CN102757502A - Fused human interleukin 28B and preparation method thereof - Google Patents

Abstract

The invention discloses a fused human interleukin 28B and a preparation method thereof. The fused protein disclosed by the invention is formed by connecting human serum albumin and lambda-interferon. Tests prove that high expression level is achieved and the expression product has higher activity when HAS-opt-hIL28B is prepared from a recombinant strain provided by the invention. Therefore, the recombinant strain and the method provided by the invention have broad application prospects in the field of preparation of long-acting human IL28B.

Description

A kind of fusion human interleukin-12 8B and preparation method thereof

Technical field

The present invention relates to a kind of fusion human interleukin-12 8B and preparation method thereof.

Background technology

Interferon, rabbit (interferon is the human cytokines of finding the earliest IFN), as far back as the thirties in 20th century, and after people have found a certain virus of organism infection, the virus generation interference that can have no to concern to another kind of antigenicity.Nineteen fifty-seven; Britain virus biologist Alick Isaacs and Switzerland researchist Jean Lindenmann recognize that the cell of virus infection can produce a kind of factor when utilizing chick chorioallantoic membrane research influenza interference; The latter acts on other cells; Duplicating of viral interference, so with its called after Interferon, rabbit, and use till today always.

Human IFN protein family is divided into three types based on their gene order, chromosomal localization and receptor-specific, i.e. I type, II type and type iii interferon, and wherein I type IFN family comprises the IFN-α non-allelic genes of at least 25 kinds of hypotypes; 1 IFN-β gene; 1 IFN-ω gene, and understand still few 1 IFN-κ and 1 IFN-ε gene, all I type INF genes all are positioned at karyomit(e) No. 9; Do not have intron, and coded albumen uses with a kind of acceptor IFN-α β R all.Only there is member of IFN-γ in II type IFN family, is called type II interferon again, and gene is positioned at karyomit(e) No. 12.Type iii interferon is a new family of Interferon, rabbit; Comprise IFN λ-1, IFN-λ 2 and 33 members of IFN-λ, IFN-λ s gene is positioned at karyomit(e) No. 19, contains a plurality of introns in the gene; Its gene structure and IL-10 family member are quite similar; And differ greatly with IFN-α, so IFN λ-1, IFN-λ 2 and IFN-λ 3 are named as IL29 again respectively, IL28A and IL28B.2003, Sheppard P etc. utilization Antibiology principle utilized information biology softwares such as Genscan, SIGNA1,4HB, WU-BLAST to identify an interferon novel family, reported first its coded product have interferon-like activity (Sheppard P, Kindsvogel W; Xu W, Henderson K, Schlutsmeyer S, Whitmore TE, Kuestner R; Garrigues U, Birks C, Roraback J, Ostrander C, Dong D; Shin J, Presnell S, Fox B, Haldeman B, Cooper E; Taft D, Gilbert T, Grant FJ, Tackett M, Krivan W; McKnightG, Clegg C, Foster D, Klucher KM.IL-28, IL-29 and their class II cytokine receptor IL-28R.Nat Immunol.2003 Jan; 4 (1): 63-8.Epub 2002 Dec 2.).Kotenko S V etc. are with they called after interferon novel families---IFN-λ s (IFN-λ 1, IFN-λ 2 and IFN-λ 3).(Human GeneomeOrganization is HUGO) temporarily with their called after IL29, IL28A and IL28B in Human Genome Organization.

