Application of recombinant human TNFR-Fc fusion protein mutant
A technology of fusion proteins and mutants, which can be used in drug combinations, peptide/protein components, medical preparations containing active ingredients, etc., and can solve problems such as weak effect
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Embodiment 1
[0042] Embodiment 1 is used for the construction of the cell strain of T0001 functional evaluation
[0043] 1.1 Construction of CHO cell lines expressing mTNFα
[0044] A CHO cell line expressing mTNFα was constructed referring to the literature method of Arora T, et al., see Cytokine.2009; 45(2):124-131. Briefly, a membrane-bound TNFα (mTNFα) mutant was constructed by site-directed mutagenesis to delete the amino acid sequence from 77 to 88, and its outer part could not be degraded by TACE enzyme. The DNA sequence of the mTNFα mutant was cloned into pIRES expression vector (Clontech Laboratories, Inc. Cat. 631605). The pIRES vector contains the CMV IE promoter, which can drive the expression of mTNFα gene and the selection of neomycin resistance marker in mammalian cells. The mTNFα cell line was constructed by transfecting mTNFα / pIRES into CHO-K1 (ATCC, CCL-61) cells, and the transfected cells were screened by 500 μg / ml G418, and the high expression of mTNFα was screened by...
Embodiment 2
[0048] Example 2 In mouse L929 cells, neutralization assay for cytotoxicity induced by soluble TNFα
[0049] Mouse L929 cells were cultured in DMEM (source culture, Cat.L170) medium containing 3% FBS (Moregate, Cat. 4 Inoculate a 96-well cell culture plate at a density of 37°C for 24 hours and then add different concentrations (0.001nM, 0.003nM, 0.009nM, 0.026nM, 0.079nM, 0.237nM, 0.711nM and 2.133nM) of the above-mentioned different TNFα Antagonist, 1 μg / ml actinomycin D (Solarbio, Cat.A8030) and 20 IU / ml sTNFα (NIBSC, 12 / 154), and continued to culture at 37°C for 18 hours. The wells without TNFα antagonist and sTNFα were used as blank control, and the cell viability was measured by MTS reagent (Promega, Cat.G5430). After adding MTS reagent to each well and incubating for 4 hours, the readings at 490 nm were collected by a microplate microplate reader SpectraMax M2e (Molecular Devices). Cell survival rate (%)=absorbance value of the drug treatment group / absorbance value of ...
Embodiment 3
[0051] Example 3 Binding of TNF antagonists to mTNFα
[0052] T0001 was biotin-labeled, and the labeling reagent was Sulfo-NHS-LC-Biotin (purchased from Thermo Scientific, Cat. 21327). Different concentrations (0.153nM, 0.61nM, 2.441nM, 9.766nM, 39.063nM, 156.25nM, 625nM, 2500nM and 10000nM) of the above-mentioned different TNFα antagonists and 13.3nM biotinylated T0001 were combined with the 5× prepared in Example 1.1 10 5CHO cells expressing mTNFα or non-transfected CHO cells were incubated at 4°C for 0.5 hours. The cells were washed with PBS to remove unbound drugs, and R-phycoerythrin-conjugated avidin (R-phycoerythrin-conjugated avidin; purchased from Life Technologies, Cat.A2660) was added and incubated at 4°C for 0.5 hours. The cells added only with biotinylated T0001 were used as the control group, and the mean fluorescence intensity (MFI) was measured by FACSCalibur flow cytometry. Percent competitive activity (%)=(MFI of control group-MFI of drug group) / MFI of c...
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