CN1500811A - Confluent protein of human interferon and human IgGFc variant without cracking ability and preparing method thereof - Google Patents

Abstract

The present invention belongs to the field of biomedicine technology, and discloses one kind of fusion protein of human interferon (HuIFN) and lysis lacking human IgGFc variant (vFc). The fusion protein has high similar bioactivity with human recombinant interferon, contains human IFN, 20 or less flexible amino acid peptide joints and human IgGFc variant. The Fc variant has no lysis and exhibits very low bad Fc interventing side effect. The present invention discloses one high level expression process of preparing the fusion protein. The HuIFN-L-vFc fusion protein of the present invention has long serum half time and extremely high bioactivity, improved pharmacodynamic performance and lowered injection frequency for patient.

Description

The fusion rotein and the preparation of the human IgG Fc variant of human interferon and no cracking performance

Technical field

The invention belongs to the biological medicine technology field.Be specifically related to the fusion rotein and the preparation of the human IgG Fc variant of a kind of human interferon and no cracking performance.

Background technology

Interferon, rabbit (IFN) is an anthropoid interior albumen that produces, and has opposing virus, stops biological activitys such as cell proliferation and adjusting immune response.Difference based on plysiochemical and biological has three kinds of Interferon, rabbit: IFN-alpha (IFN α), IFN-beta (IFN β) and IFN-gamma (IFN γ).Wherein IFN α and IFN β are referred to as first kind Interferon, rabbit.Interferon alpha/beta receptor on the cell surface that first kind Interferon, rabbit target responds.After IFN combined, the cognition that is subjected to that is activated caused tyrosine phosphorylation.Then, a series of signal transduction takes place and stimulating some gene transcription, causes the synthetic of differential protein.These by the promoted albumen of Interferon, rabbit often have Interferon, rabbit biological activity (referring to, as people such as Stark, Annu.Rev.Biochem., 67:227-264,1998).Many these classes are by the short albumen of giving birth to of Interferon, rabbit, and as 2-5 OAS, neopterin and Mx albumen etc. all are media very important in the immune response.

Recombined human interferon-alpha (rHuIFN α) comprises recombinant interferon-α _2a(rHuIFN α _2a) and interferon-' alpha ' _2b(rHuIFN α _2b), many countries be widely used in treatment viral hepatitis type b, C type hepatitis, with some cancer, comprise the Kaposi sarcoma that elytroncus (elytrophyma), hairy cell leukemia, chronic medullary cell leukemia, melanoma and AIDS cause.Recombinant human interferon alpha 2-β (rHuIFN β) has been used for the treatment of multiple sclerosis in American-European countries.IFN β is used for the treatment of the illness that cancer and some virus cause in clinical trial.As with other cytohormone after being injected into human body, it is very short that the serum of Interferon, rabbit is removed the transformation period.Because Interferon, rabbit natural in the human body only produces in the part, also short during the effectiveness, the serum of rHuIFN α and rHuIFN β remove the transformation period all have only a few hours (referring to, as Physician ' s Desk Reference).In order to reach suitable drug effect, must often inject the IFN of high dosage.Therefore among the treatment bad side effect is often arranged.Comprise being presented at nerve internal secretion and immune toxicity.At the shortcoming of these rHuIFN, we are intended to develop the product of improvement, prolong serum half-life, and keep its biological activity, thereby increase drug effect.

Existing report with polyoxyethylene glycol (PEG) be connected in range protein (comprising IFN) can obtain because of long half time render a service in vivo higher derivative (referring to, as Baker, Rev.Gastroenterol.Disord., 1:87-99,2001; People such as Zalipsky, " PEG chemistry; The application of biotechnology and biological medicine ", 347-370 page or leaf, 1992).Usually the protein puted together of PEG than the proteinic external biological activity of their not modified parental generations low (referring to, as people such as Eliason, Stem Cells, 18:40-45,2000).And render a service in these modified aleuroplasts increase to small part be because reduced by the increase of its molecular weight kidney scavenging(action) (referring to, as people such as Yamaoda, J.Pharmaceut.Sci., 83:601-606,1994).

The immunoglobulin (Ig) of IgG class is rich in protein in the human blood.Their transformation period can be up to 21 days.Existing report Fc zone with IgG combine with other protein (as various cytokines and soluble receptors) and form fusion rotein (referring to, as people such as Capon, Nature, 337:525-531,1989; People such as Chamow, Trends Biotechnol., 14:52-60,1996; U.S. Patent No. 5,116,964 and 5,541,087).The prototype fusion rotein is the heterodimeric protein matter that connects by the cysteine residues in the IgGFc hinge region, makes the protein-based IgG of being similar to molecule but does not have CH1 zone and light chain.Because structural homology, the Fc fusion rotein shows the interior medicine dynamics characteristic suitable with the human IgG of similar isotype.This method be used for some clinically very important cytokine (as IL-2, IFN β, IFN-α _2aWith IFN-α _2b) and soluble receptors (as TNF-Rc and IL-5-Rc) (referring to, as U.S. Patent No. 5,349,053,5,723,125,5,908,626 and 6,224,867).

Existing people has reported erythropoietin (EPO) derivative (as dipolymer).Compare with the EPO monomer, the fusion rotein that contains 2 complete EPO zones (3 to the 7 amino acid peptide joints of being separated by) show the activity that weakens (referring to, as people such as Qiu, J.Biol.Chem., 273:11173-11176,1998).Yet, when the length of the interregional peptide linker of these two EPO is 17 amino acid, dimer EPO molecule show improve considerable external and intravital activity (referring to, as people such as Sytkowski, J.Biol.Chem., 274:24773-24778,1999; U.S. Patent No. 6,187,564).The length of two intermolecular peptide linkers of EPO is extremely important, may be because of its flexible influence to these molecular structures, but also be not limited to this explanation.

In the Fc fusion protein molecule that major part has been reported, hinge region is as the interval between Fc zone (at carboxyl terminal) and cytokine or soluble receptors (at aminoterminal), make these two portions of this molecule can distinguish functionating (referring to, as people such as Ashkenazi, Current Opinionin Immunology, 9:195-200,1997).The flexible peptide linker that adds between people IFN and human IgG Fc may improve the external biological activity of HuIFN-L-Fc in two ways: (1) keeps Fc zone and IFN to go up the distance of IFN receptor binding site, (2) keep the distance in an IFN and another IFN zone, thus make the IFN zone can be respectively with cell surface on the IFN receptor response.

The Fc zone of human normal immunoglobulin plays an important role in the immune defense of eliminating pathogen.The effector function of IgG is main machine-processed by two kinds by the Fc mediation: (1) and cell surface Fc acceptor (Fc _γRs) combination, cause by relying on cytotoxicity (ADCC) approach of antibody, by phagolysis or the splitting action pathogenic agent of ingesting, or the C1q of (2) and first complement component C1 combining partly, cause cytotoxicity (CDC) approach that depends on complement, thus the cracking pathogenic agent.In four kinds of human IgG isotypes, IgG1 and IgG3 can be effectively in conjunction with Fc _γR.IgG4 and Fc _γThe binding affinity of R is than low order of magnitude of IgG1 or IgG3, and IgG2 and Fc _γR measured in conjunction with low being difficult to.Human IgG1 and IgG3 can also be effectively in conjunction with C1q, and the activating complement cascade reaction.The human IgG2 is fixing very weak to complement, and IgG4 as if aspect the ability of activating complement cascade reaction very defectiveness (referring to, as people such as Jefferis, Immunol.Rev., 163:59-76,1998).For being applied to people's treatment, when HuIFN-L-Fc was incorporated into IFN acceptor on the cell surface, must can not there be the ill effect subfunction in the Fc zone of fusion rotein, so can cracking or remove these cells.Therefore, the Fc zone of HuIFN-L-Fc must be non-cracking performance, promptly is incorporated into Fc _γThereby Rs and C1q trigger effect subfunction aspect, the Fc zone must be a non-activity.Obviously, there is not a kind of natural IgG isotype to be fit to produce the HuIFN-L-Fc fusion rotein.In order to obtain the Fc of non-cracking performance, must make some amino acid mutations in the natural Fc zone, to reduce its effector function.

