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A kind of Glypican GPC3 protein fragment and its application and hybridoma cell line prepared

A hybridoma cell line and cell line technology are applied in the field of monoclonal antibody hybridoma cell line GPC3-1H5 to achieve the effects of reducing production cost, improving sensitivity and accuracy, and high titer

Active Publication Date: 2016-08-17
BIOLOGY INST OF HEBEI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It shows that alpha-fetoprotein is one of the most important serum markers for the diagnosis of liver cancer at present, but at the same time, there are still more than 30% of false and missed detection rates

Method used

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  • A kind of Glypican GPC3 protein fragment and its application and hybridoma cell line prepared
  • A kind of Glypican GPC3 protein fragment and its application and hybridoma cell line prepared
  • A kind of Glypican GPC3 protein fragment and its application and hybridoma cell line prepared

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 gpc 3 Cloning of genes and construction of recombinant expression plasmids

[0030] To contain gpc The plasmids of the 3 genes were used as templates for PCR amplification.

[0031] Upstream primer: 5'-CGGAATTCCTTGGTGGTGGCGATGCT-3', inserted into the EcoRI restriction site.

[0032] Downstream primer: 5'-CGGGATCCCCCGAGGTTGTGAAAGGT-3', inserted into the BamHI restriction site.

[0033] Amplification conditions: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 1 min, annealing at 62°C for 1 min, extension at 72°C for 2 min, a total of 30 cycles; extension at 72°C for 10 min. The PCR products were detected by agarose gel electrophoresis, and the experimental results were as follows: figure 1 As shown, the resulting 1650bp nucleotide fragment was amplified.

[0034] The PCR product was recovered after agarose gel electrophoresis, ligated into pMD18-T vector, and the product was ligated with T4 ligase in a water bath at 16°C. The ligation produ...

Embodiment 2

[0037] Induced expression and purification of embodiment 2 fusion protein

[0038] HEK293 cells in good condition were treated with 2×10 5 Cells / well were cultured in 6-well plates. After culturing for 24 hours, transfection was performed when the cell confluence was 80%-90%. Rinse 3 times with PBS, and add serum-free DMEM high-glucose medium. Follow Lipofectamine TM 2000 transfection reagent instructions, the recombinant expression plasmid CMV- gpc3Transfection was carried out, and a blank control group (HEK293 cells) and a positive control group (p3XFLAG-CMV-14) were set up at the same time. The DMEM high-glucose medium containing 0.5 mg / L G418 and 10% fetal bovine serum was used for pressurized screening, and the monoclonal cells were prepared by the limiting dilution method, and finally a cell line stably expressing the recombinant protein was obtained.

Embodiment 3

[0039] Example 3 Preparation of Monoclonal Antibody Hybridoma Cell Line GPC3-1H5

[0040] Amplify the cell line expressing the recombinant protein prepared by the above steps by conventional methods, collect the bacteria by centrifugation at low temperature, perform ultrasonic crushing in an ice bath, centrifuge at 4°C, 8000r / min for 20min, collect the supernatant, and purify according to Anti-FLAG resin Kit purification to obtain GPC3 recombinant protein.

[0041] The expressed GPC3 recombinant protein was purified as an antigen, and immunized according to the standard immunization procedure, that is, the first immunization was mixed with equal volume of recombinant protein and complete Freund's adjuvant, intraperitoneally injected into mice, the dose was 100ul / mouse, which contained recombinant protein 50ug. From the second immunization, mix the same volume of recombinant protein and incomplete Freund's adjuvant, the dose of recombinant protein immunization is the same as t...

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Abstract

The present invention relates to a Glypican GPC3 protein fragment, which has an amino acid sequence such as SEQ ID No.1; the GPC3 protein fragment of the present invention has strong antibody specificity and can be used to prepare high-titer polyclonal or monoclonal Cloning antibodies, or preparing GPC3 protein standards, can be used for GPC3 protein detection, including detection of protein content, activity and protein structure. The present invention also relates to a monoclonal antibody hybridoma cell line GPC3‑1H5, the secreted GPC3 antibody has high yield, high titer, and sensitive response, which is beneficial to improving the sensitivity and accuracy of detection, and can be widely used in GPC3 protein expression. The detection reduces the production cost of the GPC3 antibody, which is conducive to the clinical promotion and use of the GPC3 detection.

Description

technical field [0001] The invention relates to a Glypican 3GPC3 protein fragment, its application and a monoclonal antibody hybridoma cell line GPC3-1H5 prepared therefrom, specifically belonging to the field of cell engineering. Background technique [0002] The most widely used detection index in the early diagnosis of liver cancer in my country is alpha-fetoprotein (AFP). Wu Mengchao et al. detected the level of alpha-fetoprotein in 5343 cases of liver cancer, and the positive rate was 69.4%. It shows that alpha-fetoprotein is one of the most important serum markers for the diagnosis of liver cancer at present, but at the same time, there are still more than 30% of false and missed detection rates. Finding new specific markers for liver cancer is one of the important ways to improve early clinical diagnosis. [0003] Glypican is a family of heparan sulfate glycoproteins involved in the regulation of individual development, cell proliferation and differentiation. Among ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/85G01N33/68G01N33/577G01N33/574C12N5/20
CPCC07K14/47G01N33/574G01N33/577G01N33/68
Inventor 程华闫静辉方向东吴萌崔硕
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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