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HA protein fusion label-containing plant expression plasmid vector and construction method of carrier

A plasmid vector, plant expression technology, applied in the field of molecular biology, can solve problems such as unfavorable research protein function, target protein function interference, unfavorable research protein, etc., and achieve the effects of ensuring accuracy, short cycle time and low cost.

Inactive Publication Date: 2018-11-20
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pCAMBIA series of vectors either do not contain protein fusion tags, and must prepare specific antibodies to the researched protein itself, which is time-consuming and laborious, which is not conducive to rapid research on protein functions; or contain larger protein fusion tags (such as GFP and GUS, etc.), These large protein fusion tags often interfere with the function of the target protein itself, so it is not conducive to accurately study the function of the protein

Method used

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  • HA protein fusion label-containing plant expression plasmid vector and construction method of carrier
  • HA protein fusion label-containing plant expression plasmid vector and construction method of carrier
  • HA protein fusion label-containing plant expression plasmid vector and construction method of carrier

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Embodiment 1

[0017] Embodiment 1: A plant expression plasmid vector containing an HA protein fusion tag disclosed in the present invention, the vector is a plant expression plasmid vector containing an HA protein fusion tag at the C-terminus of the plant expression plasmid vector. Specifically, the vector contains three gene fragments encoding the HA protein tag Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, and the nucleotide sequences of the three HA proteins are respectively shown in SEQ ID No. 1. Shown in SEQ ID No.2 and SEQ ID No.3. The connecting sequence between SEQ ID No.1 and SEQ ID No.2 is SEQ ID No.8, and the connecting sequence between SEQ ID No.2 and SEQ ID No.3 is SEQ ID No.9.

[0018] The vector also includes a transcription promoter, a transcription terminator, a kanamycin coding sequence and a replicon fragment in the prior art, and the nucleotide sequence of the vector is shown in SEQ ID No.4. The vector of the present invention, its genetic map such as figure 1 shown.

[0019] ...

Embodiment 2

[0021]Embodiment 2: A plant expression plasmid vector containing an HA protein fusion tag disclosed in the present invention, the vector is a plant expression plasmid vector containing an HA protein fusion tag at the C-terminus of the plant expression plasmid vector. Specifically, the vector contains three gene fragments encoding the HA protein tag Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, and the nucleotide sequences of the three HA proteins are respectively shown in SEQ ID No. 1. Shown in SEQ ID No.2 and SEQ ID No.3. The connecting sequence between SEQ ID No.1 and SEQ ID No.2 is SEQ ID No.8, and the connecting sequence between SEQ ID No.2 and SEQ ID No.3 is SEQ ID No.9.

[0022] The vector also includes a transcription promoter, a transcription terminator, a kanamycin coding sequence and a replicon fragment in the prior art, and the nucleotide sequence of the vector is shown in SEQ ID No.4.

[0023] Preferably, the method for constructing the plasmid vector comprises the steps o...

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Abstract

The invention discloses an HA protein fusion label-containing plant expression plasmid vector. The C end of a plant expression plasmid vector contains an HA protein fusion label. A method for constructing the plasmid vector comprises the following steps: (1) by taking pCAMBIA1302 as a start plasmid, performing enzyme digestion on the plasmid by adopting Eco065I, then filling in the sticky tail endsubjected to Eco065I enzyme digestion by using Klenow Fragment enzyme to obtain a blunt end, performing enzyme digestion by using SpeI, and cutting gel through gel electrophoresis to recycle klenow fragments; (2) performing full-sequence synthesis of a nucleotide sequence containing 3 HA proteins; (3) designing an amplification primer; (4) adding an SpeI enzyme digestion locus at the N end, adding a nucleotide G at the C end, performing enzyme digestion on the synthesized fragment by using SpeI, finally connecting products, performing heat shock conversion on DH5a, and obtaining a positive clone through PCR (Polymerase Chain Reaction) authentication. The HA protein fusion label-containing plant expression plasmid vector has the advantages that the HA protein fusion label-containing plantexpression plasmid vector is obtained to help a user to quickly research a protein function, and cannot interfere own functions of a target protein to ensure that the protein function can be accurately researched.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a plant expression plasmid vector containing an HA protein fusion tag and a method for constructing the vector. Background technique [0002] The HA tag is derived from the 9 amino acids at positions 98-106 of the hemagglutinin of human influenza virus. It has strong immunoreactivity and has little effect on the spatial structure of the target protein. The recombinant HA tagged protein can be used coupled with HA High-specificity monoclonal antibody resin for efficient purification. It is widely used in the separation, purification and detection of HA fusion target proteins. In order to scale and industrialize the HA protein tag, it is necessary to provide a plant expression plasmid vector with a commonly used fusion protein tag. The pCAMBIA family of vectors is widely used by many plant molecular biology laboratories around the world. With the rise of gene function ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/65C12N15/66
CPCC12N15/65C12N15/66C12N15/8209
Inventor 江文波庞永珍
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI