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Plasmid for detecting STAT3 dimerization

A plasmid, myc-stat3-t2a technology, applied in the field of molecular biology, can solve the problems of difficult control of experimental conditions, low detection sensitivity, false positives, etc.

Active Publication Date: 2021-08-10
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires the construction of two vectors, and it is difficult to keep the same amount of the two vectors after co-transfection into cells, then the dimer formation may be inhibited due to insufficient quantity of one of the vectors, resulting in false positives
In addition, although non-denaturing polyacrylamide gel electrophoresis combined with immunoblotting can detect protein polymers, there are problems such as low detection sensitivity and difficult control of experimental conditions.

Method used

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  • Plasmid for detecting STAT3 dimerization
  • Plasmid for detecting STAT3 dimerization
  • Plasmid for detecting STAT3 dimerization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The construction of embodiment 1 plasmid of the present invention

[0044] The construction process is: use pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro as the empty vector backbone (lifescience-market, Hong Kong) (such as figure 2 shown), at its multiple cloning site (MCS), the Flag-STAT3 fragment was inserted into pCDH-CMV-Flag-Stat3-GFP-Puro (eg image 3 shown); on the basis of pCDH-CMV-Flag-Stat3-GFP-Puro, the Myc-STAT3-T2A fragment is inserted at the upstream adjacent position of the Flag-STAT3 fragment to obtain the plasmid pCDH-CMV-Myc-STAT3 of the present invention -T2A-Flag-STAT3-GFP-Puro (eg Figure 4 shown).

[0045] T2A is the gene for 2A peptide.

[0046] The detailed steps are as follows:

[0047] 1. Construction of pCDH-CMV-Flag-Stat3-GFP-Puro

[0048] 1. Primer Design

[0049] According to the sequence of NM_139276.2 in GenBank, design the following PCR primers, wherein the upstream primer has an NheI restriction site, and adds Kozak sequence (translation enh...

Embodiment 2

[0112] Example 2 Application of plasmid of the present invention for detecting STAT3 dimerization

[0113] Transfect the pCDH-CMV-Myc-STAT3-T2A-Flag-STAT3-GFP-Puro plasmid of Example 1 into HEK293T cells, extract the total protein of the cells after 48 hours, carry out co-immunoprecipitation (IP) on Myc or Flag, and the total protein The proteins obtained by the method and IP were subjected to denaturing polyacrylamide gel electrophoresis, and then immunoblotting (IB) was used to detect Flag or Myc.

[0114] Such as Figure 6 As shown, Myc antibody was directly used for western blot detection, and there was a band (input), showing that Myc-STAT3 expressed protein; because denaturing polyacrylamide gel electrophoresis will denature dimers into monomers, the input lane is a single strip. After co-immunoprecipitation of Flag, immunoblotting with Myc antibody shows bands (IP). If no dimers are formed in the cells, STAT3 with Myc cannot be collected by co-immunoprecipitation and ...

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Abstract

The invention discloses a plasmid for detecting STAT3 dimerization and belongs to the technical field of molecular biology. The plasmid comprises the following three fragments of (1) a promoter; (2) a first STAT3 gene, wherein a 5' end of the first STAT3 gene is connected with the sequence of the tag protein A; (3) a second STAT3 gene, wherein a 3'end of the second STAT3 gene is connected with the sequence of the tag protein B; wherein (1) and (2) are connected through a gene of 2A peptide; the tag protein A and the tag protein B are different in sequence. The plasmid disclosed by the invention can translate STAT3 proteins with different tags in cells according to a ratio of 1: 1, and after the proteins are purified by one tag, the other tag is detected, so the STAT3 dimerization level can be objectively reflected.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. Background technique [0002] Signal transducer and activator of transcription 3 (STAT3) is a transcriptionally active oncogene, its monomers are first phosphorylated in the cytoplasm, and then the phosphorylated STAT3 monomers dimerize to form dimers body, becomes active STAT3. The activated STAT3 dimer translocates to the nucleus to regulate tumor proliferation, survival, metastasis, angiogenesis, immune response and other processes. [0003] STAT3 dimer has become one of the important targets for anti-tumor drug screening. Studies have shown that G-quartet oligo-nucleotides can form a complex tertiary structure to inhibit STAT3 dimerization , thereby inhibiting the growth of squamous cell carcinoma in vitro and in mouse models (Liu Jun et al. Research progress in STAT3 signaling pathway and molecular targeted therapy of tumors. Chinese Journal of Neuropsychiatric Diseases, Vol. 37,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
CPCG01N33/57484C12N15/63G01N33/68
Inventor 王紫静
Owner WEST CHINA HOSPITAL SICHUAN UNIV