By the aminoacid sequence of the IgG isotype of people and mouse relatively, show that near the Fc part the aminoterminal of CH2 zone is at IgGFc and Fc _γWork in the combination of Rs.With genetic engineering antibody proof 234 importance to 237 motifs (referring to, as people such as Duncan, Nature, 332:563-564,1988).The amino-acid residue numbering is by the described EU number system of people such as Kabat (" SEQUENCES of PROTEINS of IMMUNOLOGICALINTEREST ", the 5th edition, United States Department of Health and HumanServices, 1991).In four kinds of human IgG isotypes, IgG1 and IgG3 and Fc _γRs in conjunction with best, and have identical sequence Leu234-Leu-Gly-Gly237 (table 1 has only shown IgG1).With low-affinity and Fc _γAmong the Rs bonded IgG4, its sequence contains single amino acids and replaces, and promptly Phe replaces Leu on 234.At debond Fc _γAmong the IgG2 of Rs, two replacements and a disappearance are arranged, thereby form Val234-Ala-Gly237 (table 1).In order to reduce Fc and Fc _γThe combination of R and ADCC activity, with Ala substitute Leu235 among the IgG4 (referring to, as people such as Hutchins, Proc.Natl.Acad.Sci.USA, 92:11980-11984,1995).In this motif, substitute Glu233-Leu-Leu235 with the Pro233-Val-Ala235 in the IgG2 sequence and change IgG1.This substituting makes the IgG1 variant lose Fc in mouse _γThe ability of removing target cell of R-mediation (referring to, as people such as Isaacs, J.Immunol., 161:3862-3869,1998).

To Fc _γR combine with C1q near the CH2 zone carboxyl terminal that vital second section is positioned at human IgG (referring to, as people such as Duncan, Nature, 332:738-740,1988).In four kinds of human IgG isotypes, only have a site to show in this part and replace: Ser330 among the IgG4 and Ser331 have replaced Ala330 and the Pro331 (table 1) among IgG1, IgG2 and the IgG3.The existence of Ser330 does not influence Fc _γR combines with C1q's.Substitute Pro331 with Ser and make IgG1 lose binding affinity with C1q, and with Pro substitute the complement fixation(CF) activity that Ser331 partly kept IgG4 (referring to, as people such as Tao, J.Exp.Med., 178:661-667,1993; People such as Xu, J.Biol.Chem., 269:3469-3474,1994).

Summary of the invention

Technical problem to be solved by this invention is the transformation period of prolonged human Interferon, rabbit (HulFN) and improves its biological activity.

The inventor finds the Fc zone of human IgG derivative is connected in the carboxyl terminal of HulFN, can prolong the transformation period of HulFN, and find that flexible peptide linker can be used for therapeutic protein.

And find, can design at least three kinds of Fc variants (vFc) and be used to produce HuIFN-L-VFc fusion rotein (as shown in table 1).Human IgG2's debond Fc _γR, but demonstrate weak complement activity.Fc with Pro331Ser sudden change _{γ 2}Variant should be than natural Fc _{γ 2}The variant activity lower, and still debond in Fc _γR.Human IgG 4 Fc defectiveness in the activating complement cascade, and it and Fc _γThe low about order of magnitude of the isotype (IgG1) that the binding affinity specific activity of R is the highest.With natural Fc _{γ 4}Compare, have the Fc of Leu235Ala sudden change _{γ 4}Variant should show minimum effector function.Fc with Leu234Val, Leu235Ala and Pro331Ser sudden change _{γ 1}Also show than natural Fc _{γ 1}Much lower effector function.These Fc variants all are more suitable for preparing the IFN fusion rotein than naturally occurring human IgG Fc.In the preparation of non-cracking Fc, also can introduce the amino acid of other replacement, and not jeopardize circulating half-life or cause bad change of configuration.

The invention discloses the fusion rotein of the human IgG Fc variant of a kind of human interferon and no cracking performance.This fusion rotein contains human interferon, peptide linker and human IgG Fc variant.

Preferably, use about 20 or the flexible peptide linker of p1 amino acid length more, this peptide linker contains 2 or a plurality of following amino acid: glycine, Serine, L-Ala and Threonine.The IgGFc variant is non-cracking performance, and compares with natural IgGFc and to contain amino acid mutation.

Another object of the present invention is the preparation method of fusion rotein who discloses the human IgG Fc variant of above-mentioned human interferon and no cracking performance, and this method comprises the following steps:

(a) generate CHO-deutero-clone; (b) with allow recombination fusion protein in its growth medium during per 24 hours in, per 1,000,000 cell expressings surpass cultivates this clone under the condition of 10 μ g (preferably being 30 μ g); (c) purification step (b) expressed protein, wherein recombination fusion protein demonstrates the external biological activity of the height similar to rHuIFN on mole foundation.

When between preferable people IFN and human IgG Fc variant, have flexible peptide linker in the above-mentioned steps, and this flexible peptide linker contains or by about 20 or (but can not be less than 2) amino acid still less; And flexible peptide linker by 2 or more multiselect constitute from following amino acid: glycine, Serine, L-Ala and Threonine; And wherein human IgG Fc variant contains hinge region, CH2 and CH3 zone, and they are selected from: have Pro331Ser sudden change the human IgG2, have the human IgG 4 of Ser228Pro and Leu235Ala sudden change and have Leu234Val, Leu235Ala and the human IgG1 of Pro331Ser sudden change.

Fusion rotein of the present invention has following advantage: the activity that the HuIFN-L-vFc fusion rotein increases in the serum and the prolongation of lifetime, the frequency that can lower dosage and reduce injection.The minimizing of serum Chinese traditional medicine fluctuation of concentration is to mean the improvement of security and tolerance.The attenuating of frequency of injection can make the patient that better conformability and quality of life are arranged.Therefore the HuIFN-L-vFc fusion rotein that has non-cracking Fc variant can help treatment significantly.With the various illnesss of rHuIFN treatment, comprise multiple sclerosis, multiple cancer at present, and the viral disease that causes.

Another embodiment of the present invention behaviour IgFc, it contains hinge region, CH2 and the CH3 zone of human IgG such as human IgG1, IgG2 and IgG4.Amino acid mutation is contained 228,234,235 and 331 (by the definite positions of EU number system) in this CH2 zone, thereby reduces the effector function of Fc.

In another embodiment of the present invention, disclose a kind of from the preparation of mammal cell line such as CHO deutero-clone or the method for producing this recombination fusion protein.Cultivation cells transfected system makes recombination fusion protein express to surpass under the level of 10 (preferably being 30) 1,000,000 cells of μ g/ during 24 hours in its growth medium.These HuIFN-L-vFc fusion roteins demonstrate the biological activity and the longer serum half-life and do not have bad side effect of height, thereby have improved pharmacokinetics and drug effect, and then have reduced dosage and the frequency injection of realizing that similar drug effect is required.

Further specify by external biological analytical method and the research of rat interior medicine dynamics:

The external biological analytical method

This analytical method can measure the ability that the supernatant liquor of transfectant or purifying protein stop the pathological changes caused by virus effect (referring to, as people such as Grossberg, " Manual of ClinicalImmunology " in the book, 295-299,1985).First kind Interferon, rabbit can be protected human lung cancer cell line A549 (ATCC-CCL-185).Be unlikely to that (EMV ATCC-VR-129B) infects and dead by encephalomyocarditis virus.Proportional by protection effectiveness and survivaling cell that IFN causes.Survivaling cell can reduce the dyeing chemistry product of a kind of MTT of crying, can obtain reading soon.

A549 clone can maintain in the Dulbecco improvement Eagle substratum (DMEM) that contains 5% foetal calf serum.With every part totally 1.5 * 10 ⁴The A549 sample of individual cell (100 μ l) is added in each hole of tissue cultivating plate, 96 hole, 5% CO under 37 ℃ ₂Cultivated 24 hours in the humidified incubator.In each hole, add 100 μ l then and contain the HuIFN-L-vFc fusion rotein of 0.01 to 100pM various concentration or the substratum of rHuIFN contrast.5% CO under 37 ℃ ₂Through 24 hours, just remove the liquid in each hole in the humidified incubator, add the substratum that 100 μ l contain EMV again.5% CO under 37 ℃ ₂Cultivate in the humidified incubator after 48 hours, in each hole, add the cell that 10 μ l MTT (being mixed with 5mg/ml with PBS) dye and survive.After 4 hours, in every hole, add the 10%SDS that 100 μ l contain 0.01N HCl, with dissolved cell and Formazan.At 550nm this flat board is carried out reading then, wherein reference beam is made as 690nm.The OD reading is with respect to the concentration mapping of rHuIFN or fusion rotein.The flex point of sigmoid curve represents to produce the concentration of 50% maximum efficiency (ED50).Therefore can compare the biological activity of HuIFN-L-vFc quantitatively with respect to rHuIFN.The biological activity of rHuIFN can (Bethesda, MD) people IFN α of Gong Geiing or IFN β be as standard with the healthy office of American National.Recombination fusion protein should show the biological activity of the height similar to rHuIFN on mole foundation.

Interior medicine dynamics research in the rat

Carry out intravenous injection or by subcutaneous injection, with 10 by the tail vein ⁵The rHuIFN of unit or HuIFN-L-vFc fusion rotein be injected into the Fisher rat that mean body weight is about 500g (Harlan Bioproducts for Science, Indianapolis, IN).The PBS of injection equal volume in contrast.After the injection, depletion method is gathered the sample of a series of 0.5ml after different time points (0,0.2,1,4,24,48,96 and 168 hour) is by socket of the eye.Each time point is with 3 rats.Whole blood collection in containing the test tube of antithrombotics, is removed cell, and-70 ℃ of frozen plasma up to analyzing.

Plasma sample is used for the A549 cell analysis, and this assay determination IFN protection cell stops the activity of virus infection.In each hole of 96 hole breezing plates, add per 100 μ l and add up to 1.5 * 10 ⁴The sample of cell, 5% CO under 37 ℃ ₂Cultivate in the humidified incubator after 24 hours, in each hole, add 100 μ l and contain the various serum samples that diluted with substratum.In same incubator,, just remove the liquid in each hole, add the substratum that 100 μ l contain EMV through 24 hours.In incubator,, in each hole, add the cell that 10 μ l MTT (being mixed with 5mg/ml with PBS) dye and survive then after 48 hours.After 4 hours, add 100 μ l in every hole and contain the 10%SDS solution of 0.01N HCl with dissolved cell and Formazan.Read this flat board (reference beam is 690nm) at 550nm then.The activity of serum sample is mapped to time point, to calculate the serum clearance rate.The activity of the specific activity rHuIFN of HuIFN-L-vFc contrast reduces many slowly, shows in the rat that the transformation period of fusion rotein is longer.

Table 1 has shown the comparison of the aminoacid sequence in the hinge region of human IgG1, IgG2, IgG4 and their variants and CH2 zone.Compare these three parts: amino acid region 228,234-237 and 330-331.The amino acid mutation that shows these variants with bold Italic.The EU number system is used for amino-acid residue.

Table 2 is for being used to clone the oligonucleotide sequence of HuIFN-L-vFc fusion rotein described in the embodiment 1.

Sequence A, B, C have shown HindIII-EcoRI segmental (A) HuIFN α in the phIFP expression vector respectively _2a-L-vFc _{γ 2}, (B) HuIFN α _2a-L-vFc _{γ 4}And (C) HuIFN α _2a-L-vFc _{γ 1}Nucleotide sequence and deduced amino acid.Amino-acid residue-23 is to the-1st, people IFN α _2aLeading peptide.Maturation protein contains people IFN α _2a(amino-acid residue 1 to 165), peptide linker (amino-acid residue 166 to 181) and Fc variant (vFc _{γ 2}Amino-acid residue 182 to 409, vFc _{γ 4}Amino-acid residue 182 to 410, and vFc _{γ 1}Amino-acid residue 182 to 408).In the Fc zone, the Nucleotide of runic and corresponding amino acid variant also mark with underscore.

Table 1

The human IgG isotype	Amino acid position
		????228……234?235?236?237……330?331
	????G1		????Pro……Leu?Leu?Gly?Gly……Ala?Pro
????G2		????Pro……Val?Ala……Gly……Ala?Pro
	????G4		????Ser……Phe?Leu?Gly?Gly……Ser?Ser
The G1 variant		????pro……Val?Ala?Gly?Gly……Ala?Ser
	The G2 variant		????Pro……Val?Ala……Gly……Ala??Ser
The G4 variant		????pro……Phe?Ala?Gly?Gly……Ser?Ser

Table 2 is used to clone the oligonucleotide sequence (Sequencesof Oligonucleotides) of HuIFN-L-vFc fusion rotein

SEQ?ID?NO：1

cccaagctta?catctacaat?ggccttgacc

SEQ?ID?NO：2

cggatccttc?cttacttctt?aaactttc

SEQ?ID?NO：3

gagcgcaaat?gttgtgtcga

SEQ?ID?NO：4

ggaattctca?tttacccgga?gacaggga

SEQ?ID?NO：5

tggttttctc?gatggaggct?gggaggcct

SEQ?ID?NO：6

aggcctccca?gcctccatcg?agaaaacca

SEQ?ID?NO：7

cggatccggt?ggcggttccg?gtggaggcgg?aagcggcggt?ggaggatcag

agcgcaaatg?ttgtgtcga

SEQ?ID?NO：8

gagtccaaat?atggtccccc?a

SEQ?ID?NO：9

ggaattctca?tttacccaga?gacaggga

SEQ?ID?NO：10

cctgagttcg?cggggggacc?a

SEQ?ID?NO：11

gagtccaaat?atggtccccc?atgcccacca?tgcccagcac?ctgagttcgc

ggggggacca

SEQ?ID?NO：12

cggatccggt?ggcggttccg?gtggaggcgg?aagcggcggt?ggaggatcag?agtccaaata

tggtccccca

SEQ?ID?NO：13

gacaaaactc?acacatgccc?a

SEQ?ID?NO：14

acctgaagtc?gcggggggac?cgt

SEQ?ID?NO：15

gacaaaactc?acacatgccc?accgtgccca?gcacctgaag?tcgcgggggg

accgt

SEQ?ID?NO：16

cggatccggt?ggcggttccg?gtggaggcgg?aagcggcggt?ggaggatcag

acaaaactca?cacatgccca

Sequence A DNA and deduced amino acid sequences of HuIFN α _2a-L-vFc _{γ 2}

aag?ctt?aca?tct?aca?atg?gcc?ttg?acc?ttt?gct?tta?ctg?gtg?gcc?ctc?ctg?gtg?ctc?agc???60