Type iii interferon has the various biological function, and its irritation cell can produce hundreds of interferon-induced genes (ISGs).Can protect the human cell line to resist the cytopathy that vesicular stomatitis virus (VSV) and myocarditis virus cause at external IFN-λ s; IFN-λ 1 can suppress HBV and HCV duplicating in liver cell (Doyle SE, SchreckhiseH, Khuu-Duong K, Henderson K; Rosler R, Storey H, Yao L, Liu H; Barahmand-pour F, Sivakumar P, Chan C; Birks C, Foster D, Clegg CH; Wietzke-Braun P, Mihm S, KlucherKM.Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses inhuman hepatocytes.Hepatology.2006 Oct; 44 (4): 896-906.).Behind the macaque inoculation HIV antigen; IFN-λ s can reduce the generation of Th2 cytokines (IL-4 and IL-5 and IL-13 and IL-14 and IL-15); This has the Th1 of being beneficial to immunization route, increases regulatory T cells, improves the cytotoxicity of cd8 t cell and replys memory (Dai J; Megjugorac NJ; Gallagher GE, Yu RY, Gallagher G.IFN-lambda1 (IL-29) inhibits GATA3expression and suppresses Th2 responses in human naive and memory T cells.Blood.2009Jun 4; 113 (23): 5829-38.).Research in recent years shows that the IL28B gene pleiomorphism is replied the virusology of IFN treatment for HCV the infected has certain influence, the SNP (SNPs) relevant with cytokine IL28B; Not only replied strong correlation with chronic hcv patients polyoxyethylene glycol Interferon, rabbit/ribavirin therapy, also with acute HCV infection after spontaneous virus sweep height correlation, explain that it is on population level; Be decision HCV treatment of infection result's an important host factor (Ge D, Fellay J, Thompson AJ; Simon JS, ShiannaKV, Urban TJ; Heinzen EL, Qiu P, Bertelsen AH; Muir AJ; Sulkowski M, McHutchison JG, Goldstein DB.Genetic variation in IL28B predicts hepatitis C treatment-induced viralclearance.Nature.2009 Sep 17; 461 (7262): 399-401.).At present, people mainly concentrate on IL29 and IL28A to the biological study of type iii interferon.The PEG-IL29 cytokine of U.S. zymogenetics company and BMS cooperative research and development has got into clinical study, and shows good anti-HCV prospect.But IL28B is still blank out at present as the research of medicine.

Interferon, rabbit be find the earliest, study at most, first cloning, first is used for one type of important familial cytokine of clinical treating disease.Its at first found biological activity is the opposing virus infection, and along with going deep into of research, the immunoregulation of Interferon, rabbit, antiproliferative effect and antitumor action just are familiar with by people gradually.From 1986, U.S. FDA was at first ratified after Recomvinated Interferon and α-2b put on market, and genetically engineered β, IFN-are also got permission to put on market in nineteen ninety, 1993 in succession.Since nearly 20 years, along with the development of genetic engineering technique, existing about at present 60 state approval Interferon, rabbit listing is used to treat about more than 30 kinds of diseases such as viral hepatitis, cancer and multiple sclerosis.But the Interferon, rabbit relative molecular mass is less, is prone to by glomerular filtration, and is unstable in the body, is prone to by the serum proteins enzyme liberating, and plasma half-life is short and treatment cycle is long, need frequent drug administration by injection.Therefore; In order to overcome above shortcoming, improve reorganization IFNs dynamic metabolism and pharmacodynamic profile, developed the long-acting interferon of a lot of types in recent years; Its principle of design is mainly based on the following aspects: (1) increases the molecular weight of Interferon, rabbit, reduces glomerular filtration rate(GFR; (2) reduce the immunogenicity of heterologous protein, thereby reduce clearance rate in its body; (3) continue slowly to discharge to keep drug level prolong drug action time.The major technique means have: chemical modification technology, gene fusion technology, site-directed mutagenesis technique and Atrigel etc.

Human serum albumin (HSA) is a natural substance delivery vehicles in the blood of human body, and content is the highest in the serum, and osmotic pressure and blood plasma volume play crucial effects in the body to keeping.Its relative molecular weight is about 65kDa; The strand ball-type protein of forming by 585 amino acid; No zymetology and immunologic competence, its renal clearance is very low, and the transformation period is 14～20d in the body; Being the natural carrier of body intrinsic factor and transport of drug, is the medicine fusion rotein that ideal improves drug half-life.

Summary of the invention

An object of the present invention is to provide a kind of fusion rotein and encoding sox thereof.

Fusion rotein provided by the present invention is the fusion rotein that human serum albumin and Lambda interferon are formed by connecting.

In the above-mentioned fusion rotein, said human serum albumin is positioned at the N end of said Lambda interferon;

In the above-mentioned fusion rotein, said Lambda interferon is human interleukin-12 8B;

In the above-mentioned fusion rotein, the aminoacid sequence of said fusion rotein is shown in SEQ ID NO:1.

Said encoding sox is following 1) or 2) or 3) shown in:

1) its nucleotide sequence is a dna molecular shown in the SEQ ID NO:2;

2) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

3) with 1) or 2) dna sequence dna that limits has homology and the dna molecular of encoding said proteins more than 90%.

The recombinant vectors, reorganization bacterium, transgenic cell line, recombinant virus or the expression cassette that contain above-mentioned arbitrary said encoding sox also belong to protection scope of the present invention.

In the above-mentioned recombinant vectors, said recombinant vectors is that the MCS that gene shown in the SEQ ID NO:2 inserts expression vector pPink α-HC is obtained.

