CN107298718A

CN107298718A - UTI fusion protein and preparation method and application

Info

Publication number: CN107298718A
Application number: CN201710661652.3A
Authority: CN
Inventors: 崔俊生; 柏浩; 张彦鹏; 吴首标
Original assignee: Beijing Hundred Chinese Bacera Biotechnology Co Ltd
Current assignee: Beijing Hundred Chinese Bacera Biotechnology Co Ltd
Priority date: 2017-08-04
Filing date: 2017-08-04
Publication date: 2017-10-27
Anticipated expiration: 2037-08-04
Also published as: CN107298718B

Abstract

The present invention relates to biomedicine field, in particular to a kind of UTI fusion protein and preparation method and application.The structure of the UTI fusion protein is shown in UTI rHSA；Wherein, UTI is UTI, or part thereof fragment, mutant and deletant；RHSA is human serum albumin, or part thereof fragment, mutant and deletant.The fusion protein biological value is good, and with longer half-life period, and protein sequence derives from human body in itself, non-immunogenicity is safe.

Description

UTI fusion protein and preparation method and application

Technical field

The present invention relates to biomedicine field, in particular to a kind of UTI fusion protein and preparation method thereof With application.

Background technology

UTI is the single chain glycoprotein containing two kunitz type domains, is important urinary trypsin inhibitor (urinary trypsin inhibitor, UTI).In human body cell, UTI is by a-microglobulin precursor (AMBP) gene code, contains 10 extrons and 9 intrones.Ripe gene encoding production contains 147 amino acid.Crow Si Tading to the multiple protein enzyme including trypsase, chymotrypsin, Leukocyte elastase, fibrinolysin etc. have compared with Strong inhibitory action.In addition UTI also works in terms of anti-inflammatory, Hemorrhagic shock, suppression Nasopharyngeal neoplasms.

UTI is initially to extract to obtain from human urine.The UTI originated from human urine contains covalently bound sulphur Aching and limp ossein molecule, and high glycosylation.Initially it is believed that covalently bound chondroitin sulfate and glycosylation are to crow department His bioactivity of fourth is most important, but Bayer Altiengsellschaft companies of Germany prove glycosylation not UTI work Property institute it is required.Research then shows that the restructuring UTI expressed using Escherichia coli (E.coli) is similarly had to pancreas The inhibitory action of protease.Due to that can avoid extracting albumen presence from urine using technique for gene engineering expression recombinant protein Source limitation, sample pollution, rear extraction process complexity etc., turn into the optimal path for obtaining UTI at present.However, mesh The restructuring UTI yield that preceding utilization genetic engineering means are obtained from the microorganisms such as E.coli is still low, it is difficult to meet business Change and require.

In addition, according to the literature, 300,000 units of healthy normal male/10ml veins are given from the UTI of human urine extraction After drug administration by injection, blood concentration straight line declines in 3 hours, and it is 40 minutes to remove half-life period；The 24% of 6 hours dosages after administration Drained from urine.Dosage can be reduced, administration number of times be reduced, with good clinical practice by developing long-acting UTI Benefit.

In view of this, it is special to propose the present invention.

The content of the invention

Present invention aims at a kind of UTI fusion protein of offer, and there is provided its preparation and application.

In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：

The present invention relates to a kind of UTI fusion protein, its structure is shown in UTI-rHSA；

Wherein, UTI is UTI, or part thereof fragment, mutant and deletant；

RHSA is human serum albumin, or part thereof fragment, mutant and deletant.

1909, Beuer and Berich were reported in urine and be there is protease inhibitors (urinary trypsin first Inhibitor, UTI), now also to be referred to as UTI (ulinastatin), it is controlled in treatment acute pancreatitis, auxiliary more Shock is treated, improves operation prognosis and especially reduces in terms of complication of extracorporeal circulation and play the role of important, itself source is added In human body, non-immunogenicity is safe.

UTI fusion protein provided by the present invention, biological value is good, and has longer half-life period, and albumen sequence Row derive from human body in itself, and non-immunogenicity is safe.

Wherein, the selection of " Partial Fragment, mutant and deletant " is the polypeptide fragment still with UTI or rHSA activity, Its selection mode is well known to those skilled in the art.

It is preferred that, UTI fusion protein as described above, the amino acid sequence such as SEQ ID NO of the UTI：1 institute Show.

It is preferred that, UTI fusion protein as described above, the amino acid sequence such as SEQ ID NO of the rHSA：2 institutes Show.

It is preferred that, UTI fusion protein as described above, the UTI and rHSA also includes Linker；

It is preferred that, the amino acid number of the Linker is 1~30；Amino acid quantity can be 1,2,3,4,5,6,7, 8th, 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30；

It is preferred that, it can be 1 that the sequence of the Linker, which is selected from (GGGGS) n, (GGGS) n, (GGS) n or Gn, wherein n, 2,3,4,5 or 6；

It is preferred that, the amino acid sequence such as SEQ ID NO of the Linker：Shown in 3.

According to an aspect of the present invention, the invention further relates to encode claim UTI fusion protein as described above Nucleotide sequence.