HindIII?????????????Met?Ala?Leu?Thr?Phe?Ala?Leu?Leu?Val?Ala?Leu?Leu?Val?Leu?Ser

-23?????????-20?????????????????-15?????????????????-10

tgc?aag?tca?agc?tgc?tct?gtg?ggc?tgt?gat?ctg?cct?caa?acc?cac?agc?ctg?ggt?agc?agg???120

Cys?Lys?Ser?Ser?Cys?Ser?Val?Gly?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg

-5??????-1???1???????????????5??????????????????10

agg?acc?ttg?atg?ctc?ctg?gca?cag?atg?agg?aaa?atc?tct?ctt?ttc?tcc?tgc?ttg?aag?gac???180

Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Lys?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp

15??????????????????20??????????????????25??????????????????30

aga?cat?gac?ttt?gga?ttt?ccc?cag?gag?gag?ttt?ggc?aac?cag?ttc?caa?aag?gct?gaa?acc???240

Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr

35??????????????????40??????????????????45??????????????????50

atc?cct?gtc?ctc?cat?gag?atg?atc?cag?cag?atc?ttc?aat?ctc?ttc?agc?aca?aag?gac?tca???300

Ile?Pro?Val?Leu?His?glu?Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser

55??????????????????60??????????????????65??????????????????70

tct?gct?gct?tgg?gat?gag?acc?ctc?cta?gac?aaa?ttc?tac?act?gaa?ctc?tac?cag?cag?ctg???360

Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu

75??????????????????80??????????????????85??????????????????90

aat?gac?ctg?gaa?gcc?tgt?gtg?ata?cag?ggg?gtg?ggg?gtg?aca?gag?act?ccc?ctg?atg?aag???420

Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys

95??????????????????100?????????????????105?????????????????110

gag?gac?tcc?att?ctg?gct?gtg?agg?aaa?tac?ttc?caa?aga?atc?act?ctc?tat?ctg?aaa?gag???480

Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu

115?????????????????120?????????????????125?????????????????130

aag?aaa?tac?agc?cct?tgt?gcc?tgg?gag?gtt?gtc?aga?gca?gaa?atc?atg?aga?tct?ttt?tct???540

Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser

135?????????????????140?????????????????145?????????????????150

ttg?tca?aca?aac?ttg?caa?gaa?agt?tta?aga?agt?aag?gaa?gga?tcc?ggt?ggc?ggt?tcc?ggt???600

Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?Gly?Ser?Gly?Gly?Gly?Ser?Gly

155?????????????????160?????????????????165?????????????????170

gga?ggc?gga?agc?ggc?ggt?gga?gga?tca?gag?cgc?aaa?tgt?tgt?gtc?gag?tgc?cca?ccg?tgc???660

Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Glu?Arg?Lys?Cys?Cys?Val?Glu?Cys?Pro?Pro?Cys

175?????????????????180?????????????????185?????????????????190

cca?gca?cca?cct?gtg?gca?gga?ccg?tca?gtc?ttc?ctc?ttc?ccc?cca?aaa?ccc?aag?gac?acc???720

Pro?Ala?Pro?Pro?Val?Ala?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr

195?????????????????200?????????????????205?????????????????210

ctc?atg?atc?tcc?cgg?acc?cct?gag?gtc?acg?tgc?gtg?gtg?gtg?gac?gtg?agc?cac?gaa?gac???780

Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp

215?????????????????220?????????????????225?????????????????230

ccc?gag?gtc?cag?ttc?aac?tgg?tac?gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca?aag???840

Pro?Glu?Val?Gln?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys

235?????????????????240?????????????????245?????????????????250

cca?cgg?gag?gag?cag?ttc?aac?agc?acg?ttc?cgt?gtg?gtc?agc?gtc?ctc?acc?gtt?gtg?cac???900

Pro?Arg?Glu?Glu?Gln?Phe?Asn?Ser?Thr?Phe?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Val?His

255?????????????????260?????????????????265?????????????????270

cag?gac?tgg?ctg?aac?ggc?aag?gag?tac?aag?tgc?aag?gtc?tcc?aac?aaa?ggc?ctc?cca?gcc???960

Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Gly?Leu?Pro?Ala

275?????????????????280?????????????????285?????????????????290

tcc?atc?gag?aaa?acc?atc?tcc?aaa?acc?aaa?ggg?cag?ccc?cga?gaa?cca?cag?gtg?tac?acc???1020

Ser?Ile?Glu?Lys?hr??Ile?Ser?Lys?Thr?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr

295?????????????????300?????????????????305?????????????????310

ctg?ccc?cca?tcc?cgg?gag?gag?atg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc?aaa?????1080

Leu?Pro?Pro?Ser?Arg?Glu?Glu?Met?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys

315?????????????????320?????????????????325?????????????????330

ggc?ttc?tac?ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag?ccg?gag?aac?aac?????1140

Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn

335?????????????????340?????????????????345?????????????????350

tac?aag?acc?aca?cct?ccc?atg?ctg?gac?tcc?gac?ggc?tcc?ttc?ttc?ctc?tac?agc?aag?ctc?????1200

Tyr?Lys?Thr?Thr?Pro?Pro?Met?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu

355?????????????????360?????????????????365?????????????????370

acc?gtg?gac?aag?agc?agg?tgg?cag?cag?ggg?aac?gtc?ttc?tca?tgc?tcc?gtg?atg?cat?gag?????1260

Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu

375?????????????????380?????????????????385?????????????????390

gct?ctg?cac?aac?cac?tac?acg?cag?aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa?tga?gaa?ttc?????1320

Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys???????EcoRI

395?????????????????400?????????????????405?????????????409

Sequence B. DNA and deduced amino acid sequences of HuIFN _2a-L-vFc _{γ 4}

aag?ctt?aca?tct?aca?atg?gcc?ttg?acc?ttt?gct?tta?ctg?gtg?gcc?ctc?ctg?gtg?ctc?agc????60

HindIII?????????????Met?Ala?Leu?Thr?Phe?Ala?Leu?Leu?Val?Ala?Leu?Leu?Val?Leu?Ser

-23?????????-20?????????????????-15?????????????????-10

tgc?aag?tca?agc?tgc?tct?gtg?ggc?tgt?gat?ctg?cct?caa?acc?cac?agc?ctg?ggt?agc?agg????120

Cys?Lys?Ser?Ser?Cys?Ser?Val?Gly?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg

-5??????????????-1???1???????????????5??????????????????10

agg?acc?ttg?atg?crc?ctg?gca?cag?atg?agg?aaa?atc?tct?ctt?ttc?tcc?tgc?ttg?aag?gac????180

Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Lys?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp

15??????????????20??????????????????25??????????????????30

aga?cat?gac?ttt?gga?ttt?ccc?cag?gag?gag?ttt?ggc?aac?cag?ttc?caa?aag?gct?gaa?acc????240

Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr

35??????????????40??????????????????45??????????????????50

atc?cct?gtc?ctc?cat?gag?atg?atc?cag?cag?atc?ttc?aat?ctc?ttc?agc?aca?aag?gac?tca????300

Ile?Pro?Val?Leu?His?glu?Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser

55??????????????60??????????????????65??????????????????70

tct?gct?gct?tgg?gat?gag?acc?ctc?cta?gac?aaa?ttc?tac?act?gaa?ctc?tac?cag?cag?ctg????360

Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu

75??????????????80??????????????????85??????????????????90

aat?gac?ctg?gaa?gcc?tgt?gtg?ata?cag?ggg?gtg?ggg?gtg?aca?gag?act?ccc?ctg?atg?aag????420

Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys

95??????????????100?????????????????105?????????????????110

gag?gac?tcc?att?ctg?gct?gtg?agg?aaa?tac?ttc?caa?aga?atc?act?ctc?tat?ctg?aaa?gag????480

Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu

115?????????????120?????????????????125?????????????????130

aag?aaa?tac?agc?cct?tgt?gcc?tgg?gag?gtt?gtc?aga?gca?gaa?atc?atg?aga?tct?ttt?tct????540

Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser

135?????????????140?????????????????145?????????????????150

ttg?tca?aca?aac?ttg?caa?gaa?agt?tta?aga?agt?aag?gaa?gga?tcc?ggt?ggc?ggt?tcc?ggt????600

Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?Gly?Ser?Gly?Gly?Gly?Ser?Gly

155?????????????160?????????????????165?????????????????170

gga?ggc?gga?agc?ggc?ggt?gga?gga?tca?gag?tcc?aaa?tat?ggt?ccc?cca?tgc?cca?cca?tgc????660

Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Glu?Ser?Lys?Tyr?Gly?Pro?Pro?Cys?Pro?Pro?Cys

175?????????????180?????????????????185?????????????????190

cca?gca?cct?gag?ttc?gcg?ggg?gga?cca?tca?gtc?ttc?ctg?ttc?ccc?cca?aaa?ccc?aag?gac????720

Pro?Ala?Pro?Glu?Phe?Ala?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp

195?????????????????200?????????????????205?????????????????210

act?ctc?atg?atc?tcc?cgg?acc?cct?gag?gtc?acg?tgc?gtg?gtg?gtg?gac?gtg?agc?cag?gaa????780

Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?Gln?Glu

215?????????????????220?????????????????225?????????????????230

gac?ccc?gag?gtc?cag?ttc?aac?tgg?tac?gtg?gat?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca????840

Asp?Pro?Glu?Val?Gln?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr

235?????????????????240?????????????????245?????????????????250

aag?ccg?cgg?gag?gag?cag?ttc?aac?agc?acg?tac?cgt?gtg?gtc?agc?gtc?ctc?acc?gtc?ctg????900

Lys?Pro?Arg?Glu?Glu?Gln?Phe?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu

255?????????????????260?????????????????265?????????????????270

cac?cag?gac?tgg?ctg?aac?ggc?aag?gag?tac?aag?tgc?aag?gtc?tcc?aac?aaa?ggc?ctc?ccg????960

His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Gly?Leu?Pro

275?????????????????280?????????????????285?????????????????290

tcc?tcc?atc?gag?aaa?acc?atc?tcc?aaa?gcc?aaa?ggg?cag?ccc?cga?gag?cca?cag?gtg?tac????1020

Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr

295?????????????????300?????????????????305?????????????????310

acc?ctg?ccc?cca?tcc?cag?gag?gag?atg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc????1080

Thr?Leu?Pro?Pro?Ser?Gln?Glu?Glu?Met?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val

315?????????????????320?????????????????325?????????????????330

aaa?ggc?ttc?tac?ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag?ccg?gag?aac????1140

Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn

335?????????????????340?????????????????345?????????????????350

aac?tac?aag?acc?acg?cct?ccc?gtg?ctg?gac?tcc?gac?ggc?tcc?ttc?ttc?ctc?tac?agc?agg????1200

Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Arg

355?????????????????360?????????????????365?????????????????370

cta?acc?gtg?gac?aag?agc?agg?tgg?cag?gag?ggg?aat?gtc?ttc?tca?tgc?tcc?gtg?atg?cat????1260

Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Glu?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His

375?????????????????380?????????????????385?????????????????390

gag?gct?ctg?cac?aac?cac?tac?aca?cag?aag?agc?ctc?tcc?ctg?tct?ctg?ggt?aaa?tga?gaa????1320

Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Leu?Gly?Lys

395?????????????????400?????????????????405?????????????????410

1323

ttc

EcoRI

Preface C.DNA and deduced amino acid sequences of HuIFN _2a-L-vFc _{γ 1}

aag?ctt?aca?tct?aca?atg?gcc?ttg?acc?ttt?gct?tta?ctg?gtg?gcc?ctc?ctg?gtg?ctc?agc??60

HindIII?????????????Met?Ala?Leu?Thr?Phe?Ala?Leu?Leu?Val?Ala?Leu?Leu?Val?Leu?Ser

-23?????????-20?????????????????-15?????????????????-10

tgc?aag?tca?agc?tgc?tct?gtg?ggc?tgt?gat?ctg?cct?caa?acc?cac?agc?ctg?ggt?agc?agg?120

Cys?Lys?Ser?Ser?Cys?Ser?Val?Gly?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg

-5??????????????-1???1???????????????5??????????????????10

agg?acc?ttg?atg?ctc?ctg?gca?cag?atg?agg?aaa?atc?tct?ctt?ttc?tcc?tgc?ttg?aag?gac?180

Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Lys?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp

15??????????????20??????????????????25??????????????????30

aga?cat?gac?ttt?gga?ttt?ccc?cag?gag?gag?ttt?ggc?aac?cag?ttc?caa?aag?gct?gaa?acc?240

Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr

35??????????????40??????????????????45??????????????????50

atc?cct?gtc?ctc?cat?gag?atg?atc?cag?cag?atc?ttc?aat?ctc?ttc?agc?aca?aag?gac?tca?300

Ile?Pro?Val?Leu?His?glu?Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser

55??????????????60??????????????????65??????????????????70

tct?gct?gct?tgg?gat?gag?acc?ctc?cta?gac?aaa?ttc?tac?act?gaa?ctc?tac?cag?cag?ctg???360

Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu

75??????????????????80??????????????????85??????????????????90

aat?gac?ctg?gaa?gcc?tgt?gtg?ata?cag?ggg?gtg?ggg?gtg?aca?gag?act?ccc?ctg?atg?aag???420

Ash?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys

95??????????????????100?????????????????105?????????????????110

gag?gac?tcc?att?ctg?gct?gtg?agg?aaa?tacttc?caa?aga?atc?act?ctc?tat?ctg?aaa?gag????480

Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu

115?????????????????120?????????????????125?????????????????130

aag?aaa?tac?agc?cct?tgt?gcc?tgg?gag?gtt?gtc?aga?gca?gaa?atc?atg?aga?tct?ttt?tct???540

Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser

135?????????????????140?????????????????145?????????????????150

ttg?tca?aca?aac?ttg?caa?gaa?agt?tta?aga?agt?aag?gaa?gga?tcc?ggt?ggc?ggt?tcc?ggt???600

Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?Gly?Ser?Gly?Gly?Gly?Ser?Gly

155?????????????????160?????????????????165?????????????????170

gga?ggc?gga?agc?ggc?ggt?gga?gga?tca?gac?aaa?act?cac?aca?tgc?cca?ccg?tgc?cca?gca???660

Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala

175?????????????????180?????????????????185?????????????????190

cct?gaa?gtc?gcg?ggg?gga?ccg?tca?gtc?ttc?ctc?ttc?ccc?cca?aaa?ccc?aag?gac?acc?ctc???720

Pro?Glu?Val?Ala?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu

195?????????????????200?????????????????205?????????????????210

atg?atc?tcc?cgg?aca?cct?gag?gtc?aca?tgc?gtg?gtg?gtg?gac?gtg?agc?cac?gaa?gac?cct???780

Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro

215?????????????????220?????????????????225?????????????????230

gag?gtc?aag?ttc?aac?tgg?tac?gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca?aag?ccg???840

Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro

235?????????????????240?????????????????245?????????????????250

cgg?gag?gag?cag?tac?aac?agc?acg?tac?cgg?gtg?gtc?agc?gtc?ctc?acc?gtc?ctg?cac?cag???900

Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln

255?????????????????260?????????????????265?????????????????270

gac?tgg?ctg?aat?ggc?aag?gag?tac?aag?tgc?aag?gtc?tcc?aac?aaa?gcc?ctc?cca?gcc?tcc???960

Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Ser

275?????????????????280?????????????????285?????????????????290

atc?gag?aaa?acc?atc?tcc?aaa?gcc?aaa?ggg?cag?ccc?cga?gaa?cca?cag?gtg?tac?acc?ctg???1020

Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu

295?????????????????300?????????????????305?????????????????310

ccc?cca?tcc?cgg?gat?gag?ctg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc?aaa?ggc???1080

Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly

315?????????????????320?????????????????325?????????????????330

ttc?tat?ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag?ccg?gag?aac?aac?tac???1140

Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr

335?????????????????340?????????????????345?????????????????350

aag?acc?acg?cct?ccc?gtg?ctg?gac?tcc?gac?ggc?tcc?ttc?ttc?ctc?tac?agc?aag?ctc?acc???1200

Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr

355?????????????????360?????????????????365?????????????????370

gtg?gac?aag?agc?agg?tgg?cag?cag?ggg?aac?gtc?ttc?tca?tgc?tcc?gtg?atg?cat?gag?gct???1260

Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala

375?????????????????380?????????????????385?????????????????390

ctg?cac?aac?cac?tac?acg?cag?aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa?tga?gaa?ttc???????1317

Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?????EcoRI

395?????????????????400?????????????????405????????408

Embodiment

Embodiment 1 makes up coding HuIFN-L-vFc _{γ 2}The gene of fusion rotein

HuIFN α _2aBe an example, be used for explaining orally the method for preparing the HuIFN-L-vFc fusion rotein.Other codings people IFN (comprises IFN α _2bWith IFN β etc.) gene can also be used for preparing the fusion rotein that contains human IgG Fc variant.Coding people IFN α _2bLeading peptide and the gene of maturation protein, the available people IFN α that contains _2aDna fragmentation make template, introduce the simple point mutation (being sequence A, B, or the 152nd base of C) that an A becomes G with polymerase chain reaction (PCR).This simple point mutation influences the 23rd amino-acid residue in the HuIFN α maturation protein, with Arg (IFN α _2b) substituted Lys (IFN α _2a).For the gene of obtain to encode people IFN β leading peptide and maturation protein, the RNA of the clone of free list intelligent IFN β preparation obtains cDNA by reverse transcription, again with its template as PCR.Coding contains the gene of the people IFN β variant of Cys17Ser, and the dna fragmentation of the then available people of containing IFN β is made template, introduces two simple point mutations with PCR.These two simple point mutation coding IFN β maturation proteins the 17th amino acid whose the 2nd (G becomes C) and the 3rd (T becomes C) oligonucleotide have replaced Cys with Ser.

Fusion rotein is assembled with several dna fragmentations.In order to obtain the people IFN α that encodes _2aLeading peptide and the gene of maturation protein, the RNA of available clone KG-1 preparation is by reverse transcription and PCR.For the ease of the clone, will introduce the primer of the SEQ ID NO:1 in Restriction Enzyme inscribe site (HindIII) as 5 ' oligonucleotide.Table 2 has been listed the oligonucleotide sequence that is used to clone the HuIFN-L-vFc fusion rotein.3 ' primer (SEQ ID NO:2) has been removed the IFN termination codon and has been introduced the BamHI site.The dna fragmentation that the length that obtains is about 600bp is inserted into HindIII and the BamHI site of accepting carrier (as pUC19), obtains the pHuIFN plasmid.By dna sequencing identifier IFN α _2aThe sequence of gene.

With from the RNA of human leukocyte preparation and suitable 5 ' (SEQ ID NO:3) and 3 ' (SEQID NO:4) primer, by reverse transcription and PCR obtain encoding human IgG2's Fc zone (Fc _{γ 2}) gene.Fc with the hinge that contains IgG2 that obtains, CH2 and CH3 zone complete sequence _{γ 2}Dna fragmentation as template, produce Fc _{γ 2}Pro331Ser (vFc _{γ 2}) variant, wherein Fc _{γ 2}In 331 Pro substituted by Ser.In order to introduce this sudden change, produce two fragments, use natural Fc then _{γ 2}They are assembled up in overlapping PCR as template.Generate 5 ' fragment as 5 ' primer and with SEQ ID NO:5 as 3 ' primer with SEQ IDNO:3.Generate 3 ' fragment as 5 ' primer and with SEQ ID NO:4 as 3 ' primer with SEQID NO:6.Use then SEQ ID NO:7 as 5 ' primer and SEQ ID NO:4 as 3 ' primer, these two fragments are coupled together in the zone that covers the Pro331Ser sudden change.SEQ ID NO:7 primer contains the sequence of 16 amino acid Gly-Ser peptide linkers of coding (comprising the BamHI restriction endonuclease sites).The dna fragmentation that the length that obtains is about 700bp inserts BamHI and the EcoRI site of accepting carrier (as pUC19), obtains pL-vFc _{γ 2}Plasmid.Verify the sequence of this gene with dna sequencing.

In order to prepare HuIFN-L-vFc _{γ 2}Fusion gene downcuts the HuIFN fragment with HindIII and BamHI from the pHuIFN plasmid, uses the agarose gel electrophoresis purifying then.Then the fragment of purifying is inserted 5 ' end of pL-vFc γ 2 plasmid peptide linkers, obtained pHuIFN-L-vFc γ 2 plasmids.This fusion gene contains HuIFN, Gly-Ser peptide linker and Fc _{γ 2}Variant gene.

The peptide linker that exists between HuIFN and Fc part has increased the flexibility in HuIFN zone, thus improved its biological activity (referring to, as Sytkowski etc., J.Biol.Chem.274:24773-8,1999).For the purpose of the present invention, preferably length is about 20 or still less amino acid whose peptide linker.Can use contain 2 or more multiselect from following amino acid whose peptide linker: glycine, Serine, L-Ala and Threonine.A kind of example of peptide linker contains Gly-Ser peptide member, as GlyGlyGlyGlySer.Sequence A has shown fusion gene, and it contains coding HuIFN sequence, 16 amino acid whose peptide linkers (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) and Fc _{γ 2}The sequence of Pro331Ser variant.

HindIII and EcoRI site that the complete genome of HuIFN-L-vFc fusion rotein of will encoding then inserts mammalian expression vector such as pcDNA3 (Invitrogen).Final expression vector plasmid (being called phIFP2) contains the required cytomegalovirus early gene promoter-enhanser of high level expression in mammalian cell.This plasmid also contains alternative marker gene, thereby gives amicillin resistance in bacterium, and gives the G418 resistance in mammalian cell.In addition, when host cell DHFR genetic expression defective, the phIFP2 expression vector contains Tetrahydrofolate dehydrogenase (DHFR) gene, thus can be in the presence of methotrexate (MTX) coamplification HuIFN-L-vFc _{γ 2}Fusion gene and DHFR gene (seeing for example U.S. Patent No. 4,399,216).

Embodiment 2 makes up coding HuIFN-L-vFc _{γ 4}The gene of fusion rotein

Owing to dissociating of disulfide linkage between heavy chain in the hinge zone, human IgG 4 parts can form the molecule that is regarded as incomplete antibody.And this situation is not observed in other three-type-person IgG isotype.The monamino acid that substitutes Ser228 with Pro (residue that this position in IgG1 and IgG2 is found) replaces, cause forming the complete antibody molecule of IgG4 (referring to, as people such as Angal, Molec.Immunol., 30:105-108,1993; People such as Owens, Immunotechnology, 3:107-116,1997; U.S. Patent No. 6,204,007).Contain the Leu235Ala sudden change to fall FcR bonded Fc _{γ 4}Variant adds the Ser228Pro sudden change, also can produce this isostructural fusion rotein prepared product.