In the above-mentioned reorganization bacterium, said reorganization bacterium imports encoding sox shown in the SEQ ID NO:2 in the host yeast and obtains; Said host yeast is specially pichia spp, is specially at least a in following four kinds of bacterial strains again: ade2 protease-deficient pichia spp, ade2/pep4 protease-deficient pichia spp, ade2/prb1 protease-deficient pichia spp and ade2/prb1/pep4 protease-deficient pichia spp;

In the above-mentioned reorganization bacterium, encoding sox shown in the said SEQ ID NO:2 imports through claim 5 or 6 said recombinant vectorss.

Another object of the present invention provides a kind of method for preparing above-mentioned fusion rotein.

The method of the above-mentioned fusion rotein of preparation provided by the present invention comprises the steps:

1) above-mentioned arbitrary said recombination microzyme is cultured to logarithmic phase in advance;

2) with methyl alcohol the bacterium that step 1) obtains is carried out inducing culture, inducing culture finishes, and collects supernatant, promptly obtains said fusion rotein.

Among the above-mentioned preparation method, said step 2) in, said methyl alcohol accounts for the 1.0%-4.0% (volume percent) of inducing culture system, is specially 2%-4% or 2%-3%;

Among above-mentioned arbitrary preparation method, said step 2) in, the time of said inducing culture is 48h-72h, is specially 72h.

Among above-mentioned arbitrary preparation method, in the said step 1), said preparatory cultivation comprises the steps: the described recombination microzyme of claim 7 is inoculated in the substratum that is used for cultivating pichia spp, and the OD600 that is cultured to culture system is 5.0～6.0;

Among above-mentioned arbitrary preparation method, said step 2) in, said induce with methyl alcohol before, comprise the steps: the thalline that step 1) obtains is resuspended in and be used for inducing saccharomycetic substratum, cultivate 10-14h again;

Among above-mentioned arbitrary preparation method, the said substratum that is used for the culturing yeast bacterium is the substratum that is used to cultivate Pichia yeast, is specially the BMGY substratum; Said to be used to induce saccharomycetic substratum be the substratum that is used to induce Pichia yeast, is specially the BMMY substratum;

Among above-mentioned arbitrary preparation method, said step 1) and said step 2) in, the temperature of said cultivation is 30 ℃.

The preparation method of recombinant protein class medicine mainly comprises at present: gene engineering method such as escherichia expression system, yeast expression system, mammalian cell, insect cell, plant-animal bio-reactor.The PichiaPink yeast expression system that the present invention adopts has the following advantages: belong to eukaryotic expression system, processing and modification after translating expressing protein; The strong promotor of imitating is through the strict regulation and control of methanol induction expression of exogenous gene; The exogenous genes products expression amount is high, can reach the level of every liter of number gram expression product; Self secretory protein is few, and the secreting, expressing product is than easy separation and easy purification; Low to nutritional requirement, growth is fast, and production cost is low, but high density fermentation is fit to large-scale industrial production.The present invention uses biotechnology, through make up coding HSA and through the fusion gene cloning of codon optimized descendant IL28B in PichiaPink Yeast expression carrier pPink α-HC and obtain to efficiently express, be domestic initiation.Experiment showed, with reorganization bacterium of the present invention to prepare HSA-opt-hIL28B, expression amount is high, and purge process is easy, and expression product has stronger activity.Therefore, recombinate bacterium and the inventive method of the present invention has broad application prospects in fields such as the preparation of long-acting people IL28B and immunomodulatory, antiviral therapy.

Description of drawings

Fig. 1 cuts evaluation for the enzyme of pPink α HC-HSA-opt-hIL28B recombinant plasmid.

Fig. 2 is the screening of HSA-opt-hIL28B high-expression clone in No. 2 bacterial strains of PichiaPink strain.

Fig. 3 efficiently expresses the optimization of the expression condition of strain for the HSA-opt-hIL28B recombinant protein.This figure is 12% denaturing polyacrylamide gel electrophoresis and coomassie brilliant blue staining result.

Fig. 4 is the proteic purifying of reorganization HSA-opt-hIL28B.This figure is 12% denaturing polyacrylamide gel electrophoresis and coomassie brilliant blue staining result.

Fig. 5 is the proteic mass spectroscopy of reorganization HSA-opt-hIL28B.

Fig. 6 utilizes the present invention to prepare and opt-hIL28B of the HSA-opt-hIL28B recombinant protein of purifying and another invention preparation and purifying and available from the IFN-α 2b of the Schering Plough company activation to human hepatoma cell line HepG2's cell people myxovirus resistance protein A (MxA).

Fig. 7 is the proteic mass spectroscopy of opt-hIL28B.

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

Quantitative test in following examples all is provided with repeated experiments three times, results averaged.

Foetal calf serum and DMEM substratum are available from Invitrogen company, and plasmid extracts test kit, transfection reagent in a large number available from Giantagen company.Consumptive materials such as disposable Tissue Culture Plate and transfer pipet are available from Corning company.

The preparation of embodiment 1, fusion rotein

One, fusion gene

The nucleotide sequence of the opt-hIL28B gene Fusion gene after HSA and the optimization is shown in SEQ ID NO:2.The aminoacid sequence of this fusion gene encoded protein is shown in SEQ ID NO:1.

Synthetic human serum albumin gene (HSA).The nucleotide sequence of human serum albumin gene (HSA) is shown in 1-1755 position Nucleotide among the SEQ ID NO:2; The proteic aminoacid sequence of this genes encoding is shown in 1-585 amino acids among the SEQ IDNO:1.

The people opt-hIL28B gene of synthetic after codon optimized.Through the nucleotide sequence of people IL28B (hIL28B) gene after codon optimized shown in 1756-2343 position Nucleotide among the SEQ ID NO:2.The proteic aminoacid sequence of this genes encoding is shown in 586-780 amino acids among the SEQ ID NO:1.Gene name before codon optimized is called hIL28B, and the gene name after codon optimized is called opt-hIL28B.Compare with the hIL28B gene, the nucleotide sequence of opt-hIL28B gene changes, and aminoacid sequence is constant.

Design following primer and human serum albumin gene is fused to 5 of gene opt-hIL28B ' terminal (being about to the N-terminal that human serum albumin is fused to opt-hIL28B) through Overlapping PCR mode.The nucleotide sequence of fusion gene is shown in SEQ ID NO:2, and note is made HSA-opt-hIL28B.

The HSA gene template that is used to merge is that template amplification obtains by SEQ ID NO:3 and two pairs of primers of SEQ ID NO:4 with synthetic HAS gene, and the opt-hIL28B gene template that is used to merge is that template amplification obtains by SEQ ID NO:5 and two pairs of primers of SEQID NO:6 with synthetic opt-hIL28B gene; The HSA gene template that will be used to merge again mixes with the opt-hIL28B gene template that is used to merge, and increases with SEQ ID NO:3 and SEQ ID NO:6 primer, obtains fusion gene HSA-opt-hIL28B.

SEQ?ID?NO：3

5’-GTATCTCTCGAGAAAAGGCCTGATGCACACAAGAGTGAG-3’

SEQ?ID?NO：4

5’-GCATCCGGGAGAGCCCCGCGTAAGCCTAAGGCAGCTTGAC-3’

SEQ?ID?NO：5

5’-GTCAAGCTGCCTTAGGCTTACGCGGGGCTCTCCCGGATGC-3’

SEQ?ID?NO：6

5’-TTTAAATGGCCGGCCGGTACCTCAGACACACAGGTCCCCG-3’

Two, recombinant expression vector

PPink α-HC carrier is available from American I nvitrogen company, catalog number (Cat.No.): A11153.

(catalog number (Cat.No.): G060501) be cloned among the Stu I and Kpn I restriction enzyme site of PichiaPink yeast dedicated expression vector therefor pPink α-HC, the recombinant expression plasmid of acquisition note is made pPink α HC-HSA-opt-hIL28B through the GIANTCLONE of Giantagen company single stage method quick clone test kit with fusion gene HSA-opt-IL28B.

Recombinant plasmid pPink α HC-HSA-opt-hIL28B is carried out enzyme cut evaluation, result such as Fig. 1.Among the figure, M1:DNA molecular weight standard (TAKARA company: DL 15,000); M2:DNA molecular weight standard (TAKARA company: DL 2,000); 1:pPink α HC-HSA-opt-hIL28B plasmid is with Nco I and Kpn I double digestion; 2:pPink α HC-HSA-opt-hIL28B plasmid is with Spe I single endonuclease digestion.The result: the fragment of the visible about 1500bp of double digestion swimming lane is cut out.The recombinant expression plasmid that shows structure is correct.

With the recombinant plasmid pPink α HC-HSA-opt-hIL28B evaluation of checking order.The result is presented between Stu I and the KpnI restriction enzyme site of carrier pPink α-HC along the direction from Stu I to KpnI, and the gene order of insertion is shown in SEQ ID NO:2.The right-on plasmid of sequencing result extracts test kit in a large number with the GIANTPREP of Giantagen company plasmid, and (catalog number (Cat.No.): G060303) by specification prepares in a large number.