It is preferred that, encode the nucleotide sequence such as SEQ ID NO of the UTI：Shown in 4；

It is preferred that, encode the nucleotide sequence such as SEQ ID NO of the rHSA：Shown in 5；

It is preferred that, encode the nucleotide sequence such as SEQ ID NO of the Linker：Shown in 6.

According to an aspect of the present invention, the invention further relates to a kind of expression cassette, it contains nucleotide sequence as described above；

It is preferred that, the expression cassette also includes expressing protein disulphide isomerase PDI nucleotide sequence；

The formation of natural disulphide bonds is the rate-limiting step during many albumen are correctly folded, in stable protein conformation and holding Played an important role in terms of protein active.Disulphide isomerase (Protein disulfide isomerase, PDI) is interior A kind of important protein folding catalyst in matter net, the formation of its catalytic protein disulfide bond and the rearrangement of mistake pairing disulfide bond, And have the chaperone activity for suppressing misfolded protein aggregation.

It is preferred that, the nucleotide sequence of the PDI is located at the downstream of the rHSA；

It is preferred that, the nucleotide sequence such as SEQ ID NO of the PDI：Shown in 7.

It is preferred that, expression cassette as described above includes promoter element between the rHSA and the PDI；

It is preferred that, the nucleotide sequence such as SEQ ID NO of the promoter element：Shown in 8.

According to an aspect of the present invention, the invention further relates to a kind of recombinant vector, it contains nucleotides sequence as described above Row, or expression cassette as described above.

The present invention further includes the core construct of at least one coding nucleic acid molecules as described above, and preferably expression is carried Body, such as plasmid, can introduce the construction method of the carrier in one embodiment of the application.

According to an aspect of the present invention, the invention further relates to a kind of host cell, its by nucleotide sequence as described above, Or expression cassette as described above, or recombinant vector is converted as described above；

The host cell is eukaryotic, such as yeast, preferably Pichia pastoris.

According to an aspect of the present invention, the invention further relates to a kind of method for preparing UTI fusion protein, including：

In the medium with cultivate host cell as described above under suitable condition of culture；

Reclaim from culture medium or from the host cell cultivated the UTI fusion protein so produced.

UTI fusion protein as described above prepare for treat and/or pre- preventing tumor, autoimmune disease, Application in nerve degenerative diseases, allergy and the medicine of infection；

It is preferred that, the tumour includes：It is leukaemia, lymthoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small Cell lung cancer, nasopharyngeal carcinoma, cancer of the esophagus, stomach cancer, cancer of pancreas, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, oophoroma, cervical carcinoma, Carcinoma of endometrium, sarcoma of uterus, prostate cancer, carcinoma of urinary bladder, clear-cell carcinoma, melanoma；

It is preferred that, the autoimmune disease includes：Arthritis, rheumatic arthritis, psoriasis, multiple sclerosis Disease, ulcerative colitis, Crohn disease, systemic loupus erythematosus, glomerulonephritis, dilated cardiomyopathy sample disease, Si Yege Human relations Cotard, allergic contact dermatitis, polymyositis, chorionitis, artery week property panarteritis, rheumatic fever, leucoderma, pancreas islet Plain dependent diabetes mellitus, Behcet's syndrome and chronic thyroiditis；

It is preferred that, the nerve degenerative diseases include：Parkinson's disease, Huntington's disease, Machado-Joseph disease, Amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease.

The present invention utilizes the pichia yeast expression system successful expression fusion protein of UTI and human serum albumins, The plasmid for expressing restructuring UTI is constructed, two sulphur related with secretion to protein folding are introduced in expression plasmid Key isomerase gene (PDI), and promoter element P_GAP, and PDI genes are connected correctly to P_GAPDownstream, realizes restructuring UTI Expressed with a large amount of the fusion protein of human serum albumin, there is the suppression essentially identical with restructuring UTI after fusion protein is purified The activity of trypsase, compare with longer half-life period with restructuring UTI on mouse.The white egg of UTI human bloods that the present invention is developed White fusion protein has important application value.

Brief description of the drawings

, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid Put, other accompanying drawings can also be obtained according to these accompanying drawings.

Fig. 1 is plasmid pPICZa::UTI-rHSA builds schematic diagram；

Fig. 2 is plasmid pICZa::UTI builds schematic diagram；

Fig. 3 is plasmid pEasy-T3-P_GAP- PDI builds schematic diagram；

Fig. 4 is plasmid pPICZa::UTI-rHSA-PDI builds schematic diagram；

Fig. 5 is fermentation supernatant electrophoretogram；A：PpBUH-UH supernatants；B:PpBUH-ULH supernatants；C:PpBUH-UHP supernatants；D: PpBUH-ULHP supernatants；

Fig. 6 is fermentation supernatant and protein purification liquid SDS-PAGE；A：PpBUH-UHP supernatants；B:ppBUH-ULHP Supernatant；C：UTI-HSA refined solutions；D:UTI-L-HSA refined solutions；E：UTI refined solutions.

Embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be The conventional products that can be obtained by commercially available purchase.