With from the RNA of human leukocyte preparation and suitable 5 ' primer (SEQ ID NO:8) and 3 ' primer (SEQ ID NO:9), by reverse transcription and PCR obtain the encoding Fc zone (Fc of human IgG 4 _{γ 4}) gene.Fc with the hinge that contains IgG4 that obtains, CH2 and CH3 zone complete sequence _{γ 4}Dna fragmentation as template, produce the Fc of Ser228Pro and Leu235Ala sudden change _{γ 4}Variant (vFc _{γ 4}), wherein Ser228 and Leu235 are substituted by Pro and Ala respectively.With 3 ' primer (SEQ ID NO:9) with contain 5 ' primer (SEQ IDNO:10) the amplification CH2 and the CH3 zone of Leu235Ala sudden change.With SEQ ID NO:12 as 5 ' primer and SEQID NO:9 as 3 ' primer, in PCR be 60 bases and contain Ser228Pro and the oligonucleotide (SEQ IDNO:11) of Leu235Ala sudden change couples together the fragment of this amplification and synthetic length.SEQ ID NO:12 primer contains the sequence of 16 amino acid Gly-Ser peptide linkers of coding (comprising the BamHI site).The dna fragmentation that the length that obtains is about 700bp inserts BamHI and the EcoRI site of accepting carrier (as pUC19), obtains pL-vFc _{γ 4}Plasmid.Verify the sequence of this gene with dna sequencing.

In order to prepare HuIFN-L-vFc _{γ 4}Fusion gene downcuts the HuIFN fragment with HindIII and BamHI from the pHuIFN plasmid, inserts pL-vFc then _{γ 4}5 ' end of plasmid peptide linker obtains pHuIFN-L-vFc _{γ 4}Plasmid.This fusion gene contains HuIFN, 16 amino acid whose Gly-Ser peptide linkers and Fc _{γ 4}Variant gene is inserted into the HindIII of mammalian expression vector such as pcDNA3 (Invitrogen) and EcoRI site then (as HuIFN-L-vFc _{γ 2}Fusion rotein is described).Vector plasmid called after phIFP4 with this final expression.Sequence B has shown fusion gene, and it contains coding HuIFN α _2a, 16 amino acid whose peptide linkers (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) and have Ser228Pro and the Fc of Leu235Ala sudden change _{γ 4}The fusion gene of variant.

Embodiment 3 makes up coding HuIFN-L-vFc _{γ 1}The gene of fusion rotein

15 amino-acid residues (GluProLysSerCysAspLysThrHisThrCysProProCysPro) that comprise 3 halfcystines are contained in the hinge zone of human IgG1's heavy chain.In these 3 cysteine residues, the 2nd and the 3rd formation that participates in disulfide linkage between two heavy chains.The 1st cysteine residues participates in the disulfide-bonded of IgG heavy chain and light chain.Owing to do not have light chain in the Fc fusion protein molecule, this cysteine residues may match with other cysteine residues, and causes non-specific disulfide-bonded.Can be with Fc _{γ 1}The hinge zone prescind, to eliminate the 1st cysteine residues (AspLysThrHisThrCysProProCysPro).With from the RNA of human leukocyte preparation and suitable 5 ' primer (SEQ ID NO:13) and 3 ' primer (SEQ IDNO:4), obtain the Fc that encodes by reverse transcription and PCR _{γ 1}The gene in zone.With the Fc that contains that obtains _{γ 1}The hinge zone of brachymemma and the dna fragmentation of CH2 and CH3 complete sequence produce the Fc with Leu234Val, Leu235Ala and Pro331Ser sudden change as template _{γ 1}Variant (vFc _{γ 1}).A kind of method of introducing these sudden changes is as follows: produce two fragments, use natural Fc then _{γ 1}They are assembled up in overlapping PCR as template.Generate 5 ' fragment as 5 ' primer and with SEQ ID NO:5 as 3 ' primer with SEQ ID NO:14.This 5 ' primer contains Leu234Val, Leu235Ala sudden change, and 3 ' primer contains the Pro331Ser sudden change.Generate 3 ' fragment as 5 ' primer and with SEQ ID NO:4 as 3 ' primer with SEQ ID NO:6.Use then SEQ IDNO:14 as 5 ' primer and SEQ ID NO:4 as 3 ' primer, 5 ' and 3 ' fragment is coupled together in the zone that covers the Pro331Ser sudden change.With SEQ ID NO:16 as 5 ' primer, SEQ ID NO:4 as 3 ' primer, by PCR the oligonucleotide (SEQ ID NO:15) (containing Leu234Val and Leu235Ala) of fragment and synthetic 55 bases of the about 650bp of length of this amplification is coupled together.SEQ ID NO:16 primer contains the sequence of 16 amino acid Gly-Ser peptide linkers of coding (comprising the BamHI site).The dna fragmentation that the length that obtains is about 700bp inserts BamHI and the EcoRI site of accepting carrier (as pUC19), obtains pL-vFc _{γ 1}Plasmid.Verify the sequence of this gene with dna sequencing.

In order to prepare HuIFN-L-vFc _{γ 1}Fusion gene downcuts the HuIFN fragment with HindIII and BamHI from the pHuIFN plasmid, inserts 5 ' end of pL-vFc γ 1 plasmid peptide linker then, obtains pHuIFN-L-vFc _{γ 1}Plasmid.This fusion gene contains HuIFN, 16 amino acid Gly-Ser peptide linkers and Fc _{γ 1}Variant gene is inserted into the HindIII of mammalian expression vector such as pcDNA3 (Invitrogen) and EcoRI site then (as HuIFN-L-vFc _{γ 2}Fusion rotein is described).Vector plasmid called after phIFP1 with this final expression.Sequence C has shown fusion gene, and it contains coding HuIFN, 16 amino acid peptide joints (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) and has Ser234Val, Leu235Ala and the Fc of Pro331Ser sudden change _{γ 1}The fusion gene of variant.

Embodiment 4 is Expression of Fusion Protein and purifying and qualitative in cells transfected system

Be used for medical treatment two kinds of different rHuIFN β products are arranged: from the glycosylated protein of Chinese hamster ovary cell (CHO) generation and the sugar based albumen that contains Cys17Ser that produces from bacterial cell.Because carbohydrate chain has the effect of stable molecule configuration, the product height of the external biological specific activity sugar basedization of glycosylation rHuIFN β (referring to, as people such as Runkel, Pharm.Res., 15:641-649,1998).Carbohydrate chain in the recombinant glycoprotein molecule can have influence on the biological activity of treatment with product, immunogenicity, and protein molecular stability, and serum half-life etc.Existing report says, from Chinese hamster ovary celI deutero-rHuIFN β, its invest sugar chain that the Asn residue is connected with N-than the product of other two kinds of mammalian host cell lines all more as natural people IFN β (referring to, as people such as Kagawa, J.Biol.Chem., 263:17508-17515,1998).In order to obtain the product of the most suitable clinical use, promptly following description produces the HuIFN-vFc fusion rotein from CHO deutero-clone.

