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A kind of recombinant expression vector of fgfr3 mutant and its construction method and application

A technology for FGFR3 and expression vectors, which is applied in the field of recombinant expression vectors for FGFR3 mutants and its construction, can solve the problems of lack of effective molecular markers, the construction method and application of FGFR3 mutant recombinant expression vectors, and lag, etc., to achieve transformation High dyeing efficiency, good application value, and stable expression

Inactive Publication Date: 2018-11-09
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of clinical diagnosis and treatment, my country currently lacks effective, specific, and guiding molecular markers, as well as corresponding diagnostic, treatment, and prognostic detection methods, especially in the research of specific drugs.
However, there are no specific reports on the recombinant expression vectors of FGFR3 mutants and their construction methods and applications.

Method used

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  • A kind of recombinant expression vector of fgfr3 mutant and its construction method and application
  • A kind of recombinant expression vector of fgfr3 mutant and its construction method and application
  • A kind of recombinant expression vector of fgfr3 mutant and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The construction of embodiment 1 recombinant expression vector

[0051] 1. Construction of pWPI.1-FGFR3IIIc-IRES2-EGFP, pWPI.1-FGFR3AT-I-IRES2-EGFP and pWPI.1-FGFR3Δ7-9-IRES2-EGFP vectors and pcDNA3.0-FLAG-FGFR3, pcDNA3.0- HA-FGFR3, pcDNA3.0-FLAG-FGF1, pcDNA3.0-HA-FGF1, pcDNA3.0-FLAG-FGF2, pcDNA3.0-HA-FGF2 vector construction (the above vectors are FGFR3 wild type IIIc, FGFR3 mutant Construction of AT I and mutant Δ7-9-associated vectors, and vector construction of tagged proteins in some experiments)

[0052] 1. PCR primer design and target gene amplification

[0053] Primers were designed by Primer5.0 software, sense strand: FGFR3-F 5'-gac GGATCCatgggcgcccctgcctgcgccctc-3', as shown in SEQ ID NO.2; antisense strand: FGFR3-R 5'-gac ACTAGTtcacgtccgcgagcccccactgct-3', as shown in SEQ ID NO.3 shown; primers were synthesized by Shanghai Sangon Bioengineering Company. The restriction site for the 5' end of the sense strand is: BamHI, and the restriction site for the 5' e...

Embodiment 2

[0183] Embodiment 2, identify the affinity of FGF1 / FGF2 and FGFR3 and mutants thereof

[0184] 1. Identification of binding ability of FLAG-FGF1, FLAG-FGF2 and HA-FGFR3 and their mutants by immunoprecipitation reaction

[0185] 1. Routinely culture 293T cells co-transfected with HA-FGFR3 and FLAG-FGF1 for two days, harvest the cells, add an appropriate amount of RIPA lysis buffer (millipore) containing protease inhibitors (roche, protease inhibitor tablets), on ice or at 4°C Lyse for 30min, then centrifuge at 12,000g for 30min and take the supernatant;

[0186] 2. Take a small amount of lysate (generally 5% of the total amount) as input for Western blot analysis. Add 1-2 μg HA probe (santacruz) to the remaining lysate, incubate at 4°C for 2 hours and then add 20-40 μl protein A / G-beads (santacruz), incubated overnight at 4°C with slow shaking, this step is mainly to immunoprecipitate HA-FGFR3;

[0187] 3. After the immunoprecipitation reaction, centrifuge at 3,000g for 5 min...

Embodiment 3

[0212] Embodiment 3 identifies the ability of FGFR3 and its mutants to form dimers

[0213] 1. 293T cells were transiently transfected with pcDNA3.0 empty load, pcDNA3.0-FLAG-FGFR3IIIc, pcDNA3.0-FLAG-FGFR3Δ7-9 and pcDNA3.0-FLAG-FGFR3AT-I, divided into two groups, one group without FGF1, Another group was routinely cultured with medium containing 10 ng / ml FGF1 for 2 days. Add an appropriate amount of RIPA lysis buffer (millipore) containing protease inhibitors (roche, protease inhibitortablets), lyse on ice or at 4°C for 30min, then centrifuge at 12,000g for 30min and take the supernatant.

[0214] 2. Load the sample to non-denaturing polyacrylamide gel electrophoresis (Native-PAGE) without SDS, and perform Western Blotting.

[0215] Depend on Figure 7 It can be seen that FLAG-FGFR3IIIc hardly forms a dimer without the stimulation of FGF1, but can form a dimer under the stimulation of FGF1; and FLAG-FGFR3Δ7-9 can form a dimer regardless of whether it is stimulated by FGF1; ...

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Abstract

The invention discloses a recombinant expression vector of FGFR3 mutant. The recombinant expression vector contains mutant FGFR3 delta7-9 of deletion exons 7, 8 and 9. The nucleotide sequence of the deletion part of the mutant FGFR3 delta7-9 is shown as SEQ ID NO.1. In addition, the invention further discloses a construction method of the recombinant expression vector of the FGFR3 mutant. The construction method comprises the following steps: (1) amplifying DNA fragments of the FGFR3 mutant through an SOE PCR (gene splicing by overlap extension PCR) method; (2) carrying out gel electrophoresis on the amplification product, and carrying out rubber cutting and recycling on target genes; (3) carrying out double enzyme digestion on the PCR product; and (4) connecting the target fragments and a carrier fragment. In addition, the invention further discloses an application of the recombinant expression vector of FGFR3 mutant. The experiment shows that the recombinant expression vector has good application values in identifying the affinity of FGF1 / FGF2 and FGFR3 as well as the mutant thereof, and the capacity of forming a dimer of FGFR3 and the mutant thereof, as well as in identifying activation of tyrosine kinase areas of FLAG-FGFR3 IIIc, FLAG-FGFR3 delta 7-9, and FLAG-FGFR3 AT-I.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a recombinant expression vector of FGFR3 mutant, its construction method and application. Background technique [0002] Chronic liver disease is one of the major public health problems that endanger the health of our people. It ranks sixth among the main causes of death in our country. The carrier rate of hepatitis B virus HBV in the population is 9.09%, of which about 5000 are chronic hepatitis B patients. Ten thousand, recurrent attacks of liver inflammation lead to 10% to 20% of patients developing liver cirrhosis, of which 10% to 15% of patients with liver cirrhosis eventually develop into hepatocellular carcinoma. In addition to the hazards of liver cancer, each stage in the transformation process of hepatitis-cirrhosis-hepatocarcinoma will cause serious harm to the health and life of patients, and has caused a heavy economic burden and social problems in our country. The current...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66C12N1/21G01N33/68
Inventor 邱伟华李科沈柏用彭承宏景晓乾刘心玉马丁程兮
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE