CN1523040A - Fusion protein of human thymosin alpha1 and human composite interferon and preparation thereof - Google Patents

Abstract

The present invention relates to a fusion protein of human thymosin alpha 1and human compound interferon, and said protein expression and purification method and application of said protein which can be used as medicine for resisting virus and resisting tumor.

Description

The fusion rotein and the preparation thereof of human thymosin alpha 1 and people's Interferon alfacon-1

Technical field:

The present invention relates to a kind of people's Interferon alfacon-1 (IFN-con) and thymosin (Thymosin alpha1) fusion rotein and this protein expression, purifying and use this albumen as method antiviral, antitumor drug.

Background technology:

The protein polypeptide of the Interferon, rabbit relative small molecular weight that to be a class be made up of single chain molecule has antiviral, antiproliferative and immunoregulatory function, is used for the treatment of virus disease, tumour and multiple scleroderma etc. clinically.It is α, β and γ type that human interferon is divided into three major types, is mainly produced by white corpuscle, inoblast and lymphocyte respectively and names.Human alpha interferon is made up of 166 or 165 amino acid, and molecular weight is about 19000 dalton, and the inferior member of human alpha interferon who has cloned and determined at present aminoacid sequence has more than 20 kinds.Though interferon formulation has very big clinical use value, result of treatment is still undesirable clinically, and its antiviral specific activity only is 1 * 10 ⁸About IU/mg, the bigger shortcoming of side effect when existing heavy dose of the use.For this reason, human alpha interferon is suddenlyd change and transform, generate various analogues is a focus to improve its active research always.Wherein the most noticeable is the research of the relevant Interferon alfacon-1 of U.S. Amgen company.Its main thought is the brand-new interferon molecule that the gene order homology of 13 kinds of alpha-interferons having reported according to nineteen eighty-three is designed, describe this kind Interferon, rabbit is existing in the United States Patent (USP) 4695623,4897471, proved that IFN-con has the interferon activity of wide spectrum, its antiviral specific activity reaches 1 * 10 ⁹More than the IU/mg, be more than 10 times of any natural alpha-interferon, wherein IFN-con1 FDA approval 1997 is gone on the market in the U.S., commodity are called INFERGEN, are used for the treatment of chronic hepatitis C, and are evident in efficacy, it is higher to have demonstrated the virus sweep rate, the characteristics that side effect is littler.On the basis of INFERGEN, Chinese patent CN1208731 has reported a kind of the Hans' of being more suitable for Interferon alfacon-1 Val ¹⁵⁸IFN-con1, its clinical effectiveness is consistent with INFERGEN, but side reaction is lower.

1961, Miller etc. at first found the function of thymus gland, and newborn mouse is after thymus gland is cut, and its growth and adenoid growth all are subjected to serious obstruction.Later some studies show that thymus gland is a kind of important immune organs, and it plays conclusive effect in lymphsystem is grown.Find that in these researchs a kind of thymus gland prepared product TF5 (thymosin fraction5) has very strong immune enhancing function.This prepared product can be corrected some immune deficiency that causes owing to the shortage thymus function, and treats the cancer that some immunity system is suppressed.TF5 prepared product thermostability is fine, can anti-80 ℃ reaches 25min, and it is made up of the polypeptide of one group of molecular weight 1000-15000, also contains the non-protein ingredient of trace such as carbohydrate etc.Obtaining thymosin (being called for short TM-alpha1) after TF5 further separated, is an Acid polypeptide, exceeds 10-1000 doubly than the biological activity of original TF5 prepared product.It is made up of 28 amino acid, does not contain methionine(Met), halfcystine and die aromatischen Aminosaeuren, and iso-electric point is 4.2, and the N end is acetylation, but it is not remarkable to activity influence.TM-alpha1 is a kind of biological respinse regulatory factor, at first be a kind of at the lymphoid immunostimulant of T, can impel the maturation and the differentiation of T cell, can impel sophisticated T cell, the various lymphokines of NK emiocytosis such as interleukin-2 and IFN-, also promote the generation of interleukin-2 acceptor simultaneously.TM-alpha1 has huge value on medical market, can be used for treating that some immunity systems are suppressed or the destructive disease, comprises chronic viral hepatitis B, third hepatitis, cancer, AIDS etc.Present TM-alpha1 (trade(brand)name: Zadaxin) comprising a plurality of countries listing of China, but be chemical synthetic drug, the cost height, expensive, existing report is attempted genetically engineered and is produced TM-alpha1, but because its peptide chain is shorter,, purification difficult low and be difficult to form industrialization in the expression in escherichia coli amount.

TM-alpha1 derives from precursor prophymosin-alpha (prothymosin α), a kind of on evolving the strongly-acid albumen of high conservative, TM-alpha1 is in its 2-29 position, prophymosin-alpha has similar biological activity to thymosin, and prophymosin-alpha will be higher than thymosin to the adjusting activity of immunologic function.The combined utilization of IFN-α and TM-alpha1

IFN-α and TM-alpha1 have complementary biological activity, and the combined utilization of IFN-α and TM-alpha1 extensively proves the independent use that obviously is better than both to hepatitis with to tumor treatment.As in the chronic hepatitis C treatment, Moscarella etc. find 34 routine chronic hepatitis C patient random research: 17 routine patient's combined utilization TM-alpha1 (1mg, twice weekly) and IFN-α treatment after 6 months, 71% patient ALT level is reduced to normally, and hepatitis C virus RNA clearance rate is 65%.And 17 routine patients only use after the IFN-α treatment 35% patient ALT level separately and reduce to normally, and hepatitis C virus removing rate is 29% (P＜0.05).Sherman etc. to 109 routine chronic hepatitis C patients carry out at random, double blinding, multicenter study show: combined utilization TM-alpha1 (1.6mg, twice weekly) and IFN-α (3MU, 3 times weekly) treatment 6 months after the normal person of ALT level be 37.1%, use IFN-α (3MU separately, 3 times weekly) the patient ALT level of treatment back 16.2% is reduced to normally, and 2.7% patient ALT level is reduced to normally (P＜0.05) behind the application placebo treatment.Evidence suggests: the increase of Th1 relevant cell factor (IL-2, IFN-γ) helps the removing of hepatitis C virus, and the Th-2 relevant cell factor (IL-4, increase IL-10) then impels viral long-term existence.Andreone etc. studies show that: use the generation of TM-alpha1 treatment can increasing IL-2, IFN-α also can increase the generation of IL-2 slightly, and both share the generation that then obviously promotes IL-2.TM-alpha1 can reduce the generation of IL-4 simultaneously, and IFN-α then promotes the generation of IL-4, and TM-alpha1 can reverse the effect that IFN-α promotes that IL-4 generates when both share.This shows that TM-alpha1 and IFN-α combined utilization can improve the biological effect of IFN-α, obtain better result of treatment.Can improve treatment of diseases effects such as comprising viral hepatitis, tumour greatly though proved TM-alpha1 and IFN-α combined utilization, because two kinds of medicines need to produce respectively, drug cost is high, has limited its application clinically.Therefore press for and develop a kind of TM-alpha1 of having and the bioactive medicine of IFN-α.U.S. Pat 6319691 discloses the fusion rotein of a kind of TM-alpha1 and IFN-α 2b, and it had both had the antiviral activity of IFN-α, has the immunologic competence of TM-alpha1 again, and the work simplification that will produce two kinds of medicines is a technology.But find after deliberation, though no matter IFN-α is can not cause both noticeable changes on conformation at N end or C end with TM-alpha1 fusion back, but for Interferon, rabbit, may be owing to there be the sterically hindered avidity that reduces itself and associated receptor after the fusion, cause antiviral specific activity to descend greatly, when its antiviral specific activity is reduced to a certain degree, then can cause the difficulty in the clinical application.

Interferon alfacon-1 has been widely used in multiple virus disease, and its antiviral specific activity is better than any natural IFN-α, has especially shown the result of treatment that significantly is better than natural IFN-α in the treatment of third liver.The present invention utilizes this characteristics, on the fusion rotein manufacturing technology basis of existing Interferon, rabbit and TM-alpha1, produced the fusion rotein of a kind of Interferon alfacon-1 and TM-alpha1, unexpected simultaneously this fusion rotein of finding has than the better stability of the fusion rotein of known Interferon, rabbit and TM-alpha1, hypotoxicity and biological activity.

The present invention selects the codon of intestinal bacteria preference, synthetic IFN-con and TM-alpha1 fusion gene, in intestinal bacteria, realize efficiently expressing of fusion rotein, because IFN-con and the complementary action of TM-alpha1 on function, make that its antivirus action is stronger, the virus sweep rate is higher, it is more extensive to use.

Summary of the invention:

The invention provides a kind of the have IFN-con of antiviral and enhancing body immunologic function and fusion rotein of TM-alpha1 and preparation method thereof.

Fusion rotein of the present invention is made of TM-alpha1 and IFN-con two big functional zone, has dual function, and it is formed by covalent bonds by polypeptide with TM-alpha1 function and the polypeptide with IFN-con function.

Fusion rotein of the present invention, the mode of connection between its two kinds of functional zone can be direct connection, also can be provided with connection peptides, comprise with mode of connection as polypeptide: I-T, T-I, T-I-T with polypeptide of IFN-con function by the TM-alpha1 function, I-L-T, T-L-I, T-L-I-L-T.Wherein I representative: IFN-con, T representative: TM-alpha1, the L representative: connection peptides, wherein preferred mode of connection is T-L-I.

Fusion rotein of the present invention, preferred amino acids sequence are at the aminoacid sequence shown in the sequence table 2.Fusion rotein of the present invention, its DNA sequences encoding can be the dna sequence dnas of any fusion rotein of the present invention with TM-alpha1 and two kinds of functions of IFN-con of can encoding, and comprise their prolongation sequence.

Fusion rotein of the present invention, its DNA sequences encoding is the nucleotide sequence shown in the sequence table 1 preferably.

The present invention also comprises, fusion rotein preparation method of the present invention.

Fusion rotein of the present invention is made by gene recombination technology.

The host cell that uses in the fusion rotein manufacturing processed of the present invention comprises prokaryotic cell prokaryocyte and eukaryotic cell, as bacterium, yeast, zooblast and vegetable cell etc.

The preparation process of fusion rotein of the present invention comprise the steps: engineering bacteria structure, preparation seed liquor, shake-flask culture, slightly carry, essence is carried, work in-process preparation, packing, freeze-drying.

The present invention also provides the pharmaceutical composition of fusion rotein, and said composition comprises the fusion rotein of the present invention and the pharmaceutically acceptable carrier of significant quantity on the physiology.

Fusion rotein of the present invention can also be under the prerequisite that does not change described fusion rotein characteristic, the polypeptide that replacement, disappearance or the adding of the indivedual amino-acid residues of fusion rotein obtained.

Below describe the manufacturing of fusion rotein of the present invention in detail.

The design of IFN--con and TM-alpha1 fusion rotein

TM-alpha1 does not generally show the fixed structure in the aqueous solution, IFN-alpha molecule N end and C end all extend in the molecule outside, TM-alpha1 merges the N end by a hydrophilic and flexible connection peptides with IFN-α or the fusion of C end can not cause both noticeable changes on conformation, but the document of relevant interferon fusion protein proves, the N that one small peptide is blended in interferon molecule holds the reservation that more helps its antiviral activity, hold first amino acid position because the disulfide linkage of fusion rotein no longer is in N simultaneously, ten minutes helps the formation of disulfide linkage and stablizes.For the TM-alpha1 molecule, it is consistent with its precursor prophymosin-alpha N end structure to be blended in N end back, helps the reservation of its immunologic competence on the one hand, prolongs the transformation period of TM-alpha1 molecule, because it does not show the fixed structure in the aqueous solution, immunogenicity is also very weak on the other hand.Therefore TM-alpha1 and IFN fusion optimal way is T-L-I, and the aminoacid sequence of fusion rotein is seen sequence table 2.

The synthetic design of fusion gene

Adopt the habitual codon synthetic TM-alpha1-Linker-IFN-con gene fragment of intestinal bacteria earlier, add initial code ATG and clone recognition sequence at TM-alpha1 gene 5 ' end, at terminal series connection stop code TAATAG and the restriction endonuclease BamHI recognition sequence of adding of IFN-con gene fragment with restriction endonuclease NdeI.

