Recombinant expression vector of FGFR3 mutant as well as construction method and application of recombinant expression vector

A technology for FGFR3 and expression vectors, which is applied in the field of recombinant expression vectors of FGFR3 mutants and its construction, can solve the problems of the construction method and application of FGFR3 mutant recombinant expression vectors, lack of effective molecular markers, hysteresis, etc., and achieve good results. Application value, high transfection efficiency, and stable expression

Inactive Publication Date: 2016-03-02
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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  • Recombinant expression vector of FGFR3 mutant as well as construction method and application of recombinant expression vector
  • Recombinant expression vector of FGFR3 mutant as well as construction method and application of recombinant expression vector
  • Recombinant expression vector of FGFR3 mutant as well as construction method and application of recombinant expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of 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 vector construction 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 all FGFR3 wild type IIIc, FGFR3 mutant ATI and mutant Δ7-9-related vector construction, as well as the vector construction of the tag protein in some experiments)

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

[0053] The primers were designed by Primer5.0 software, the sense strand: FGFR3-F5ˊ-gacGGATCCatgggcgcccctgcctgcgccctc-3ˊ, as shown in SEQ ID NO.2; the antisense strand: FGFR3-R5ˊ-gacACTAGTtcacgtccgcgagcccccactgct-3ˊ, as shown in SEQIDNO. Synthesized by bioengineering company. The 5'end restriction site of the sense strand is BamHI, and the 5'end restriction site of the antisense strand is SpeI. KODplus ...

Embodiment 2

[0183] Example 2. Identification of the affinity of FGF1 / FGF2 and FGFR3 and their mutants

[0184] 1. Co-immunoprecipitation to identify the binding ability of FLAG-FGF1, FLAG-FGF2, HA-FGFR3 and its mutants

[0185] 1. Routinely cultivate 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), and lyse on ice or at 4°C for 30 minutes , Then centrifuge at 12,000g for 30min and take the supernatant;

[0186] 2. Take a small amount of lysate (usually 5% of the total amount) as input for Westernblot analysis, add 1-2μg HAprobe (santacruz) to the remaining lysate, incubate at 4°C for 2h and then add 20-40μl proteinA / G- Beads (santacruz), incubate overnight with slow shaking at 4°C. This step is mainly to immunoprecipitate HA-FGFR3;

[0187] 3. After the immunoprecipitation reaction, centrifuge at 3,000g at 4°C for 5 minutes, and cent...

Embodiment 3

[0212] Example 3 Identification of the ability of FGFR3 and its mutants to form dimers

[0213] 1. 293T cells were transiently transferred with pcDNA3.0 empty, pcDNA3.0-FLAG-FGFR3IIIc, pcDNA3.0-FLAG-FGFR3Δ7-9 and pcDNA3.0-FLAG-FGFR3AT-I, divided into two groups, one group did not add FGF1, The other group was routinely cultured with medium containing 10ng / ml FGF1 for 2 days. Add an appropriate amount of RIPA lysis buffer (millipore) containing protease inhibitors (roche, protease inhibitor tablets), lyse on ice or 4°C for 30 min, then centrifuge at 12,000 g for 30 min and take the supernatant.

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

[0215] by Figure 7 It can be seen that FLAG-FGFR3IIIc hardly forms dimers without FGF1 stimulation, but can form dimers under FGF1 stimulation; and FLAG-FGFR3Δ7-9 can form dimers regardless of whether FGF1 is stimulated or not; Regardless of FGF1 stimulat...

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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 an FGFR3 mutant and a construction method and application thereof. Background technique [0002] Chronic liver disease is one of the major public health problems that endanger the health of our people. It is the sixth leading cause of death in our country. The HBV carrier rate of the hepatitis B virus in the population is 9.09%, of which about 5000 patients are chronic hepatitis B Wan, repeated episodes of liver inflammation cause 10% to 20% of patients to develop cirrhosis, and 10% to 15% of cirrhosis patients eventually develop hepatocellular carcinoma. In addition to the hazards of liver cancer, all stages of the hepatitis-cirrhosis-liver cancer transformation process will cause serious harm to the health and life of patients, and cause heavy economic burdens and social problems to our country. The current research on the pathological mechanism of hepa...

Claims

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Application Information

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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
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