Three, reorganization bacterium

(1) linearizing of pPink α HC-HSA-opt-hIL28B DNA

Get 10 μ g pPink α HC-HSA-opt-IL28B recombinant plasmids, carry out linearizing, after the DNA after the linearizing is purified, be dissolved in the aseptic deionized water of 10ul with the reaction system that restriction enzyme Spe I advises to specifications.

(2) preparation of yeast competent cell

The PichiaPink expression system is available from American I nvitrogen company, catalog number (Cat.No.): A11154.

Yeast strain 1#, 2#, 3# and 4# are all available from American I nvitrogen company, catalog number (Cat.No.): A11154.This four strains bacterium all belongs to the test kit that catalog number (Cat.No.) is A11154, and different strains all has mark clearly, and the user is easy to distinguish.

Yeast strain 1#, 2#, 3# and 4# are respectively protease-deficient in the PichiaPink expression system, and the gene of defective is respectively ade2, ade2/pep4, ade2/prb1 and ade2/prb1/pep4.Ade2, prb1, pep4 are known, and yeast strain 1#, 2#, 3# and 4# indicate on its specification sheets that all available from American I nvitrogen company they are the defective strains of constructing through the gene knockout mode.

1. single bacterium colony of the bacterial strain in the difference picking PichiaPink yeast expression system is seeded in the 125ml triangular flask that contains 10ml YPD substratum, and 30 ℃, 260r/min cultivated 1 day;

2. get four kinds of strain cultures, 100～500 μ l after the recovery in the above-mentioned steps respectively, be seeded to the 1L triangle that contains the fresh YPD substratum of 100ml and shake in the bottle, adjustment connects bacteria concentration makes initial OD ₆₀₀=0.2,30 ℃, the 260rpm/min overnight cultures is to OD ₆₀₀Reach 1.3～1.5;

3. yeast cell culture in the step 2 is transferred in the aseptic centrifugal bottle of 300ml, 4 ℃, the centrifugal 5min of 1500g abandons supernatant, and bacterial sediment is resuspended with the sterilized water of 200ml ice precooling;

4. 3 centrifugal set by step, with the sterilized water of the ice precooling of 50ml that bacterial sediment is resuspended;

5. 3 centrifugal set by step, with the 1M Sorbitol Solution USP of the ice precooling of 10ml that bacterial sediment is resuspended;

6. 3 centrifugal set by step, with the 1M Sorbitol Solution USP of the ice precooling of 300ul that bacterial sediment is resuspended, its final volume is about 500ul.The competence for preparing is put on ice and is placed, and used the same day.

(3) saccharomycetic electricity transforms

PAD, YPDS are all available from American I nvitrogen company, catalog number (Cat.No.): A11156.

1. linearizing DNA and the competent cell of four kinds of bacterial strains of 80 μ l got respectively behind the 5 μ g pPink α HC-HSA-opt-hIL28B purifying mix, and are transferred in the electric revolving cup of 0.2cm ice precooling ice bath 5min;

2. the MicroPulser Electroporator of utilization Bio-Rad company carries out electricity and changes, and electric commentaries on classics condition is 2kV, 25 Ω, 200 μ F; The electric shock time is about 5ms.After electric shock finished, the YPDS substratum that adds the precooling of 1ml ice mixed thalline, is transferred to 30 ℃, left standstill and cultivated 2h;

3. every pipe is got 100～300 μ l thalline suspensions and is coated on the PAD selection culture plate, and flat board is placed 30 ℃ of cultivations, occurs until single bacterium colony;

From step 3 every plate respectively picking 3-8 white be cloned into fresh PAD and select in the culture plate, the separation and purification of ruling again is cultured to and single bacterium colony occurs.

Four, proteic expression

BMGY substratum composition and the concentration of each composition in substratum are following: 1% yeast powder (yeast extract); 2% peptone (peptone); 1.34% YNB; 0.0004% vitamin H (biotin), 1% glycerine is supplied volume with the phosphate buffered saline buffer (potassium phosphate) of 100mM, pH 6.0.Said percentage composition is except that glycerine is volume percent, and all the other are the quality percentage composition.

BMMY substratum composition and the concentration of each composition in substratum are following: 1% yeast powder (yeast extract); 2% peptone (peptone); 1.34% YNB; 0.0004% vitamin H (biotin), 0.5% methyl alcohol is supplied volume with the phosphate buffered saline buffer (potassium phosphate) of 100mM, pH 6.0.Said percentage composition is except that methyl alcohol is volume percent, and all the other are the quality percentage composition.