The cloning and expression of fusion gene

The gene fragment of encoding fusion protein is cloned among the plasmid pET-22b (+) with recombinant DNA technology, meets fully with original design through the dna sequencing proof.The recombinant expression plasmid carrier is converted in BL21 (DE3)-codon plus-RPX host bacterium expresses.It can solubility high expression level recombination target protein TM-alpha1-Linker-IFN-con1 in the LB substratum.Bacterial strain is: E.coli BL21 (DE3)-codon plus-RPX/pET-TIN, this bacterial classification be Chinese typical culture collection center in 2003.8.21 is preserved in Wuhan University, is numbered CCTCC No.M203066

The preparation of fusion rotein

Colibacillus engineering strain E.coli BL21 (the DE3)-codon plus-RPX/pET-TIN that has the recombinant expression plasmid of TM-alpha1-Linker-IFN-con fusion gene by shake-flask culture, can obtain a large amount of TM-alpha1-Linker-IFN-con fusion roteins, through some purification steps, as saltout, chromatography (comprises ion exchange chromatography, the gel chromatography chromatography, hydrophobic chromatography, affinity chromatography, high pressure liquid chromatography (HPLC) etc.), ultrafiltration etc. in conjunction with using, can obtain the fusion rotein with high purity (＞95%) of uniform component.

The fusion rotein biologic activity detects

Adopt cytopathic-effect inhibition assay to measure the antiviral activity of fusion rotein, the result shows that this fusion rotein and standard reference material have stronger antiviral activity, and specific activity reaches 4 * 10 ⁸IU/mg.

Adopt cell proliferation experiment to detect the influence of fusion rotein to mice spleen lymphocytes proliferation, experimental result shows, the synthetic TAl of different concns and fusion rotein promote the proliferation rate of mice spleen lymphocytes proliferation, compare and do not have significant difference for two groups within scope of experiment.

The application of fusion rotein

IFN-con of the present invention and TM-alpha1 fusion rotein can be used to regulate immunologic function, and be ripe and carry out function as strengthening the propagation of T cell, and the antiviral and antineoplastic ability of enhancing immunity system is used to improve former or insecondary immune deficiency etc.As a kind of medicine, may be used for some virus disease and tumor treatment, comprise the acute and chronic disease of viral infection as third liver, hepatitis B, herpesvirus infection, Genital warts etc., the diseases such as Kaposi's sarcoma, Fei Hejiejinshi disease, gastroenteric tumor and renal cell carcinoma that malignant tumour such as hairy cell, melanoma, chronic myeloid leukemia, multiple myeloma, AIDS are correlated with.

Content of the present invention comprises that also a kind of fusion rotein with IFN-con and TM-alpha1 of preparation is the pharmaceutical preparation of activeconstituents, this pharmaceutical preparation is mainly with the parenteral route administration, as injection of normal injection agent, freeze drying injection, lipidosome injection, target administration etc., also can be made into the preparation of gi tract administration, as tablet, capsule, sprays, gelifying agent, gel inhalation, oral liquid, suspensoid, electuary, patch, pill, powder, injection, infusion solution, suppository, sustained release preparation, controlled release preparation etc.

The pharmaceutical preparation of the present invention wherein content of the fusion rotein of IFN-con and TM-alpha1 is 0.1-99.9%, and other are the medicine acceptable carrier.These medicine acceptable carriers can be, starch, dextrin, sucrose, lactose, cellulose family, Magnesium Stearate, tween-80, carbomer, methylcellulose gum, Xylo-Mucine, sodium alginate, glycerine, gelatin, polyoxyethylene glycol, Thiomersalate, propylben, Tegosept M, ethyl p-hydroxybenzoate, trichloro-butyl alcohol, Sodium Benzoate, borax, bromogeramine, sorbyl alcohol, the propylene glycol Virahol, bay nitrogen table ketone, Virahol/propylene glycol (1: 1), Virahol/(1: 2) and Virahol/propylene glycol (2: 1), trolamine, sodium hydroxide Vaseline, stearic acid, Liquid Paraffin, lanolin, stearyl alcohol, hexadecanol.Glyceryl monostearate, sodium laurylsulfate, sorbester p18, peregal 0, N.F,USP MANNITOL, sorbyl alcohol etc.

Pharmaceutical preparation of the present invention can be by the method preparation of this area routine.

