable of quickly determining titer and construction and application thereof

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Experimental program
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Effect test

Embodiment 1

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

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

[0053] Construct a recombinant linear fragment with enhanced green fluorescent protein, including: the downstream fragment L of the baculovirus plasmid p74 gene...

Embodiment 2

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

[0077] 2. Preparation of cell plate: Sf9 cells were cultured in suspension in a shaker flask with SF900Ⅱ serum-free medium, the rotating speed of the shaker was 100 rpm, and the culture temperature was 27°C. Sf9 cells in the logarithmic growth phase in suspension culture are diluted with SF900Ⅱ serum-free medium, and the diluted cell liquid is 2×10 4 Spread the insect cell suspension into 96-well cell culture plate, 100 μl / well, and place it in a 27°C incubator for static culture, so that Sf9 cells are attached to the bottom of the well for use.

[0078] 3. After the recombinant baculovirus is diluted gradually, take 10 -1 ~10 -6 A total of 6 dilutions of baculovirus solution wer...

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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 that can quickly determine the titer and its construction and application. Background technique [0002] The baculovirus expression system is a widely used eukaryotic expression system. Compared with other protein expression systems, it has many advantages, such as protein post-translational modification. There are many commercialized baculovirus expression systems. Among them, Life's Bac-to-Bac system is the most commonly used, which has successfully expressed thousands of proteins. [0003] Compared with traditional methods, the use of Bac-to-Bac expression system to obtain recombinant baculovirus can reduce the time of two weeks. However, according to the Bac-to-Bac system operating manual, it takes nearly a week to determine the titer of recombinant baculovirus. Determining virus titer is a very important s...

Claims

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Application Information

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Patent Type & Authority Applications(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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