Chemical conjugate of recombinant deleted human keratinocyte growth factor type I

Abstract

The invention discloses a coupling matter formed by chemical modification of polyethylene glycol of a deletion-recombination human keratinocyte growth factor I type. The coupling matter is characterized in that covalent chemical coupling is performed on activated polyethylene glycol molecules with the molecular weight being 5K to 40K and free sulfhydryl in 79-site cysteine of the deletion-recombination human keratinocyte growth factor I type (delta 23KGF-I) with the deletion of 23-site amino acid at the N terminal, and then column chromatography purification is performed to obtain the pegylation deletion-recombination human keratinocyte growth factor I type. The coupling matter retains the biological effects of human keratinocyte growth factors, changes the internal metabolic dynamics characteristic of the factors and has values of prevention and therapy on digestive tract mucosa damage or ulcerative mucosal inflammation caused by radiotherapy and chemotherapy as well as on acute chemical liver damage.

Description

technical field

[0001] The invention belongs to the field of bioengineering, and specifically relates to the recombinant preparation of deleted human keratinocyte growth factor type I, the polyethylene glycol chemical modification of deleted human keratinocyte growth factor type I, as well as the purification before and after modification and its application. Background technique

[0002] Keratinocyte growth factor (KGF) belongs to the fibroblast growth factor (FGF) family. At present, two types of keratinocyte growth factor (KGF) have been discovered, namely keratinocyte growth factor I (KGF-I) and keratinocyte growth factor type II (KGF-II), respectively named as FGF-7 and FGF-10 (Aaronoson SA. et al. J. Annals of the New York Academy of science, 1991, 638: 62-77.). Rubin et al. (Rubin, J.S., et al.Proc Natl Acad Sci USA, 1989.86 (3): p.802-6.) isolated and purified KGF-1 from the culture supernatant of human fetal lung fibroblasts, and It was found that it can promote t...

Examples

example 1

[0036] Example 1. Recombinant Expression of Human Keratinocyte Growth Factor Type I (Δ23hKGF) Deleting N-terminal 23 Amino Acids

[0037] According to the amino acid sequence of Δ23hKGF (SEQ ID No: 1), the corresponding cDNA sequence of Escherichia coli biased codon was designed and included the 5' end and 3' respectively design restriction endonuclease Nde I and BamH I site (SEQ ID No: 2 ). Entrust Bao Bioengineering (Dalian) Co., Ltd. to artificially synthesize the whole gene, insert it into the pUC18 vector and name it pUC18-Δ23hKGF / DH5α.

[0038] Plasmid pUC18-Δ23hKGF DH5α and expression vector plasmid pET-3c were double-digested with restriction endonucleases Nde I and BamH I, and subjected to 1% agarose electrophoresis to recover the fragment of about 4638bp after digestion of pET-3c and pUC18- After Δ23hKGF / DH5α digestion, the fragment of about 450bp was digested, and the two fragments were connected with T4 ligase into plasmid pET-3c-Δ23hKGF, and then the plasmid pET-...

example 2

[0040] Example 2, the purification and preparation of Δ23hKGF and r-Δ23hKGF

[0041] Take out the bacteria from the refrigerator at -20°C, put them into the bacterial lysate (50mmol / L Tris-HCl, 5mmol / LEDTA, 10μg / ml DNase, 2mmol / L MgCl) at 1:15w / v 2 , pH 8.5-9.0), stirred at 37°C and added trisodium citrate with a final concentration of 0.1M when the bacteriostasis solution was not viscous, and continued to stir for 0.5h. Adjust the pH to 7.5, centrifuge at 10,000 rpm for 15 min, and collect the supernatant. Put the supernatant on the SP-Sepharose Fast Flow column (equilibrium solution: 50mmpb, pH7.5), rebalance, and elute with a linear gradient of 50-500mmNacl, collect the elution peak, and detect by SDS-PAGE to determine the content of the main target protein. Elution peak. The target sample eluted by SP is put on the Heparin column (equilibrium liquid: 20mm pb, pH7.5), rebalanced, and eluted with a linear gradient of 50-500mm Nacl, and the elution peak is collected, detect...

example 3

[0042] Example 3, mPEG-MAL-20K modification of r-Δ23hKGF and purification after modification (PEG-20K-Δ23KGF)

[0043] The r-Δ23hKGF solution with a purity greater than 95% was diluted with 100mmol / L phosphate buffer, pH 6.5, to 2 mg / mL, and mixed with r-Δ23hKGF and mPEG-MAL-20KD (linear type, purchased from Beijing Jiankai Technology Co., Ltd. Co., Ltd.) with a mass ratio of 1:3, weighed mPEG-MAL-20KD and added it to r-Δ23hKGF solution, stirred and reacted at 100 r / min at 25°C for 2 hours, and lowered the pH with hydrochloric acid to terminate the reaction. The modified r-Δ23hKGF (PEG-20K-Δ23KGF) solution was dialyzed overnight (dialysate: 20mmNaAc, pH 5.0), and the dialyzed PEG-20K-Δ23KGF solution was applied to SP-Sepharose Fast Flow column (equilibrium: 20mmNaAc, pH5.0) .0), rebalanced, eluted with a linear gradient of 50-500mm Nacl, collected the eluted peak, and detected by SDS-PAGE and HPLC, obtained PEG-20K-Δ23KGF with a purity greater than 95% ( figure 2 , image 3 )...