The antiviral activity of fusion rotein of the present invention

Adopt cytopathic-effect inhibition assay to measure the antiviral activity of fusion rotein.With WISH cell (human amniotic cell) suspension inoculation in 96 orifice plates, every hole 3.5 * 10 ⁴Cell/100ul, 37 ℃, 5%CO ₂Cultivate 4～6h under the condition, get sample behind the fusion rotein purifying, do serial dilution after every hole add 100ul, cultivate the 24h. suction for 37 ℃ and abandon supernatant, to working concentration, every hole adds 100ul with the DMEM substratum dilution VSV (vesicular stomatitis virus) that contains 3% bovine serum, cells infected, 37 ℃, 5%CO ₂Continue to cultivate 24h, every sample is done three multiple holes.Violet staining is measured the A578nm absorbance value in every hole, and that calculates each test sample respectively partly imitates extension rate, promptly from sample solution to the extension rate that is equivalent to standard substance 50% maximum effect point, and be calculated as follows test-results:

Sample to be checked tires=standard crystalline substance X (the pre-extension rate of the pre-extension rate/standard substance of the sample to be checked) X (sample to be checked is partly imitated extension rate/standard substance and partly imitated extension rate) that tires

The result shows that fusion rotein and standard reference material have stronger antiviral activity, see the following form 1:

Table 1 TM-alpha1-IFN-con antiviral activity

Sample specific activity (1U/mg)

IFN-alpha1b 0.9×10 ⁷

IFN-alpha2a 2.5×10 ⁸

TM-alpha1-IFN-con 4×10 ⁸

The immunologic competence of fusion rotein

Adopt cell proliferation experiment to detect of the influence of TM-alpha1-IFN-con fusion rotein to mice spleen lymphocytes proliferation.With ³H-TdR mixes method, gets mouse spleen and isolate monocyte with lymphocyte separation medium after grinding on the steel sieve, after washing with PBS, is suspended from the RPMI-1640 that contains 10% calf serum, adds 5 * 10 in 96 orifice plates ⁵The splenocyte in/hole and the ConA.CO of 5ug/ml ₂Cultivate 6h for 37 ℃ in the incubator, add the fusion rotein and the synthetic TM-alpha1 standard substance of different concns again, continue to cultivate 72h, every hole adds ³H-TdR2 μ ci cultivates 6h, and harvested cell dries the back and measures the cpm value on glass fiber filter paper, is calculated as follows proliferation rate:

Proliferation rate (%)=[(experimental group cpm value-control group cpm value)/control group cpm value] * 100%

Experimental result shows, adds the synthetic TM-alpha1 and the fusion rotein of different concns, compares and do not have significant difference (p＞0.5) for two groups within scope of experiment, and all there were significant differences (p＜0.01) and compare with negative control group, the results are shown in Table 2.

The short cell-proliferation activity of table 2 fusion rotein TM-alpha1-IFN-con

Sample concentration (mg/L) cpm proliferation rate (%)

Synthetic Tal 12.5 293701 ± 10,562 99

25 306307±13825 108

50 316844±17896 115

100 287925±9682 95

TM-alpha1-IFN-con1 12.5 297750±13825 102

25 336692±11485 128

50 310508±29036 110

100 294380±17253 100

Physiological saline 147463 ± 4681

Excellent results of the present invention

The employing intestinal bacteria have been habitually practised the codon synthetic IFN-con and TM-alpha1 fusion gene fragment, made up the prokaryotic expression carrier pET-TIN of fusion gene, conversion contains the e. coli host cell of t7 rna polymerase, but induces expressed fusion protein through IPTG.

Engineering bacteria LB21 (the DE3)-RPX/pET-TIN that makes up can be efficient, solubility ground expressed fusion protein, and it is proteic more than 20% that its expression amount reaches escherichia coli host.This bacterial classification now has been preserved in Chinese typical culture collection center in the Wuhan University.

Set up the purifying process of easy fusion rotein, the SDS-DAGE collection of illustrative plates gray scale scanning of purifying protein shows that purity is greater than 95%.

The external activity experiment shows that fusion rotein has and uses the similar antiviral activity of IFN separately and the independent similar stimulation lymphocyte proliferation activity of thymosin of using that wherein antiviral specific activity is greater than IFN-α 2b that has reported and TM-alpha1 fusion rotein.

Specific embodiments:

The present invention is further illustrated by the following examples.

Embodiment 1

Synthesizing of fusion gene

Synthetic segmental design is with synthetic

According to the habitual sign indicating number of intestinal bacteria, artificially design IFN-con-Linker-TM-alpha1 antigen-4 fusion protein gene sequence (seeing figure), entrust Shanghai to give birth to worker's biotechnology company limited complete genome sequence and synthesize.

