Expression vector containing detection label IgG (immunoglobulin g), construction method and application thereof

An expression vector and detection label technology, applied in the field of molecular biology experiments, can solve the problems of cumbersome detection and detection process of target protein, inability to complete antibodies, etc., to reduce the probability of false negative detection results, improve accuracy and sensitivity, use handy effect

Inactive Publication Date: 2014-07-02
SHENZHEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide an expression vector containing a detection tag IgG for the problems in the prior art that there may be false negative experimental results when detecting the target protein, the detection of the target protein cannot be completed due to the lack of antibodies, and the detection process is cumbersome. , so as to provide a detection method for the target protein that is easy to operate, accurate in results, and widely used

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  • Expression vector containing detection label IgG (immunoglobulin g), construction method and application thereof
  • Expression vector containing detection label IgG (immunoglobulin g), construction method and application thereof
  • Expression vector containing detection label IgG (immunoglobulin g), construction method and application thereof

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Embodiment 1

[0100] This embodiment uses pET28a prokaryotic expression vector. Rat blood samples were extracted, total RNA was extracted and reverse transcribed into cDNA. Design the upstream primers and downstream primers shown in 2-2, use the mouse cDNA as a template, and use the above-mentioned amplification primers to perform PCR amplification to obtain the desired constant region gene of mouse IgG. The constant region gene of mouse IgG was double-digested with Hind III and Xho I, and the pET28a expression vector was double-digested with Hind III and Xho I, and the two fragments were recovered by DNA electrophoresis at the same time. Fragments and gene fragments were ligated with T4 DNA ligase, reacted at 16°C for 2 hours, took 10 microliters of the ligated products and added them to 100 microliters of E. Medium heat shock for 90 seconds, then let stand on the ice-water mixture for 1 minute, add 100 microliters of SOC medium at 37°C, incubate in a shaker at 37°C for 1 hour, spread on ...

Embodiment 2

[0106] This embodiment uses pCDNA3.1 / His C eukaryotic expression vector. Rat blood samples were extracted, total RNA was extracted and reverse transcribed into cDNA. Design the upstream primers and downstream primers shown in 1-2, use the mouse cDNA as a template, and use the above-mentioned amplification primers to perform PCR amplification to obtain the desired constant region gene of mouse IgG. The mouse IgG gene was double-digested with Xho I and Xba I restriction sites, and the pCDNA3.1 / His C vector was double-digested with Xho I and Xba I at the same time, and the two restriction fragments were simultaneously recovered by DNA electrophoresis, with 1: Carry out the ligation reaction between the carrier fragment and the gene fragment with T4 DNA ligase at a molar ratio of 4, react at 16°C for 2 hours, take 10 microliters of the ligation product and add it to 100 microliters of Escherichia coli DH5α competent cells, and place it in the ice-water mixture After 30 minutes, h...

Embodiment 3

[0109] This embodiment uses pET28a prokaryotic expression vector. Rabbit blood samples were extracted, total RNA was extracted and reverse transcribed into cDNA. Design the upstream primers and downstream primers shown in 2-3, use the rabbit cDNA as a template, and use the above-mentioned amplification primers to perform PCR amplification to obtain the desired constant region gene of rabbit IgG. The constant region gene of rabbit IgG was double-digested with Hind III and Xho I, and the pET28a vector was double-digested with Hind III and Xho I at the same time, and the two fragments were recovered by DNA electrophoresis at the same time, and the vector was digested at a molar ratio of 1:4 Fragments and gene fragments were ligated with T4 DNA ligase, reacted at 16°C for 2 hours, took 10 microliters of the ligated products and added them to 100 microliters of E. Medium heat shock for 90 seconds, then let stand on the ice-water mixture for 1 minute, add 100 microliters of SOC med...

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Abstract

The invention discloses an expression vector containing detection label IgG (immunoglobulin g), a construction method, and an application thereof in expression detection of target protein. The detection label IgG is the constant region gene of at least one species of IgG, and the expression detection of the target protein is realized through direct reaction of the constant region gene of at least one species of IgG and at least one species of secondary antibody. The construction method for the expression vector containing detection label IgG comprises the steps of designing an upstream primer and a downstream primer of the constant region gene of at least one species of IgG; taking at least one species of cDNA (complementary deoxyribonucleic acid) as a template, carrying out PCR (polymerase chain reaction) amplification through the upstream primer and the downstream primer so as to obtain the constant region gene of at least one species of IgG; and connecting the constant region gene of at least one species of IgG with the expression vector so as to construct the expression vector containing detection label IgG. When being used for detection of target protein, the expression vector can lower the probability of false negative detection result, and improve the accuracy and sensitivity of the detection.

Description

technical field [0001] The invention belongs to the field of molecular biology experiment techniques and methods, and relates to a molecular biology experiment technique. More specifically, the present invention relates to an expression vector containing detection tag IgG, a construction method and application thereof. Background technique [0002] In molecular biology experiments, the expression of foreign proteins by genetic engineering is a common technical means, and the process must detect the expression of the target protein. At present, the usual method for detecting the target protein is to carry out antigen-antibody reaction through the antibody corresponding to the protein, and then indirectly determine the target protein through the color development of the secondary antibody. This will cause two problems: (1) The binding ability between the target protein and the antibody cannot be effectively guaranteed, and the secondary antibody cannot effectively develop col...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66G01N33/68
Inventor 易俊波苏庆宁买制刚卢海蓉周兆平林枫
Owner SHENZHEN UNIV
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