Fc fusion protein and application thereof
A technology of fusion protein and antigen, applied in Fc fusion protein and its application field, can solve the problem of lack of fusion protein, etc., and achieve the effect of improving curative effect
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Embodiment 1
[0032] Example 1 Synthetic DNA
[0033] The deoxyribonucleic acid fragment FcεRIα (see SEQ ID NO.3 in the sequence listing for details, can also be found in GenBank: E07699.1; the corresponding protein sequence is shown in SEQ ID NO.1 in the sequence listing for details, and can also be found in NCBI ReferenceSequence: NP_001992.1) and hIL5Rα (see SEQ ID NO.4 for details, also found in NCBI ReferenceSequence: NM_000564.4; for the corresponding protein sequence, see SEQ ID NO.2 for details, also found in ACCESSION NP_000555) and human IgG1 Hinge (hinge) connection as arms of Fc (CH2 and CH3 of human IgG1). The name of the recombinantly expressed protein after ligation is FcepsilonRIalph-hIL5Rα-Fc.
[0034] The synthesized DNA was amplified by PCR (primers had enzyme cutting sites), purified, digested, and purified again, and the digested fragments were combined with the expression vector PGKpuro-EF (purchased from Biofeng, http: / / www.biofeng .com / ) to connect. The expression...
Embodiment 2
[0035] Embodiment 2 fusion protein expression
[0036] Using Fugene, 5 μg of the expression plasmid prepared in Example 1 was transformed into 5×10 5CHO cells (purchased from ATCC). After 24 hours, select with 5 μg / ml puromycin (puromycin) (the operation is routine in the field, specifically, replace the cell culture medium containing 5 μg / ml puromycin every 2 to 3 days) until colonies are obviously formed. About 8-10 clones were picked and further amplified. When the number of cells reaches approximately 1 x 10 6 , use 5×10 5 Cells were cultured in 10 ml of antibiotic-free culture medium. After 24 hours, the culture medium was collected to check the production of related proteins. Select 2 to 3 high-expressing clones, continue to select for 1 month in culture medium containing puromycin, select for 1 month without puromycin, and finally select the highest-expressing clone as a stable cell line.
[0037] For the cell culture solution expressing Fc fusion protein, the prot...
Embodiment 3F
[0038] The performance measurement of embodiment 3Fc fusion protein
[0039] 1. Detect the content and activity of FcεRIα or hIL-5Rα in the Fc fusion protein by ELISA. For the detection of FcεRIα, Human FcERI / FcERI ELISA Kit (SandwichELISA) from Lifespan BioSciences, Inc. was used. The operation steps are carried out according to the requirements of its kit. For the detection of hIL5Rα, Interleukin 5 ReceptorΑ, ELISA Kit (#MBS073569) from Mybiosource.com was used. The operation steps are carried out according to the requirements of its kit.
[0040] Test results such as figure 2 As shown in Table 1, it can be seen that after the stable cell line culture fluid is purified by Protein A column, the fusion protein components contained in it are detected by ELISA, and contain the activities of FcεRIα and hIL5Rα. It shows that each protein component of the fusion protein produced by the cell line retains the structure and function of the original protein.
[0041] Table 1 The ...
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