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Method for constructing recombinant rhabdovirus expression vector for EGFP (enhanced green fluorescent protein) gene

A technology of baculovirus and expression vector, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problems of large gap in expression efficiency, short expression time, low transduction efficiency of recombinant baculovirus plasmid vector, etc.

Inactive Publication Date: 2012-07-04
HEILONGJIANG UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0003] The present invention aims to solve the problems of low transduction efficiency of the current recombinant baculovirus plasmid vector, large gap between expression efficiency and short expression time compared with industrial production requirements, and provides a method for constructing EGFP gene recombinant baculovirus expression vector

Method used

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  • Method for constructing recombinant rhabdovirus expression vector for EGFP (enhanced green fluorescent protein) gene
  • Method for constructing recombinant rhabdovirus expression vector for EGFP (enhanced green fluorescent protein) gene
  • Method for constructing recombinant rhabdovirus expression vector for EGFP (enhanced green fluorescent protein) gene

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specific Embodiment approach 1

[0008] Specific embodiment one: the construction method of the EGFP gene recombinant baculovirus expression vector of the present embodiment is carried out according to the following steps: one, with the plasmid peglk as a template, WK2 and WK3 as primers, carry out PCR amplification, and carry out 1% PCR product Agarose gel electrophoresis detection, using a gel recovery kit for purification and recovery, to obtain the EF1α gene; 2. Link the EF1α gene to the pMD18-T vector to obtain the plasmid pT-EF1α; 3. Separate the plasmids pT-EF1α and pFB-VSVGED -WPRE was subjected to double enzyme digestion, and the target fragment EF1α obtained after enzyme digestion was connected with pFB-VSVGED-WPRE after enzyme digestion to obtain plasmid pWK; 4. Use pAAV-LacZ as a template and La and Lb as primers for PCR amplification 1% agarose gel electrophoresis was performed on the PCR product, and the gel recovery kit was used to purify and recover the L-ITR gene to obtain the L-ITR gene. The ...

specific Embodiment approach 2

[0020] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is that the reaction system of PCR amplification in step 1 is a 50 μL reaction system, which consists of the following components:

[0021]

[0022] PCR amplification conditions were: denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1.2 min, a total of 30 cycles, extension at 72°C for 10 min, and incubation at 4°C. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0023] Specific embodiment three: the difference between this embodiment and specific embodiment one is: the system connected in step two is as follows:

[0024]

[0025] The ligation reaction condition was 16°C for 12h. Others are the same as in the first embodiment.

[0026] The Solution I described in this embodiment is a pMD18-T vector matching buffer containing T4 DNA ligase, which was purchased from Dalian Bao Biological Engineering Co., Ltd.

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Abstract

The invention provides to a method for constructing a recombinant rhabdovirus expression vector for EGFP (enhanced green fluorescent protein) gene, relating to a method for constructing the recombinant rhabdovirus expression vector for the EGFP gene. The invention aims to solve the problems of low transduction efficiency, large distance in expression efficiency from requirement of industrial production, short expression time and the like of the traditional recombinant rhabdovirus expression vector. The method comprises the following steps: firstly, amplifying EF1 alpha gene through PCR (polymerase chain reaction); secondly, constructing a plasmid pT-EF1 alpha; thirdly, constructing a plasmid pWK; fourthly, amplifying L-ITR (L-inverted terminal repeat) gene through PCR, and constructing pWK-L-ITR; fifthly, amplifying R-ITR gene through PCR, and constructing pWK-ITR; sixthly, amplifying the EGFP gene through PCR, and constructing the recombinant rhabdovirus expression vector pWK-ITR-EGFP for the EGFP gene. In the invention, through the addition of elements of VSV (vesicular stomatitis virus)-GED, WPRE (Woodchuck Posttranscriptional Regulatory Element) and ITR, the expression efficiency of exogenous genes is enhanced, the transduction efficiency of virus is improved, and the expression time is prolonged.

Description

technical field [0001] The invention relates to a method for constructing an EGFP gene recombinant baculovirus expression vector. Background technique [0002] With the development and application of molecular biology techniques and methods, baculovirus has been developed into an efficient eukaryotic expression vector system for the expression of various foreign genes, and has been accepted as a new type of vaccine and gene therapy vector. People attach great importance to it, but there is a large gap between the exogenous gene expression mediated by the existing baculovirus and the requirements of industrialized mass production. Moreover, the duration of sustained expression is relatively short, and the transduction efficiency is low. These factors limit the application of the baculovirus expression system. Therefore, the key point of the research on the baculovirus expression system is to prolong the expression time of foreign genes, and improve the expression efficiency ...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/66
Inventor 葛菁萍高冬妮平文祥楼庄伟
Owner HEILONGJIANG UNIV
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