Fusion gene is expressed the structure of engineering bacteria

The structure of fusion gene expression plasmid

Get TM-alpha-Linker-IFN-con1 fusion gene plasmid and behind restriction endonuclease Nde I and BamH I double digestion, reclaim small segment,, behind Nde I and BamHI double digestion, reclaim big fragment the prokaryotic expression plasmid pET-22b (+) of band T7 promotor.Connect transformed into escherichia coli DH5 α, screening recon by the T4 ligase enzyme.Clone's called after pET-TIN after enzyme is cut evaluation and sequential analysis conclusive evidence.

Fusion gene is expressed the structure of engineering bacteria

The pET-TIN expression plasmid is transformed in BL21 (DE3)-Codon plus-RP-X host bacterium, LB cultivates, an amount of IPTG abduction delivering, expression product is analyzed through SDS-PAGE, but expression amount reaches more than 20% of intestinal bacteria dsred protein, and the expression product major part is soluble proteins, with this engineering bacteria called after BL21 (DE3)-RPX/pET-TIN, this bacterial classification now has been preserved in Chinese typical culture collection center in the Wuhan University, is numbered CCTCC No.M203066.

Engineering bacteria Ecoli Bi21 (DE3)-RPX/pET-TIN cultivates and the purifying of fusion rotein in batches

The shake-flask culture of engineering bacteria

The single bacterium colony of picking BL21 (DE3) from the LB flat board-RPX/pET-TIN, be seeded to (50ml cultivates triangular flask) in the 10mlLB substratum that contains the 100ug/ml penbritin, 37 ℃ of 220rpm joltings were cultivated 12 hours, 1% is forwarded in the 1L LB substratum, the nectar degree is about 0.1 OD600nm, 37 ℃ of shaking tables, the 220rpm cultivation adds IPTG after 3 hours and induces, and express tracking with SDS-PAGE, after 3 hours, fusion protein expression is near 20% of e. coli total protein, and cell density OD 600nm reaches 4, sees Fig. 3.Centrifugal collection thalline, frozen in-20 ℃, be ready for use on the purifying of fusion rotein.

The separation and purification of fusion rotein

Ultrasonication → centrifugal reservation supernatant → ammonium sulfate precipitation → dissolving back is crossed Phenyl Sepharose 6Fast Flow → mistake Q Sepharose Fast Flo → mistake SP Sepharose Fast → Flow and is crossed molecular sieve Super → lex 75 reorganization TM-alpha-Linker-IFN-con1

Reorganization TM-alpha-Linker-IFN-con1 can reach more than 95% by aforesaid method purifying purity, is lyophilized into white powder pin after adding 5% N.F,USP MANNITOL after the Lowry method is measured protein concentration in its buffered soln PBS.

Description of drawings:

Fig. 1 makes up synoptic diagram for expression plasmid pET-TIN.

Fig. 2 is that plasmid pET-TIN double digestion is identified electrophorogram.1 road is a molecular weight standard, the 2-5 road be recombinant plasmid pET-TIN through NdeI and BamHI double digestion rear electrophoresis result, all cut out the DNA band of 634bp.

Fig. 3 is an expressing fusion protein SDS-PAGE electrophorogram.1 road is 30 ℃ and induces inclusion body behind 4 hours broken bacterium, 2 roads are 30 ℃ and induce supernatant behind 4 hours broken bacterium, 3 roads are 30 ℃ and induce inclusion body behind 2 hours broken bacterium, 4 roads are 30 ℃ and induce supernatant behind 2 hours broken bacterium, 5 roads are 25 ℃ and induce inclusion body behind 4 hours broken bacterium, and 6 roads are 25 ℃ and induce supernatant behind 4 hours broken bacterium, and 7 roads are 25 ℃ and induce inclusion body behind 2 hours broken bacterium, 8 roads are 25 ℃ and induce supernatant behind 4 hours broken bacterium that 9 roads are molecular weight standard.

