Human interferon alpha derivative and polyethylene glycol modified substance thereof

[0034] Example 1: gly-gly-gly-gly-gly-IFNα-2a (hereinafter referred to as Gly(5)-IFN

[0035] α) Secreted expression in Pichia pastoris

[0036] 1. Design and acquisition of target gene:

[0037] The amino acid sequence of IFNα-2a is shown in SEQ ID No. 1. After obtaining the cDNA of IFNα-2a through Genebank, change the corresponding codon to yeast preference, and add the corresponding nucleotide sequence of gly-gly-gly-gly-gly at the N-terminal. This cDNA sequence was used for the construction of the pGAPZα expression plasmid (purchased from invitrogen) of Pichia pastoris GS115 (purchased from invitrogen). Secreted expression is achieved after transformation of GS115 host bacteria. Therefore, the KEX2 enzyme recognition site CTC GAG AAA AGA was added to the design, where CTC GAG is the XhoI restriction site. At the same time, a double stop codon TGA TAA and NotI digestion sequence GCG GCCGC was introduced at the 3'end. The cDNA sequence of IFN used in the yeast expression vector is

[0054] Example 2: Purification of Gly(5)-IFNα

[0055] Step 1: Cationic gel column (CM Sepharose F.F. gel purchased from Amersham Biosciences) chromatography:

[0056] Use pH3.8-4.6 acetate buffer for column loading, elution, and electrophoresis monitoring to collect the target. Then, the target was dialyzed with a pH 7.5-8.5 Tris-HCl buffer solution.

[0057] Step 2: Anion gel column (DEAE Sepharose F.F. gel purchased from Amersham Biosciences) chromatography:

[0058] Use pH 7.5-8.5 Tris-HCl buffer solution for column loading, elution, and collection of the target substance. Then dialyze the target with a pH 7.5-8.5 phosphate buffer solution.

[0059] Example 3: Preparation and purification of PEG coupling modified samples

[0060] 1. The Gly(5)-IFNα sample obtained in Example 1 was dialyzed with phosphate buffer (pH 6.0), and then ALD-PEG 40KD (purchased from Beijing Jiankai Technology Co., Ltd.) with a molar ratio of 1:3 was added. Modification is carried out at 2-15°C, and the reaction time is 40 hours. The modified samples obtained were tested by SDS-PAGE. The test results showed that after PEG coupling modification, the molecular weight increased from 19,000 Daltons to nearly 90,000 Daltons, and the modification rate reached about 60%. The target product was obtained. mPEG-Gly(5)-IFNα.

[0061] 2. Purification of mPEG-Gly(5)-IFNα:

[0062] The mPEG-Gly(5)-IFNα was adjusted with acetate buffer (pH value 4.0-5.0) to terminate the reaction, and then purified on a cationic gel column SP Sepharose F.F. gel column. Use NaCl (0.15M) solution for elution, collect the target, and the purity is more than 95%. The result is image