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Construction method and applications of multifunctional plant binary plasmid pPBEL-BiFC

A construction method and a multi-functional technology, applied in the direction of introducing foreign genetic material using carriers, biological testing, material inspection products, etc., to achieve the effect of simplifying experimental procedures and improving work efficiency

Inactive Publication Date: 2018-09-18
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
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Problems solved by technology

[0002] The basic skeleton of this plasmid is based on the pCambia1302 vector, but pCambia1302 can only express a single gene, without HA, MYC tags, and cannot use universal tag sequences for Western blot analysis

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Embodiment Construction

[0018] The technical solutions of the present invention will be further described in detail below in conjunction with specific drawings and implementations.

[0019] Separation of YFP fluorescent proteins into YFPN 173 and YFPC 155 Two fragments were constructed into HA-PmlI-EcoRI-SpeI-HindIII-BamHI-NcoI-YFPN 173 -BstEII and Myc-SmaI-KpnI-SalI-PstI-BglII-XbaI-SacI-YFPC 155 -XhoI two cassettes replaced the GFP and Hygromycin genes on the original plasmid. The constructed plasmid can use HA and Myc as protein translation initiation and as a universal label for Western blot, and was identified by biochemical co-immunoprecipitation (Co-IP) method The interaction between two proteins. At the same time, by using the subsequent YFPN 173 and YFPC 155 The split fragments can be analyzed by bimolecular fluorescence complementation, so that the dynamic process and the strength of the protein interaction relationship in cells can be detected.

[0020] The multi-cloning site EcoRI-Hi...

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Abstract

The invention discloses a construction method and applications of a multifunctional plant binary plasmid pPBEL-BiFC, and belongs to the technical field of gene engineering. According to the construction method, the original multiple cloning site is modified, wherein the original MCS region is knocked out, and the two new MCS regions are introduced, such that the more enzyme digestion sites can beselected; the original GFP is modified, wherein the GFP is replaced, and the YFP-C fragment and the YFP-N fragment are introduced at different positions of the vector and are respectively positioned at two vectors; the HA tag and the MYC tag are introduced, such that the western-blot protein detection can be performed, wherein the protein A is linked to the HA, the protein B is linked to the MYC,and the western-blot analysis is performed by detecting the protein A so as to detect the presence of the protein B, and vice versa. According to the present invention, with the contructed plasmid, the co-immunoprecipitation (Co-IP) reaction can be performed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a construction method and application of a multifunctional plant binary plasmid (pPBEL-BiFC) for the expression and interaction of two different purpose proteins. Background technique [0002] The basic skeleton of this plasmid is based on the pCambia1302 vector, but pCambia1302 can only express a single gene, without HA, MYC tags, and cannot use universal tag sequences for Western blot analysis. The newly constructed vector can simultaneously express two different foreign proteins with HA and Myc tags respectively and YFPN 173 and YFPC 155 In the same frame (in-frame) to express the fusion protein, the original carrier cannot perform Co-IP immunoprecipitation reaction, bimolecular fluorescence complementation (BiFC) test, redistributed, increased and arranged multiple cloning sites, two multiple Each cleavage point in the cloning site region (MCS) is un...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82G01N33/68
CPCC12N15/82G01N33/6845
Inventor 丁晓东宋雨尤宏光刘圆明张航李强朱延明
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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