Fig. 4 is a fusion rotein HIC column chromatography electrophorogram.1 road is for splitting supernatant behind the bacterium, and 2 roads are for passing the peak, and 3 roads are the washing peak, and the 4-9 road is that gradient washing substep is collected the peak, and 10 roads are molecular weight standard

Fig. 5 is a fusion rotein Q column chromatography electrophorogram.1 road is a molecular weight standard, and 2 roads are dilution back, the active peak of HIC chromatography sample, and 3 roads are for passing liquid, and 4 roads are the washing peak, and the 5-8 road is that gradient washing substep is collected active peak.Fig. 6 is a fusion rotein purifying SP column chromatography electrophorogram.1 road is a molecular weight standard, and the 2-7 road is that the result is collected in the gradient elution distribution, and wherein 6,7 for collecting liquid.

Fig. 7 is fusion rotein purifying Superdex 75 column chromatography electrophorograms.The 1-3 road is for collecting the peak, and 4 roads are molecular weight standard.

Sequence table

＜110〉Kangerwei Pharmaceutical Co., Ltd., Chongqing

＜120〉fusion rotein and the preparation thereof of human thymosin alpha 1 and people's Interferon alfacon-1

<160>3

<210>1

<211>621

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)…(621)

＜223〉according to the aminoacid sequence of human thymosin alpha 1 with human interferon-con1, and the aminoacid sequence that is used as connection peptides between two polypeptide, with habitual yard dna sequence dna that carries out code Design of intestinal bacteria.

<400>1

atgtctgacg?ctgctgttga?cacctcttct?gaaatcacca?ccaaagacct?gaaagaaaaa 60

aaagaagttg?ttgaagaagc?tgaaaacggt?ggtggtggtt?ctggaggtgg?tggtggttgc 120

gacctgccgc?aaacccacag?cctgggtaac?cgtcgtgccc?tgatcctgct?ggcacaaatg 180

cgtcgtatct?ctcctttctc?ctgtctgaaa?gaccgtcacg?acttcggttt?cccgcaggag 240

gagtttgatg?gcaaccagtt?ccagaaggct?caagccatct?ctgtcctcca?tgagatgatc 300

cagcagacct?tcaacctctt?cagcaccaaa?gattcctctg?ctgcttggga?cgagtccctg 360

ctggagaaat?tctacaccga?actctaccag?cagctgaacg?acctggaagc?ctgtgtgatc 420

caggaggtcg?gtgtggaaga?aaccccgctg?atgaatgtcg?actccatctt?ggctgtgaag 480

aaatacttcc?agcgtatcac?tctctatctg?acagagaaga?aatacagccc?ttgtgcctgg 540

gaggttgtcc?gtgcagaaat?catgcgttcc?ttctctctgt?ccaccaacct?gcaggaacgt 600

ctgcgtcgta?aggaataata?g 621

<210>2

<211>205

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide sequence that human interferon-con1 and 1 two peptide molecules of human thymosin alpha is formed by connecting with one section connection peptides that comprises ten amino-acid residues.

<400>2

Met?Ser?Asp?Ala?Ala?Val?Asp?Thr?Ser?Ser?Glu?Ile?Thr?Thr?Lys?Asp

1 5 10 15

Leu?Lys?Glu?Lys?Lys?Glu?Val?Val?Glu?Glu?Ala?Glu?Asn?Gly?Gly?Gly

20 25 30

Gly?Ser?Gly?Gly?Gly?Gly?Gly?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu

35 40 45

Gly?Asn?Arg?Arg?Ala?Leu?Ile?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser

50 55 60

Pro?Phe?Ser?Cys?Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu

65 70 75 80

Glu?Phe?Asp?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Gln?Ala?Ile?Ser?Val?Leu

85 90 95

His?Glu?Met?Ile?Gln?Gln?Thr?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser

100 105 110

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

115 120 125

Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Glu?Val?Gly

130 135 140

Val?Glu?Glu?Thr?Pro?Leu?Met?Asn?Val?Asp?Ser?Ile?Leu?Ala?Val?Lys

145 150 155 160

Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser

165 170 175

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

180 185 190

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

195 200 205

<210>3

<211>205

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide sequence that human interferon-con1 and 1 two peptide molecules of human thymosin alpha is formed by connecting with one section connection peptides that comprises ten amino-acid residues.

