CN104974263A - Human recombinant [beta]-interferon fusion protein - Google Patents

Abstract

The invention relates to human recombinant [beta]-interferon fusion protein, which is a fusion protein of human [beta]-interferon and an anti-human serum albumin heavy chain single-domain antibody. The amino acid sequence of the fusion protein is represented as the Seq ID No.1 and the amino acid sequence of the anti-human serum albumin heavy chain single-domain antibody is represented as the Seq ID No.2. In the invention, by means of construction of the fusion expression cloning of the anti-human serum albumin heavy chain single-domain antibody and the [beta]-interferon and introduction into a host cell for expression, a heavy chain single-domain antibody-[beta]-interferon fusion protein having a long acting time is obtained.

Description

A kind of human recombinant interferon B fusion rotein

Technical field

The invention belongs to biomedicine field, relate to a kind of human recombinant interferon B fusion rotein.

Background technology

Interferon beta (is also called interferon-β, Interferon-β, IFN-β) be cytokine by fibrocyte in body and macrophages secrete, be a kind of glycoprotein, containing 166 amino acid, molecular weight is about 20,500 dalton, have anti-inflammatory function, antiviral, antiproliferative and immunoregulatory activity, it can be widely used in anti-infective, antitumor and antagonism autoimmune disorder as medicine.But interferon beta is short for plasma half-life, only have frequent drug administration could obtain gratifying curative effect.But so frequent drug administration is adding longer treatment cycle, makes patient be difficult to bear, in the urgent need to the interferon beta that the trituration time is long.

Human serum albumin (HSA) is natural substance delivery vehicles in blood of human body, in serum, content is the highest, and its relative molecular weight is about 65kDa, the strand ball-type protein be made up of 585 amino acid, without zymetology and immunologic competence, Half-life in vivo reaches 19 days.

Heavy chain antibody (the heavy-chainantibody of natural deletions light chain is there is in camel and shark class animal body, HCAb), this antibody only comprises a variable region of heavy chain (variable domain of heavychain of HCAb, VHH) and two conventional CH2 and CH3 districts, independent clone the VHH district expressed, be called single domain heavy chain antibody, there is good structural stability and antigen-binding activity, molecular weight only has 15kDa, solubility is high, not easily assemble, high temperature resistant etc. can cause Denaturing.

Bacteriophages display technology is a kind of most widely used Antibody library, this technology is by building antibody library, then antibody gene is expressed in phage surface, carries out washing in a pan sieve to the carrier of expression product with immobilization antigen, the antibody of high degree of specificity can be obtained.

Summary of the invention

The object of the present invention is to provide a kind of recombinant interferon B fusion rotein, this fusion rotein has the longer transformation period.

A kind of human recombinant interferon B fusion rotein of the present invention, be the fusion rotein of people's interferon beta and AHS's albumin single domain heavy chain antibody, its aminoacid sequence is as shown in SEQ ID NO.1.

According to the further feature of the fusion rotein that the present invention states, the aminoacid sequence of described AHS's albumin single domain heavy chain antibody is as shown in SEQ ID NO.2.

The present invention adopts alpaca as immune animal, uses bacteriophages display technology, and collect lymphocyte from immune alpaca peripheral blood, constructing storage capacity is 5 × l0 ⁷aHS's albumin (HSA) heavy chain antibody library.Screened by HSA, obtain the heavy chain antibody of the anti-HSA of 1 strain.The VHH cloning this antibody obtains single domain heavy chain antibody, and then structure single domain heavy chain antibody and interferon beta amalgamation and expression are cloned, and import to host cell and express, and obtains the single domain heavy chain antibody-interferon beta fusion rotein with longer action time.

Accompanying drawing explanation

Fig. 1 is at the recombinant expressed single domain heavy chain antibody of yeast cell-people's interferon beta fusion rotein schema.

Fig. 2 is after pPicZ α-6His-Zipper1-CTHS-VHH-IFN transformed yeast bacterium, the abduction delivering result of the single bacterium colony of picking 4.

Fig. 3 is single domain heavy chain antibody-people's interferon beta fusion rotein cytopathic effect inhibition result.

Embodiment

Below in conjunction with specific embodiment, the present invention will be further described, and object is the protection domain explaining content of the present invention and unrestricted invention.The test method of unreceipted actual conditions in embodiment below, usual conveniently condition, as people's works such as Sambrook, molecular cloning: test guide (New York, Cold SpringHabour Laboratory Press) described in condition, or according to manufacturer suggestion condition carry out.