technical field

[0001] The invention belongs to the field of bioengineering, and specifically relates to the recombinant preparation of deleted human keratinocyte growth factor type I, the polyethylene glycol chemical modification of deleted human keratinocyte growth factor type I, as well as the purification before and after modification and its application. Background technique

[0002] Keratinocyte growth factor (KGF) belongs to the fibroblast growth factor (FGF) family. At present, two types of keratinocyte growth factor (KGF) have been discovered, namely keratinocyte growth factor I (KGF-I) and keratinocyte growth factor type II (KGF-II), respectively named as FGF-7 and FGF-10 (Aaronoson SA. et al. J. Annals of the New York Academy of science, 1991, 638: 62-77.). Rubin et al. (Rubin, J.S., et al.Proc Natl Acad Sci USA, 1989.86 (3): p.802-6.) isolated and purified KGF-1 from the culture supernatant of human fetal lung fibroblasts, and It was found that it can promote t...

Examples

example 1

[0036] Example 1. Recombinant Expression of Human Keratinocyte Growth Factor Type I (Δ23hKGF) Deleting N-terminal 23 Amino Acids

[0037] According to the amino acid sequence of Δ23hKGF (SEQ ID No: 1), the corresponding cDNA sequence of Escherichia coli biased codon was designed and included the 5' end and 3' respectively design restriction endonuclease Nde I and BamH I site (SEQ ID No: 2 ). Entrust Bao Bioengineering (Dalian) Co., Ltd. to artificially synthesize the whole gene, insert it into the pUC18 vector and name it pUC18-Δ23hKGF / DH5α.

[0038] Plasmid pUC18-Δ23hKGF DH5α and expression vector plasmid pET-3c were double-digested with restriction endonucleases Nde I and BamH I, and subjected to 1% agarose electrophoresis to recover the fragment of about 4638bp after digestion of pET-3c and pUC18- After Δ23hKGF / DH5α digestion, the fragment of about 450bp was digested, and the two fragments were connected with T4 ligase into plasmid pET-3c-Δ23hKGF, and then the plasmid pET-...

example 2

[0040] Example 2, the purification and preparation of Δ23hKGF and r-Δ23hKGF

[0041] Take out the bacteria from the refrigerator at -20°C, put them into the bacterial lysate (50mmol / L Tris-HCl, 5mmol / LEDTA, 10μg / ml DNase, 2mmol / L MgCl) at 1:15w / v 2 , pH 8.5-9.0), stirred at 37°C and added trisodium citrate with a final concentration of 0.1M when the bacteriostasis solution was not viscous, and continued to stir for 0.5h. Adjust the pH to 7.5, centrifuge at 10,000 rpm for 15 min, and collect the supernatant. Put the supernatant on the SP-Sepharose Fast Flow column (equilibrium solution: 50mmpb, pH7.5), rebalance, and elute with a linear gradient of 50-500mmNacl, collect the elution peak, and detect by SDS-PAGE to determine the content of the main target protein. Elution peak. The target sample eluted by SP is put on the Heparin column (equilibrium liquid: 20mm pb, pH7.5), rebalanced, and eluted with a linear gradient of 50-500mm Nacl, and the elution peak is collected, detect...

example 3

[0042] Example 3, mPEG-MAL-20K modification of r-Δ23hKGF and purification after modification (PEG-20K-Δ23KGF)

[0043] The r-Δ23hKGF solution with a purity greater than 95% was diluted with 100mmol / L phosphate buffer, pH 6.5, to 2 mg / mL, and mixed with r-Δ23hKGF and mPEG-MAL-20KD (linear type, purchased from Beijing Jiankai Technology Co., Ltd. Co., Ltd.) with a mass ratio of 1:3, weighed mPEG-MAL-20KD and added it to r-Δ23hKGF solution, stirred and reacted at 100 r / min at 25°C for 2 hours, and lowered the pH with hydrochloric acid to terminate the reaction. The modified r-Δ23hKGF (PEG-20K-Δ23KGF) solution was dialyzed overnight (dialysate: 20mmNaAc, pH 5.0), and the dialyzed PEG-20K-Δ23KGF solution was applied to SP-Sepharose Fast Flow column (equilibrium: 20mmNaAc, pH5.0) .0), rebalanced, eluted with a linear gradient of 50-500mm Nacl, collected the eluted peak, and detected by SDS-PAGE and HPLC, obtained PEG-20K-Δ23KGF with a purity greater than 95% ( figure 2 , image 3 )...

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