PEG interleukin 15
A technology of human interleukin and conjugates, applied in peptide/protein components, chemical instruments and methods, medical preparations of non-active ingredients, etc., can solve problems such as no research
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Embodiment 1
[0045] Example 1 Preparation of rhIL-15
[0046] Test materials: DNA restriction endonuclease, T4DNA ligase, TaqDNA polymerase, and M-MLV reverse transcriptase are all products of GIBCO; Escherichia coli host strain DH5a, BL21 (DE3) (purchased from Invotrogen); pVB220 vector (purchased from Novagen)
[0047] 1 Design and synthesis of primers: design a pair of primers according to the gene sequence of hIL1-15 reported, upstream primer P1: 5'-C ctcgagTTCATGAACTGGGTGAATGTAATAAG-3', including the start codon ATG, peripheral xho I restriction site; downstream primer P2: 5'-CGGATCCttaTTAAGAAGTGTTGATGAAGATTTG-3', change the stop codon into a strong terminator TAA frequently used in prokaryotic systems, peripheral Bam H I restriction site, primers were synthesized by Beijing Sanbo Polygala Biotechnology Co., Ltd.
[0048] 2 Preparation of mononuclear cells rich in rhIL-15mRNA and extraction of total RNA, normal human peripheral blood leukocytes were separated by conventional dens...
example 2
[0052] Example 2, recombinant integrated interferon variant PEGylated conjugate (mPEG 20 -rhIL-15) modification conditions
[0053] rhIL-15 solution (rhIL-15, concentration 10 mg / mL, buffered in 100 mM PB, pH 7.0) was prepared by Beijing Kain Technology Co., Ltd., mPEG-ALD-20KD (purity>95%, relative molecular weight Mass is 20×10 3 ) was purchased from Beijing Kaizheng Bioengineering Development Co., Ltd., sodium cyanoborohydride (CH3BrNa) solution was purchased from Sigma Company, SDS-PAGE related reagents and solutions and various analytical grades were from Shanghai Sangon. Constant temperature magnetic stirrer (Jiangsu Zhongda Instrument Factory), electrophoresis instrument, Q Sepharose F.F., Superdex75 molecular sieve, AKTA explore chromatography system, and SDS-PAGE electrophoresis system are all products of Pharmacia.
[0054] 1. The influence of reaction pH value on the modified product
[0055] The rhIL-15 solution was made up to 2 mg / mL with 100 mmol / L PB (p...
Embodiment 3
[0065] Example 3 Purification of mPEG-rhIL-15
[0066] The reaction mixture was first dialyzed against 50mM Tris-HCl buffer (pH 6.5), and then the sample was loaded on a Q Sepharose FF cation exchange column equilibrated with the same buffer. The adsorbed protein was eluted with a 0-1M NaCl gradient. PEG-interferon was further purified by Superdex 75, and the mobile phase was 50mM Tris-HCl buffer (pH 6.5) containing 0.15M sodium chloride. The eluted active fractions were collected, and the SDS-PAGE test results showed that PEG-interleukin had been purified to a single electrophoresis band.
[0067] The process of coupling rhIL-15 with 10kDa and 40kDa is the same as above, and the electrophoresis of the purified PEG interleukin is a single band, see the attached image 3 .
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