1. the screening of anti-HSA single domain heavy chain antibody (VHH)

(1) immunity of alpaca

With human serum albumin (HSA) immunity two alpacas, immunization protocol is as follows:

Immunity alpaca antigen consumption: 0.5mg, 0.5mg, 1.0mg, 1.0mg, 2.0mg, 2.0mg, 4.0mg, 4.0mg.

The immunization interval time: 2 weeks.

Immunization method:

First time immunity: antigen Jia Fushi Freund's complete adjuvant is ground, subcutaneous multi-point injection.

Follow-up immunization: antigen Jia Fushi Freund's incomplete adjuvant, subcutaneous multi-point injection, 0.5ml/ point, 4 points/time.Take a morsel before each immunity blood, detects the titre of anti-HSA antibody in alpaca blood by ELISA method.

(2) structure of anti-HSA immunity phage antibody library

Collect immune alpaca peripheral blood, be separated lymphocyte, extract total serum IgE, reverse transcription synthesis cDNA.With Auele Specific Primer, adopt Chao Shi PCR method amplification variable region of heavy chain fragment.Structure storage capacity is 5 × l0 ⁷anti-HSA immunity phage antibody library, enzyme is cut and is identified that recombination fraction is 90%.

First round PCR primer:

5 ' primer: 5 '-GATGTGCAGCTGCAGGCGTCTGGRGGAGG-3 ' SEQ ID NO.3

5’-GTCCTGGCTCTCTTCTACAAGG-3’SEQ ID NO.4

3 ' primer: 5 '-CGCCATCAAGGTACCAGTTGA-3 ' SEQ ID NO.5

5’-GGTACGTGCTGTTGAACTGTTCC-3’SEQ ID NO.6

Second takes turns PCR primer:

5 ' primer:

5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTGTGG-3’SEQ ID NO.7

5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTRCAGCTGGTGGAGTCTGG-3’SEQ ID NO.8

5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGTCTGG-3’SEQ ID NO.9

5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGATGTGCAGCTGGTGGAGTCTGG-3’SEQ ID NO.10

5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGCCGTGCAGCTGGTGGATTCTGG-3’SEQ ID NO.11

5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGCGGTGCAGCTGGTGGAGTCTGG-3’SEQ ID NO.12

3 ' primer:

5’-GGACTAGTGCGGCCGCGTGAGGAGACGGTGACCTG-3’SEQ ID NO.13

5’-GGACTAGTGCGGCCGCTATGAGGAGACGGTGACCTG-3’SEQ ID NO.14

First round PCR reaction conditions: 94 DEG C of 5 minutes denaturations, 94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 50 seconds, 35 circulations, 72 DEG C 10 minutes.

Second takes turns PCR reaction conditions: 94 DEG C of 5 minutes denaturations, 94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 50 seconds, 30 circulations, 72 DEG C 10 minutes.

(3) enrichment isolation of anti-HSA immunity phage antibody library

Screening antigen is HSA, and with the carbonate buffer solution of pH9.6 dilution antigen, concentration is 5 μ g/ml, gets 2ml bag by immunotubes, antagonist storehouse carry out 3 take turns enrichment isolation after, with phage-ELASA method screening positive clone.

(4) screening of anti-HSA immunity phage antibody library

Select the single bacterium colony after 192 enrichment isolation, 37 DEG C of joltings are spent the night.Then transfer in fresh 200ul2YTGA by 1:100,37 DEG C of joltings 2.5 hours, M13K07 superingection.3,500rpm, 10min collect thalline, then use the resuspended bacterial precipitation of 200ul2YTGAK.30 DEG C of joltings are spent the night, and centrifugal 15min, got supernatant and identified next day 11,000rpm.With HSA bag by elisa plate, add above-mentioned supernatant, it is two Hangzhoupro that HRP marks mouse-anti phage M13 monoclonal antibody (GE Healthcare), and TMB develops the color.M13K07 is negative control, and immune alpaca serum is positive control.Antagonist storehouse is screened, and obtains 1 strain positive colony.Then check order to positive colony, obtain the VHH sequence of anti-HAS, its sequence is SEQ ID NO.2.