(1) screening that relatively reaches the high expression level strain of reorganization HSA-opt-hIL28B expression amount in four kinds of different strains

Picking contain HSA-opt-hIL28B single colony inoculation to the 125ml triangular flask that contains 10ml BMGY substratum, 30 ℃, 260rpm/min cultivated 1 day;

2. culture is transferred in the 50mL awl end centrifuge tube, the centrifugal 5min of 1500g room temperature abandons supernatant, and deposition is resuspended in the 1ml BMMY substratum, and 30 ℃, the 260rpm/min overnight cultures;

3. add 100ul 40% (volume percent) methanol aqueous solution next day, 30 ℃, 260rpm/min continues overnight cultures;

4. second day centrifugal collection supernatant of 1500g * 10min got 20ul and analyzed the relatively variation of HSA-opt-hIL28B expression amount in PichiaPink expression system strain1#, strain2#, strain3#, four kinds of different strains of strain4# through SDS-PAGE.Picking each 32 clone of yeast strain 1#, 2#, 3# and 4# of changing HSA-opt-hIL28B over to carry out abduction delivering respectively, collect supernatant, with each proteic expression amount of Bicinchoninic acid (BCA) method detection.The result; The proteic average expression amount of HSA-opt-IL28B is respectively 0.158ug/ul, 0.176ug/ul, 0.073ug/ul, 0.057ug/ul in strain1#, strain2#, strain3#, four kinds of different strains of strain4#; Show that the expression amount of HSA-opt-IL28B in strain2# is higher than other three kinds of bacterial strains.

Contrast 1: change the expression that does not all detect target protein in the supernatant of yeast strain 1#, 2#, 3# and 4# of empty carrier pPink α-HC over to.

5.HSA-opt-hIL28B the screening of high-expression clone in PichiaPink expression system strain2# bacterial strain.Be higher than on the basis of other three kinds of bacterial strains at the definite expression amount of HSA-opt-hIL28B in strain2#; Further extensive picking changes the single bacterium colony of the strain2# that HSA-opt-hIL28B is arranged and carries out abduction delivering; Analyze relatively (Fig. 2) through SDS-PAGE; Electrophoresis is 12% denaturing polyacrylamide gel electrophoresis and coomassie brilliant blue staining, filters out the high expression level strain of 9 strain HSA-opt-hIL28B, and what wherein expression amount was the highest is No. 6 bacterial strains; Select this bacterial strain to carry out the optimization of expression condition, and this bacterial strain is protected kind.

(2) reorganization HSA-opt-hIL28B efficiently expresses the expression condition optimization of strain.

1. the HSA-opt-hIL28B that filters out is efficiently expressed No. 6 inoculation to the 125ml triangular flask that contains 10ml BMGY substratum, 30 ℃, 260rpm/min cultivated 1 day;

2. the culture in the step 1 is seeded to respectively in 4 1L triangular flasks that contain the 50mLBMGY substratum, 30 ℃, 260rpm/min cultivated 1 day;

3. second day 1500g * 5min is centrifugal, abandons supernatant, and deposition is resuspended in the 10mLBMMY substratum, and 30 ℃, the 260rpm/min overnight cultures;

4. adding 100% methyl alcohol to final concentration (being the volume percent that methyl alcohol accounts for culture system) next day respectively is 1.0%, 2.0%, 3.0%, 4.0%; 30 ℃; 260rpm/min induces, and when induction time is 24h, 48h, 72h, collects supernatant respectively and carries out expressing quantity detection and SDS-PAGE detection.

The result is as shown in Figure 3, and under identical induction time, 3% is higher relatively with the target protein expression amount of the methanol induction of 4% final concentration: under the methanol induction of identical final concentration, the target protein expression amount was higher relatively when induction time was 72h.Electrophoresis is 12% denaturing polyacrylamide gel electrophoresis and coomassie brilliant blue staining.The amount of embodying is following:

Expressing quantity (ug/ul) under table 1, the various condition

	?24h	48h	72h
				1.0％	?0	0	0.086
2.0％	?0	0	0.106
				3.0％	?0	0	0.187
4.0％	?0	0.019	0.177

(3) the proteic great expression of reorganization HSA-opt-hIL28B

1. the HSA-opt-hIL28B that filters out is efficiently expressed No. 6 inoculation to the 125ml triangular flask that contains 10ml BMGY substratum, 30 ℃, 260rpm/min cultivated 1 day; The culturing purposes of this step is to recover, improve active so that next step preparatory cultivation to No. 6 bacterial strains.