To recombinate phIFP1, phIFP2 or phIFP4 expression vector plasmid transfection gone into mammalian host cell line, to express HuI FN-L-vFc fusion rotein.In order to stablize high-caliber expression, preferred host cell system be the DHFR enzyme defect Chinese hamster ovary celI (referring to, for example U.S. Patent No. 4,818,679).A kind of preferred transfection method is an electroporation.Also can use other method, comprise that coprecipitation of calcium phosphate, fat transfection and protoplasma merge.In electroporation, be set to 250V electric field and 960 μ Fd electric capacity Gene Pulser Electroporator (Bio-RadLaboratories, Hercules, CA), the 2-5 in cuvette * 10 ⁷Add the 10 μ g linearizing plasmid DNA of BspCI in the individual cell.After 2 days, substratum is contained instead the growth medium of 0.2mg/ml G418 in transfection.With anti-human IgG Fc ELISA, whether test secretes fusion rotein to the transfectant that selected medicine has resistance.Also available anti--HuIFN test, the quantitative analysis of the fusion rotein of expressing by ELISA.By diluting in 96 orifice plate limes superiors, subclone produces the hole of high-level Fc fusion rotein.

In order to realize the expression of fusion rotein higher level, should carry out coamplification with the DHFR gene that suppressed by the MTX medicine.In the growth medium that contains progressive concentration MTX, the antigen-4 fusion protein gene of transfection and DHFR gene coamplification.The transfectant that can grow in up to 1 μ g/ml MTX substratum carries out subclone by the Method of Limited Dilution method once more.By measuring secretion rate, the clone of subclone is further analyzed.Secretion rate horizontal exceeding about 10 (preferably about 30) μ g/10 ⁶The clone of (promptly 1,000,000) individual cell/24 hour is fit to use the suspension culture of serum-free growth medium.Use the conditioned medium purified fusion protein then.

The conditioned medium that will contain fusion rotein with 1N NaOH is titrated to pH7 to 8, filters with 0.45 micron nitrocellulose strainer then.With the filtrate application of sample to phosphate buffered saline (PBS) (PBS) equilibrated Prosep A post.After treating that fusion rotein is incorporated into Prosep A, discard stream and wear component.Wash this post with PBS, be lower than 0.01 up to the OD at 280nm place value.Use the fusion rotein of the citrate buffer solution elution of bound of 0.1M pH3.75 then.1M K with 0.4 volume ₂HPO ₄Neutralization merges the component that contains purifying protein, and dialyses with PBS.Solution filters with 0.22 micron nitrocellulose strainer then, and is stored in 4 ℃.Under non-reduced condition, the proteinic molecular weight ranges of HuIFN-L-vFc that records purifying by SDS-PAGE is 110 to 120kDa.Under reductive condition, the albumen of purifying migrates to about 50 to 60kDa.With BSA as standard, by quantitative this fusion rotein of BCA protein analysis.

Claims (17)

1. a reorganization HuIFN-L-vFc fusion rotein is characterized in that described fusion rotein contains people IFN, peptide linker and human IgG Fc variant.

2. reorganization HuIFN-L-vFc fusion rotein as claimed in claim 1 is characterized in that, described people IFN contains and is selected from human interferon, is IFN α _2a, IFN α _2b, IFN β or contain the IFN β of Cys17Ser variant.

3. reorganization HuIFN-L-vFc fusion rotein as claimed in claim 1 or 2 is characterized in that described peptide linker contains have an appointment 20 or amino acid still less, and this peptide linker is present between people IFN and the human IgG Fc variant; And described peptide linker contains two or more amino acid that are selected from glycine, Serine, L-Ala and Threonine.

4. as claim 1 or 2 or 3 described reorganization HuIFN-L-vFc fusion roteins, it is characterized in that described human IgG Fc variant is hinge, CH2 and the CH3 zone of containing the human IgG2 of Pro331Ser sudden change.

5. as claim 1 or 2 or 3 described reorganization HuIFN-L-vFc fusion roteins, it is characterized in that described human IgG Fc variant is hinge, CH2 and the CH3 zone of containing the human IgG 4 of Ser228Pro and Leu235Ala sudden change.

6. as claim 1 or 2 or 3 described reorganization HuIFN-L-vFc fusion roteins, it is characterized in that described human IgG Fc variant is hinge, CH2 and the CH3 zone of containing the human IgG1 of Leu234Val, Leu235Ala and Pro331Ser sudden change.

7. a CHO deutero-clone is characterized in that, described clone in its growth medium during per 24 hours in, produce to surpass 1,000,000 cells of 10 μ g/ as the arbitrary described HuIFN-L-vFc fusion rotein of claim 1-6.

8. CHO-deutero-clone as claimed in claim 7 is characterized in that, in growth medium, during per 24 hours in, produce to surpass the HuIFN-L-vFc fusion rotein of 1,000,000 cells of 30 μ g/.

9. method for preparing the CHO-deutero-clone of the described HuIFN-L-vFc fusion rotein of claim 1, it is characterized in that, wherein human IgG Fc variant contains hinge, CH2 and the CH3 zone of the human IgG that is selected from IgG1, IgG2 and IgG4, and IgGFc contains the amino acid mutation that reduces effector function.Between people IFN and human IgG Fc variant, exist and contain have an appointment 20 or still less amino acid whose flexible peptide linker, and people IFN contains, and to be selected from human interferon be IFN α _2a, IFN α _2b, IFN β or contain the IFN β of Cys17Ser variant.

10. a method for preparing the recombination fusion protein that contains people IFN, flexible peptide linker and human IgG Fc variant is characterized in that, this method comprises the following steps: that (a) generates CHO deutero-clone; (b) express to surpass under the condition of 1,000,000 cells of 10 μ g/ in during per 24 hours in its growth medium at recombination fusion protein, cultivate this clone; (c) purification step (b) expressed protein.

11. method as claimed in claim 10 is characterized in that wherein said the existence contain 20 or an amino acid whose flexible peptide still less between people IFN and human IgG Fc variant; And described flexible peptide linker contains 2 or a plurality of amino acid that is selected from glycine, Serine, L-Ala and Threonine.

12. method as claimed in claim 10 is characterized in that wherein said human IgG Fc variant is hinge region, CH2 and the CH3 zone of containing the human IgG2 who comprises the Pro331Ser sudden change.

13. method as claimed in claim 10 is characterized in that wherein said human IgG Fc variant is hinge region, CH2 and the CH3 zone of containing the human IgG 4 that comprises Ser228Pro and Leu235Ala sudden change.

14. method as claimed in claim 10 is characterized in that, described human IgG Fc variant is hinge region, CH2 and the CH3 zone of containing the human IgG1 who comprises Leu234Val, Leu235Ala and Pro331Ser sudden change.

15. method as claimed in claim 10 is characterized in that wherein said people IFN contains that to be selected from human interferon be IFN α _2a, IFN α _2b, IFN β or contain the IFN β of Cys17Ser variant.

16. as the arbitrary described method of claim 10-15, it is characterized in that in the wherein said step (b) during per 24 hours in per 1,000,000 cell expressings surpass 30 μ g albumen.

17. a method for preparing the recombination fusion protein that contains people IFN, flexible peptide linker and human IgG Fc variant is characterized in that this method comprises the following steps: that (a) generates CHO deutero-clone; (b) express above under the condition of 1,000,000 cells of 10 μ g/ in during per 24 hours in its growth medium with recombination fusion protein, cultivate this clone; (c) (b) expressed protein in the purification step; Wherein between people IFN and human IgG Fc variant, exist and contain 20 or still less amino acid whose flexible peptide linker and flexible peptide linker contains 2 or a plurality of amino acid that is selected from glycine, Serine, L-Ala and Threonine; And people IFN contains and is selected from human interferon for as IFN α _2a, IFN α _2b, IFN β or contain the IFN β of Cys17Ser variant; Contain the human IgG2 of the hinge region that is selected from following human IgG, CH2 and CH3 zone: Pro331Ser sudden change with human IgG Fc variant; The human IgG 4 of Ser228Pro and Leu235Ala sudden change; Human IgG1 with Leu234Val, Leu235Ala and Pro331Ser sudden change.