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Method used for rapidly identifying generation of recombinant virus particles in eukaryotic expression

A technology of recombinant virus and eukaryotic expression vector, applied in the field of biochemistry and molecular biology, can solve the problems of labor consumption, waste of reagents, time-consuming and so on

Inactive Publication Date: 2013-08-28
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the past, in the experiment process of BEVS expressing foreign genes, the expression of the target protein was not identified by Western blot, we would doubt the experimental design and each step in the experimental process, especially whether the recombinant Ac-Bacmid transfection was successful, repeated It is time-consuming, labor-intensive, and wastes reagents to check each step in the experiment by techniques such as RT-PCR and Western blot

Method used

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  • Method used for rapidly identifying generation of recombinant virus particles in eukaryotic expression
  • Method used for rapidly identifying generation of recombinant virus particles in eukaryotic expression
  • Method used for rapidly identifying generation of recombinant virus particles in eukaryotic expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. Recombinant plasmid build

[0017] Design two specific primers IE1-F-:5'- by Primer Premier5.0 software TACGTA GTAGGTTATTGAT AAAATGA-3'(SnaBI) (SEQ ID NO. 1) and IE1-R:5'- GGATCC AGTCACTTGGTTGTT-3'(BamH I)(SEQ ID NO.2), amplify the ie1 early promoter from the baculovirus AcMNPV genome: a DNA fragment with a length of 579bp, and connect the amplified DNA fragment to the pMD18-T vector And it was sequenced, and the correct DNA fragment was subcloned into the pFastHTB vector to generate a recombinant plasmid: pFastHTB-ie1; through the specific primer EGFP-F:5'- GGATCC ATGGTGAGCAAGGGC-3'(BamHI) (SEQ ID NO.3) and EGFP-R:5'- GGTAC C TTACTTGTACAGCTCGTCCATG-3'(KpnI)(SEQ ID NO.4) amplifies the full length of the egfp gene sequence, connects the amplified target fragment to the pMD18-T vector, and subclones the correctly sequenced target fragment into the pFastHTB-ie1 vector , and name the resulting recombinant plasmid:

[0018] Such as figure 1 As shown,...

Embodiment 2

[0020] Example 2. Recombinant plasmid Identification

[0021] constructed by pair Carry out KpnI / PstI double enzyme digestion, the product is and the digested vector fragment, specifically amplified by PCR Fragment; right Carry out Pst I / Hind III double enzyme digestion, the product is and the digested vector fragment, specifically amplified by PCR fragment; through Carry out Kpn I / Hind III double enzyme digestion, the product is and the digested vector fragment, specifically amplified by PCR Fragments; electrophoretic analysis of these digested products and corresponding PCR products, such as figure 2 , the results showed that the PCR product and the digested product were located on the same horizontal line, and the sequence of the identified recombinant plasmid was determined, and the results showed that: The sequence is completely consistent with the theoretical sequence and can be used in the knockout experiment of the ac68 gene in Ac-Bacmid in the nex...

Embodiment 3

[0023] Example 3. The tandem expression cassette replaces the 120-bp DNA fragment at the 3' end of the ac68 gene on Ac-Bacmid

[0024] Streak the cryopreserved Escherichia coli DH10B (containing Ac-Bacmid and pBAD-gbaA plasmids) onto a freshly prepared LB plate containing Amp and Kan resistance, and culture it upside down at 37°C for 48 hours. Pick a single clone colony from the plate and put it in 3ml LB liquid medium containing Amp and Kan resistance, culture overnight at 37°C, 245rpm shaker, transfer 1ml bacterial solution to 100ml LB containing Amp and Kan resistance the next day Culture in liquid salt-free medium at 37°C with vigorous shaking. When the cultured bacteria solution OD 600 When the value reaches 0.2-0.3, directly add 0.1g L-arabinose powder to 100ml LB liquid salt-free medium, shake well to fully dissolve in the medium, and continue to cultivate for 45-60min to make the bacterial liquid OD 600 Reach 0.4-0.5. Cool the culture on ice for 15-30 minutes, the...

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Abstract

The invention belongs to a field of biochemistry and molecular biology, and relates to a method used for rapidly identifying generation of recombinant virus particles in an eukaryotic expression. The method comprises using an eukaryotic expression vector Ac-Bacmid as a molecular carrier to construct an integrated recombinant bacmid for integratedly expressing egfp, wherein 120 bps at a 3' end of an ac68 gene of the molecular carrier is replaced by an egfp gene expression box and a tandem Cm expression box, transfecting the integrated recombinant bacmid to insect cells through mediation of liposome, and directly monitoring production situation of budding virus particles through visualization signals of green fluorescent. According to the invention, through observing the green fluorescent protein signals, whether the recombinant budding virus particles BV is generated in the transfected sf-9 insect cells can be rapidly determined, thereby further providing basis for expression of exogenous target genes transfected to the Ac-Bacmid and subsequent functional studies.

Description

technical field [0001] The invention mediates green fluorescent protein into a eukaryotic expression system and belongs to the fields of biochemistry and molecular biology. Specifically, it refers to the introduction of a green fluorescent protein (EGFP) gene expression cassette at the ac68 gene site when constructing a recombinant baculovirus shuttle vector (Ac-Bacmid) in vitro, and quickly determines the transfected sf- 9 Whether the recombinant budding virion BV is produced in insect cells, and then provides the basis for the expression of exogenous target genes transposed to Ac-Bacmid and its subsequent functional studies. Background technique [0002] The baculovirus-insect cell expression system (BEVS) is a eukaryotic expression system, which has been widely used in the expression of foreign target genes and the production of high-titer vaccines. It has the following advantages (i) the expression product has the correct folding conformation and post-translational modi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64C12N7/01C12N15/866
Inventor 李国辉王鹏胡朝阳
Owner JIANGSU UNIV
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