TNFR-Fc fusion protein and usage thereof
A fusion protein, human immunoglobulin technology, applied in the field of new recombinant TNFR-Fc fusion protein, can solve the problems of low biological activity, low binding ability, short effective time and so on
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Embodiment 1
[0095] Example 1. Obtaining of hTNFRII-Fc fusion gene expression plasmid
[0096] 1. Acquisition of hTNFRII-hIgG2 / Fc gene
[0097] hTNFRII-hIgG2 / Fc is a fusion protein, its gene sequence has 1389bp, and it encodes a protein of 463 amino acids. Among them, the N-terminal hTNFRII extracellular region soluble fragment (mature protein) gene (705bp / 235aa), the sequence is as SEQ ID NO: 1 (derived from Genebank NM001066); the C-terminal hIgG2-Fc gene (684bp / 228aa), the sequence is SEQ ID NO : 2 (derived from Genebank AK131045).
[0098] Design oligonucleotide fragments for synthetic genes (Table 1): the length of the fragments is generally about 70 bases, and there are about 15 bases complementary between two adjacent oligonucleotide fragments. Try to ensure that each oligonucleotide The annealing temperature of the complementary bases of the fragments is the same. The hTNFRII gene was assembled and synthesized by overlap extension PCR.
[0099] Table 1 The artificially synthesi...
Embodiment 2
[0130] Expression and purification of embodiment 2.hTNFRII-Fc fusion gene
[0131] 1. Transfection and screening of CHO cells
[0132] Cell line and culture conditions: FreeStyle CHO-S cells (Invitrogen), culture conditions are freestyle medium (Invitrogen), containing 10% dialyzed serum (Hyclone), cultured in 10% CO 2 , 37 ℃ thermostat. G418, MSX (Sigma).
[0133] Lipofectamine produced by Invitrogen was used for transfection TM 2000 liposome transfection kit, operate according to the instructions. As a control, CHO-S cells were transfected with pIRES empty vector. G418 (200 μM) and MSX (50 μg / ml) were used for combined pressurization screening, and the medium was replaced once every 3 days. After 20 days of pressurization, transfer to a 96-well plate for culture and screening by the limiting dilution method. After 1 week of culture, the culture supernatant was taken for ELISA Determination.
[0134] Mouse anti-human TNFR primary antibody (Sigma) was coated with ELISA ...
Embodiment 3
[0141] Embodiment 3. Physicochemical activity detection of hTNFRII-Fc fusion protein
[0142] 1. SDS-PAGE protein electrophoresis and Western blotting
[0143] Take 20 μl of purified protein for SDS-PAGE electrophoresis, the concentration of the electrophoresis separation gel is selected as 10%, the result is as follows Figure 4 . Please refer to the second edition of "Molecular Cloning" and the experimental technique of protein electrophoresis for the specific operation steps.
[0144] The samples were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. The membrane was blocked with 5% milk solution containing 0.5% Tween and bound to antibodies, the primary antibody was mouse anti-human TNFR (Sigma), the secondary antibody was goat anti-mouse IgG (Calbiochem) labeled with horseradish peroxidase, PBS / 0.5% Tween washed 3 times, TMB color development, the results are as follows Figure 5 .
[0145] 2.L929 Cytotoxicity Neutralization Test
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