<400>3

atg?tct?gac?gct?gct?gtt?gac?acc?tct?tct?gaa?atc?acc?acc?aaa?gac 48

Met?Ser?Asp?Ala?Ala?Val?Asp?Thr?Ser?Ser?Glu?Ile?Thr?Thr?Lys?Asp

1 5 10 15

ctg?aaa?gaa?aaa?aaa?gaa?gtt?gtt?gaa?gaa?gct?gaa?aac?ggt?ggt?ggt 96

Leu?Lys?Glu?Lys?Lys?Glu?Val?Val?Glu?Glu?Ala?Glu?Asn?Gly?Gly?Gly

20 25 30

ggt?tct?gga?ggt?ggt?ggt?ggt?tgc?gac?ctg?ccg?caa?acc?cac?agc?ctg 144

Gly?Ser?Gly?Gly?Gly?Gly?Gly?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu

35 40 45

ggt?aac?cgt?cgt?gcc?ctg?atc?ctg?ctg?gca?caa?atg?cgt?cgt?atc?tct 192

Gly?Asn?Arg?Arg?Ala?Leu?Ile?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser

50 55 60

cct?ttc?tcc?tgt?ctg?aaa?gac?cgt?cac?gac?ttc?ggt?ttc?ccg?cag?gag 240

Pro?Phe?Ser?Cys?Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu

65 70 75 80

gag?ttt?gat?ggc?aac?cag?ttc?cag?aag?gct?caa?gcc?atc?tct?gtc?ctc 288

Glu?Phe?Asp?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Gln?Ala?Ile?Ser?Val?Leu

85 90 95

cat?gag?atg?atc?cag?cag?acc?ttc?aac?ctc?ttc?agc?acc?aaa?gat?tcc 336

His?Glu?Met?Ile?Gln?Gln?Thr?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser

100 105 110

tct?gct?gct?tgg?gac?gag?tcc?ctg?ctg?gag?aaa?ttc?tac?acc?gaa?ctc 384

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

115 120 125

tac?cag?cag?ctg?aac?gac?ctg?gaa?gcc?tgt?gtg?atc?cag?gag?gtc?ggt 432

Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Glu?Val?Gly

130 135 140

gtg?gaa?gaa?acc?ccg?ctg?atg?aat?gtc?gac?tcc?atc?ttg?gct?gtg?aag 480

Val?Glu?Glu?Thr?Pro?Leu?Met?Asn?Val?Asp?Ser?Ile?Leu?Ala?Val?Lys

145 150 155 160

aaa?tac?ttc?cag?cgt?atc?act?ctc?tat?ctg?aca?gag?aag?aaa?tac?agc 528

Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser

165 170 175

cct?tgt?gcc?tgg?gag?gtt?gtc?cgt?gca?gaa?atc?atg?cgt?tcc?ttc?tct 576

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

180 185 190

ctg?tcc?acc?aac?ctg?cag?gaa?cgt?ctg?cgt?cgt?aag?gaa?taa?tag 621

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

195 200 205

Claims (10)

1, a kind of fusion rotein is characterized in that: it is formed by covalent bonds by polypeptide with TM-alphal function and the polypeptide with IFN-con function.

2, the described fusion rotein of claim 1, it is characterized in that: the polypeptide with TM-alphal function with the mode of connection with polypeptide of IFN-con function is: I-T, T-I, T-I-T, I-L-T, T-L-I, T-L-I-L-T, wherein, I represents IFN-con, T represents TM-alphal, and L represents connection peptides.

3. the described fusion rotein of claim 2, it is characterized in that: mode of connection is T-L-I, and wherein I represents IFN-con, and T represents TM-alphal, and L represents connection peptides.

4, the described fusion rotein of claim 3 is characterized in that: its aminoacid sequence is the aminoacid sequence of sequence table 2.

5, the DNA of the fusion rotein of coding claim 1-4.

6, the described DNA of claim 5 is characterized in that: its nucleotide sequence is the nucleotide sequence in the sequence table 1.

7, a kind of expression vector is characterized in that: it contains the DNA of claim 5.

8, a kind of host cell is characterized in that: it contains the carrier of claim 7.

9, the preparation method of the fusion rotein of claim 1 is characterized in that, comprise the steps: engineering bacteria structure, preparation seed liquor, shake-flask culture, slightly carry, essence is carried, work in-process preparation, packing, freeze-drying.

10, contain the pharmaceutical composition of the described fusion rotein of claim 1-4, it is characterized in that: the fusion rotein and the pharmaceutically acceptable carrier that comprise the claim 1 of significant quantity on the physiology.

Images