2. express the structure of the Yeast expression carrier of VVH-IFN

The DNA sequence dna of His-Zipper2-CTHS is by being inserted in pPicZ α respectively by EcoRI and XhoI site, the VHH of anti-HAS is inserted again by BsaI and Xhol site, interferon beta IFN is inserted finally by XhoI and XbaI site, build expression plasmid of yeast pPicZ α-6His-Zipper1-CTHS-VHH-IFN, wherein, the sequence of 6His-Zipper1-CTHS-VHH-IFN is SEQ ID NO.1.

Wherein, CTHS is writing a Chinese character in simplified form of C-terminal (C-SUMO) of SUMO.The N end of corresponding SUMO is referred to as NTHS(or NT), Zipper refers to leucine zipper.

Embodiment 2. yeast expression and protein purification

Plasmid pPicZ α-6His-Zipper1-CTHS-IFN is transformed into pichia spp, picking mono-clonal, is seeded in the 1L dividing plate shaking flask containing 100ml BMGY substratum.28-30 DEG C in shaking table, 250-300rpm grows to OD600=2-6, with the centrifugal 5min collecting cell of 1500-3000g, remove supernatant, with the BMMY re-suspended cell (about 10-20ml) of the former culture volume of 1/5-1/10, being inoculated in l00ml dividing plate shaking flask, covering bottleneck with 2 layers of sterile gauze or cheese cloth, putting into shaking table and continuing to cultivate.Every 24 hours, add methyl alcohol to final concentration be 0.5% with induction.

After induction terminates, with the centrifugal 30min of 12,000rpm, get supernatant, be 0.45 μm of membrane filtration with aperture, then carry out Ni-NTA affinity chromatography, adopt continuous gradient type of elution (balance liquid: 50mM Tris(pH8.0), 50mM NaCl, 2.5%Glycerol; Elutriant: 50mM Tris(pH8.0), 50mM NaCl, 2.5%Glycerol, 500mM Imidazol), collect by peak, each collection sample carries out SDS-PAGE electrophoresis detection.

Merged by collection sample containing target protein, add the mixing of 6His-NTHS-Zipper2 albumen, 6His-Zipper1-CTHS and 6His-NTHS-Zipper2 can rebuild SUMO protein molecular (the method is shown in patent ZL200810036879.X).Then mixture is carried out SUMO enzyme to cut, it is carry out according to the suggestion of Genecopoeoia company CoolcutTM protease product description that excision restructuring becomes the reaction conditions that complete SUMO molecule enzyme cuts.

Then, digestion products is carried out Ni-NTA affinity chromatography (balance liquid: 50mM Tris(pH8.0), 50mM NaCl, 2.5%Glycerol, elutriant: 50mM Tris(pH8.0), 50mM NaCl, 2.5%Glycerol, 500mM Imidazol), collect and penetrate liquid, carry out SDS-PAGE electrophoresis detection subsequently, obtain VHH-IFN albumen.

Embodiment 3. interferon beta cytopathic-effect inhibition assay (cytopathic effect inhibition, CPEI)

Dilution for difference interferon beta sample is added in 96 hole flat-bottomed plates, 100 μ l/ holes; Dilution for difference standard substance are added in 96 hole flat-bottomed plates, 100 μ l/ holes; Every hole adds 4X106/ml Wish cell, 100 μ l/ holes; Except Wish cell control well, every hole adds VSV virus, 50 μ l/ holes; Put 5%CO2,48-52 hour cultivated by 37 DEG C of incubators; When cytopathy appears in Microscopic observation virus control wells, abandon supernatant, add Viola crystallina dye liquor and dye 5 minutes; Use running water culture plate, until washing fluid is colourless, dry culture plate; Every hole adds 70% ethanol 150 μ l/ hole.Viola crystallina is fully dissolved; Colorimetric in microplate reader, reads the numerical value of 600nm wavelength.

Adopt the interferon beta that BIOGEN company of the Avonex(U.S. produces) in contrast, the curative for multiple sclerosis that this medicine is used in nineteen ninety-five by U.S. FDA approval, result shows, and human recombinant interferon B fusion rotein of the present invention (VHH-IFN albumen) has and contrasts similar activity.

Claims (2)

1. a human recombinant interferon B fusion rotein, be the fusion rotein of people's interferon beta and AHS's albumin single domain heavy chain antibody, its aminoacid sequence is as shown in SEQ ID NO.1.

2. fusion rotein according to claim 1, is characterized in that: the aminoacid sequence of described AHS's albumin single domain heavy chain antibody is as shown in SEQ ID NO.2.