2. the culture in the above-mentioned steps is seeded in the 1L triangular flask that contains the 250mLBMGY substratum, 30 ℃, about 260rpm/min incubated overnight to OD600=5.0～6.0; The culturing purposes of this step is to logarithmic phase, so that next step abduction delivering with No. 6 strain culturing after the recovery.

3.1500g * 5min is centrifugal, abandons supernatant, deposition is resuspended in the 50mL BMMY substratum, 30 ℃, and the 260rpm/min overnight cultures.This step is that No. 6 bacterial strains that are cultured to logarithmic phase are carried out abduction delivering.

4. adding methyl alcohol to final concentration (being the volume percent that methyl alcohol accounts for culture system) next day is 3.0%; 30 ℃; After 260rpm/min induced 72h, it was subsequent use to receive bacterium centrifuging and taking supernatant (being protein crude extract), got the part supernatant simultaneously and carried out expressing quantity detection and SDS-PAGE detection.

As a result, proteic expression amount is 0.223ug/ul.

(4) the proteic purifying of reorganization HSA-opt-hIL28B

The solvent of 20mM PB is a water, and solute and the concentration of each solute in 20mM PB are following: K ₂HPO ₄3H ₂O20mM, KH ₂PO ₄20mM; The PH of 20mM PB is 7.0.

1. column equilibration: (available from U.S. GE company) is filled in the chromatography column with cationic exchange coloum SP-Agarose filler, with 5 times of column volumes sterilization purified water balance purifying, to remove 20% residual ethanol.Use buffer A (20mM PB, PH=7.0) balance SP post again;

2. go up appearance: with 2ml/ minute flow velocity sample introduction article, treat that sample advances post fully after, with the balance buffer A of 5 times of volumes (20mM PB, PH=7.0) removal is not conjugated protein;

3. wash-out: behind the solution of 5 times of column volumes of washing, fall baseline after rise to 280 nanometer absorbancys.Use the solution of 5 times of column volumes of elution buffer wash-out more respectively instead and collect elutriant; Elution buffer is made up of KCl and 20mM PB, and the concentration of KCl in elution buffer is respectively 0.5M, 1.0M, 1.5M.

Different salt concentration wash-out is collected liquid carry out SDS-PAGE electrophoresis and coomassie brilliant blue staining evaluation, the result is as shown in Figure 4, and target protein mainly concentrates in the 1.0M elutriant, and after SDS-PAGE identified, the recombinant protein freeze-drying was preserved.Electrophoresis is 12% denaturing polyacrylamide gel electrophoresis and coomassie brilliant blue staining.

Five, the checking of recombinant protein---mass spectroscopy

For identifying the proteic attribute of reorganization HSA-opt-hIL28B of preparation; Target protein band among Fig. 4 is cut; Send Xi Nongsheng (Beijing) Bioisystech Co., Ltd to carry out matrix assisted laser desorption ionization flying time mass spectrum analysis (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry; MALDI-TOF-MS); The result is as shown in Figure 5, with the peptide finger printing of gained (Peptide Mass Fingerprinting, PMF) the up-to-date NCBI albumen database of utilization MASCOT software search comparison is to identify target protein; Find that peptide section sequence and HSA or hIL28B mate, show that the aminoacid sequence of recombinant protein of preparation is correct.

The intracellular signaling pathway analysis that the functional verification of embodiment 2, fusion rotein---HSA-opt-IL28B fusion rotein is regulated

The HepG2 cell is purchased in Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

People's myxovirus resistance protein A (Myxovirus resistance protein A; MxA) be one of interferon inducible protein; This albumen has the activity of anti-multiple virus; Gene is not expressed under the normal circumstances, but when multiple virus infection, can pass through virus infection inductive I type (α β) and III type (λ s) Interferon, rabbit path is expressed by rapid induction.MxA is I type and the active mark of type iii interferon in virus infection and IFN treatment, so can be through measuring the biological activity that MxA albumen or its mRNA reflect I type and type iii interferon.

The present invention with the HepG2 cell inoculation after 6 orifice plates cover with individual layer; Add 1 respectively, the reorganization HSA-opt-hIL28B albumen behind the 10ng/ml purifying; The HepG2 groups of cells of handling with people IL28B albumen, with IFN-α 2b (purchasing in Schering Plough company) and the PBS of 1ng/ml is simultaneously hatched for 37 ℃ as contrast, receives cell behind the 24h; It is resuspended that every hole adds 100 μ L, 1 * SDS loading buffer; Boil 10min, the centrifugal 5min of 12000rpm room temperature gets the 10ul supernatant detects MxA through western Blotting expression level.MxA antibody is available from U.S. R&D company.Detection method is an immunoblotting.

