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Reporter gene assay for measuring biological activity of recombinant human keratinocyte growth factors

A biological activity and reporter gene technology, applied in the field of recombinant drug activity detection, can solve the problems of high initial drug concentration, large result variability, long test period, etc., and achieve the effects of simple operation, high accuracy and small variation.

Pending Publication Date: 2019-07-19
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are certain limitations in the application of the cell proliferation method by the applicant company. For example, the cells used for activity measurement are KGF-independent cell lines, which are derived from rhesus monkeys and mice respectively; the initial concentration of the drug is relatively high (KGF-2>3000ng / mL) , long test period (3d-6d), low signal-to-noise ratio and large variability of results

Method used

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  • Reporter gene assay for measuring biological activity of recombinant human keratinocyte growth factors
  • Reporter gene assay for measuring biological activity of recombinant human keratinocyte growth factors
  • Reporter gene assay for measuring biological activity of recombinant human keratinocyte growth factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Screening HEK293 and HaCat stable cell lines expressing SRE and KGFR2IIIb receptors

[0039] 1. Materials and methods

[0040] 1.1 cells

[0041] HEK293 cells were derived from ATCC; HaCat cells were derived from the Cell Bank of the Chinese Academy of Sciences.

[0042] 1.2 Reagents and materials

[0043] pGL4.33[luc2p / SRE / Hygro] plasmid and ViaFect TM Transfection reagents were purchased from Promega; KGFR2IIIb gene overexpression lentivirus was constructed and packaged by Shanghai Jikai Gene Technology Co., Ltd.; DMEM and fetal bovine serum (FBS) were purchased from GIBCO; puromycin was purchased from Invitrogen; hygromycin B was purchased from Suolaibao Biotechnology Co., Ltd.; Britelite plus luciferase substrate was purchased from PerkinElmer; white bottom light-transmitting 96-well plate was purchased from CORNING; recombinant KGF-1 and KGF-2 were retained by the Recombinant Drug Department of China National Institutes for Food and Drug Control.

[004...

Embodiment 2K

[0062] The methodological optimization of embodiment 2 KGF activity assay

[0063] 1. KGF-1 concentration optimization

[0064] According to the monoclonal cell strain screening conditions in 2.5 of Example 1, the KGF-1 pre-diluted concentration was 100,000 ng / ml, and 20 gradients were serially diluted by 3 times, and 2 duplicate holes were set for each. Add equal volumes of different concentrations of recombinant KGF-1, act for 4-6h to detect the fluorescence signal value and fit the four-parameter curve ( image 3 ).

[0065] The results show that the pre-diluted concentrations of HEK293-Luc and HaCat-Luc cell lines are about 137ng / mL and 411ng / mL, and the 3-fold serial dilution of 8 concentrations is on the lower plateau of the curve. Therefore, choose 150ng / mL and 400ng / mL The concentration of recombinant KGF-1 was used as the pre-diluted concentration of HEK293-Luc and HaCat-Luc cell lines, and was diluted 3 times.

[0066] 2. Optimization of heparin sodium concentrati...

Embodiment 3K

[0090] The methodological verification of embodiment 3KGF activity assay

[0091] 1. Precision

[0092] The precision of the method was evaluated using the experimental conditions determined in Example 2. One batch of recombinant KGF-1 samples was taken for activity determination, which was measured 3 times a day for 4 consecutive days, with 3 replicate wells for each dilution.

[0093] The results in Table 7 show that the intraday coefficient of variation (CV) of the HEK293-Luc cell line is 1.49-3.75%, and the intraday CV value is 2.82% after continuous measurement for 4 days; the intraday coefficient of variation (CV) of the HaCat-Luc cell line is 0.52-5.78 %, the intraday CV value is 5.57%. The intraday and interday CV values ​​of the two cell lines are both less than 6%, which shows that the precision of the method is good and can be used to measure the biological activity of recombinant KGF-1.

[0094] Table 7 The precision of HEK293-Luc and HaCat-Luc cell lines to mea...

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Abstract

The present invention relates to a reporter gene assay for measuring biological activity of recombinant human keratinocyte growth factors. The method utilizes plasmids comprising a SRE response element and overexpressed lentiviruses comprising a KGFR2IIIb gene to respectively transfect and infect HEK293 and HaCat cells to obtain a cell line stably expressing the SRE and KGFR2IIIb, after the cell line is stimulated by recombinant KGF-1 / KGF-2, an expression of a downstream luciferase reporter gene of a SRE-containing promoter, and a four-parameter curve is fitted according to reporter gene signal values to determine the biological activity of the recombinant KGF-1 / KGF-2. The rapid, sensitive and accurate quantitative detection method is established aiming at the biological activity measurement of the KGF, is short in test period and simple in operation, and has important guiding roles for quality controls in research, development and production of the recombinant KGF-1.

Description

technical field [0001] The invention relates to the field of recombinant drug activity detection, and establishes a rapid, sensitive and accurate method for measuring the biological activity of recombinant human keratinocyte growth factor (Keratinocyte growth factor, KGF) 1 and 2 (KGF-1 and KGF-2). Reporter gene activity assay method. Background technique [0002] KGF belongs to the fibroblast factor family, among which KGF-1 (FGF-7) is a growth factor that can stimulate the proliferation of epithelial cells discovered in 1989; KGF-2 (FGF-10) was discovered in 1996, and its structure and mitogenic activity are related to KGF-1 is similar. After KGF binds to its receptor, it can promote the proliferation, differentiation and migration of epithelial cells. On December 15, 2004, the US FDA approved Kepivance (palifermin) for the treatment of oral mucositis in bone marrow transplant patients receiving high-dose chemotherapy drugs. Kepivance is a recombinant human keratinocyte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/66
CPCC12Q1/66
Inventor 王军志饶春明姚文荣郭莹于雷秦玺史新昌刘兰
Owner NAT INST FOR FOOD & DRUG CONTROL
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