Concrete outcome is as shown in Figure 6; The proteic processing of HSA-opt-hIL28B albumen and hIL28B all can stimulate the expression of HepG2 cell induction MxA; And its expression level increases with the rising of HSA-opt-hIL28B albumen and hIL28B albumen concentration of treatment, and the same BA with the expression of stimulation interferon inducible protein with reorganization hIL28B albumen of reorganization HSA-opt-hIL28B albumen is described.

The proteic preparation of people IL28B: the preparation method of method and HSA-opt-hIL28B is basic identical, and different is: the foreign gene that changes in the reorganization bacterium is nucleotide sequence gene shown in 1756-2343 position Nucleotide among the SEQ ID NO:2.

The proteic checking of people IL28B---mass spectroscopy

Send Xi Nongsheng (Beijing) Bioisystech Co., Ltd to carry out matrix assisted laser desorption ionization flying time mass spectrum analysis (Matrix-Assisted Laser Desorption Ionization-Time of Flight MassSpectrometry the target protein of preparation; MALDI-TOF-MS); The result is as shown in Figure 7; Peptide finger printing (Peptide MassFingerprinting with gained; PMF) utilization MASCOT software search is compared up-to-date NCBI albumen database to identify target protein, finds peptide section sequence and hIL28B coupling, shows that the proteic aminoacid sequence of preparation is correct.

Claims (10)

1. a fusion rotein is the fusion rotein that human serum albumin and Lambda interferon are formed by connecting.

2. fusion rotein according to claim 1 is characterized in that: said human serum albumin is positioned at the N end of said Lambda interferon;

And/or said Lambda interferon is human interleukin-12 8B;

And/or the aminoacid sequence of said fusion rotein is shown in SEQ ID NO:1.

3. the encoding sox of claim 1 or 2 said fusion roteins.

4. encoding sox according to claim 3 is characterized in that: said encoding sox is following 1) or 2) or 3) shown in:

1) its nucleotide sequence is a dna molecular shown in the SEQ ID NO:2;

2) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

3) with 1) or 2) dna sequence dna that limits has homology and the dna molecular of encoding said proteins more than 90%.

5. the recombinant vectors, reorganization bacterium, transgenic cell line, recombinant virus or the expression cassette that contain claim 3 or 4 said encoding soxs.

6. recombinant vectors according to claim 5 is characterized in that: said recombinant vectors is that the MCS that gene shown in the SEQ ID NO:2 inserts expression vector pPink α-HC is obtained.

7. reorganization bacterium according to claim 5 is characterized in that: said reorganization bacterium imports encoding sox shown in the SEQ ID NO:2 in the host yeast and obtains; Said host yeast is specially pichia spp, is specially at least a in following four kinds of bacterial strains again: ade2 protease-deficient pichia spp, ade2/pep4 protease-deficient pichia spp, ade2/prb1 protease-deficient pichia spp and ade2/prb1/pep4 protease-deficient pichia spp;

And/or encoding sox shown in the said SEQ ID NO:2 imports through claim 5 or 6 said recombinant vectorss.

8. a method for preparing the said fusion rotein of claim 1 comprises the steps:

1) the described recombination microzyme of claim 7 is cultured to logarithmic phase in advance;

2) with methyl alcohol the bacterium that step 1) obtains is carried out inducing culture, inducing culture finishes, and collects supernatant, promptly obtains said fusion rotein.

9. method according to claim 8 is characterized in that: said step 2), said methyl alcohol accounts for the 1.0%-4.0% (volume percent) of inducing culture system, is specially 2%-4% or 2%-3%;

Said step 2) in, the time of said inducing culture is 48h-72h, is specially 72h.

10. according to Claim 8 or 9 described methods; It is characterized in that: in the said step 1); Said preparatory cultivation comprises the steps: the described recombination microzyme of claim 7 is inoculated in the substratum that is used for the culturing yeast bacterium, and the OD600 that is cultured to culture system is 5.0～6.0;

Said step 2) in, said induce with methyl alcohol before, comprise the steps: the thalline that step 1) obtains is resuspended in and be used for inducing saccharomycetic substratum, cultivate 10-14h again;

The said substratum that is used for the culturing yeast bacterium is the substratum that is used to cultivate Pichia yeast, is specially the BMGY substratum; Said to be used to induce saccharomycetic substratum be the substratum that is used to induce Pichia yeast, is specially the BMMY substratum;

Said step 1) and said step 2) in, the temperature of said cultivation is 30 ℃.

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