Embodiment

1. yeast expression vector pPICZa::UTI-rHSA and pPICZa::UTI-L-rHSA structure

According to the codon preference of Pichia pastoris, the gene order SEQ ID NO of UTI are designed：4 and human blood it is white The gene order SEQ ID NO of albumen：5 and Linker (bonding pad) gene order SEQ ID NO：6, and submit Hua Da company Full genome is synthesized, the UTI-L- of the UTI-rHSA sequences of the fusion protein without Linker and the fusion protein containing Linker RHSA sequences.XhoI and KpnI restriction enzyme site are introduced respectively at the two ends of antigen-4 fusion protein gene, by being inserted into after double digestion In the Yeast expression carrier pICZa of same double digestion, after being connected through T4 ligases, transformed competence colibacillus Escherichia coli TOP10 is positive Clone through sequence verification it is correct after extract plasmid be respectively designated as：pICZa::UTI-rHSA,pICZa::UTI-L-rHSA.

Plasmid pICZa::UTI-rHSA collection of illustrative plates is as shown in Figure 1.

2.UTI expression plasmids are built

With pUTIF:gttactcgaggtccatgtATGGGTATGACTTCTAGATA C；And pUTIR: GattGGTACCTCAGAGACCGAGAGCAGC is upstream and downstream primer (restriction enzyme site for introducing XhoI and KpnI respectively), to close It is that template enters performing PCR amplification UTI genes into gene UTI-rHSA, PCR primer is double with XhoI and KpnI after being reclaimed through glue kit Digestion is reclaimed, and is attached with the Yeast expression carrier pICZa of same double digestion, after being connected through T4 ligases, transformed competence colibacillus Escherichia coli TOP10, positive colony through sequence verification it is correct after extract plasmid be respectively designated as：pICZa::UTI, as UTI tables Up to plasmid, its plasmid map is as shown in Figure 2.

3. clone and the pPICZa of disulfide isomerase gene (PDI)::UTI-rHSA-PDI and pPICZa::UTI-L- RHSA-PDI structure

The disulfide isomerase gene (PDI) related with secretion to protein folding is cloned from Pichia pastoris GS115, together Shi Kelong high efficient expression starter element P_GAP, and PDI genes are connected correctly to P_GAPDownstream, builds PDI Expression elements.Carry Take the genome of Pichia pastoris GS115, by template of genome with PpGAPF (agatctATGTCTTGGTGTCCTCGTC) and PpGAPR (aagcttAATACCGACAGTGATAGCC) be respectively upstream and downstream primer (two ends separately designed BglII and HindIII restriction enzyme site) PCR amplification acquisition promoter elements P_GAP(sequence such as SEQ ID NO：Shown in 8), by this PCR primer With being inserted into after BglII and HindIII double digestions in the plasmid pEasy-T3 of same double digestion, after transformed competence colibacillus Escherichia coli Select the correct rear plasmid that extracts of monoclonal sequencing standby, as plasmid pEasy-T3-P_GAP.With the genome of Pichia pastoris GS115 Template is with PpPDIF (aagcttAAAGAACTGCCCGATGAAC) and PpPDIR (gcggccgcAGGCCAACCACAAGATGAA) Respectively upstream and downstream primer (two ends have separately designed HindIII and NotI restriction enzyme site) PCR amplifications obtain PDI gene (sequences Row such as SEQ ID NO：Shown in 7), by this PCR primer with the plasmid that same double digestion is inserted into after HindIII and NotI double digestions pEasy-T3-P_GAPIn, monoclonal sequencing is selected after transformed competence colibacillus Escherichia coli, and correctly the rear plasmid that extracts is standby, as plasmid pEasy-T3-P_GAP- PDI, plasmid map is as shown in Figure 3.

It will contain P with NotI_GAP- PDI DNA fragmentation is from pEasy-T3-P_GAPScaled off in-PDI plasmids, and before being inserted into The pPICZa that phase work is built::UTI-rHSA and pPICZa::The NotI restriction enzyme sites of UTI-L-rHSA plasmids, obtain plasmid pPICZa::UTI-rHSA-PDI and pPICZa::UTI-L-rHSA-PDI.Plasmid pPICZa::UTI-rHSA-PDI plasmid figure Spectrum is as shown in Figure 4.

4. the screening of the structure of expressed fusion protein recombinant bacterial strain and the recombinant bacterial strain of high efficient expression fusion protein

By the expression plasmid pPICZa of structure::UTI-rHSA, pPICZa::UTI-L-rHSA, pPICZa::UTI-rHSA- PDI and pPICZa::UTI-L-rHSA-PDI and pPICZa::UTI obtains the expression matter of linearisation with Bgl II digestions respectively Grain, is imported in Pichia pastoris GS115, and be coated on the YPD flat boards of the Zeocin containing 100 μ g/mL by electroporated.28 DEG C culture 3 days, the random multiple monoclonals of picking are transferred on the fresh Zeocin containing 100 μ g/mL YPD flat boards, and point Genomic DNA is indescribably taken to enter performing PCR checking.With pUHF (ATGGGTATGACTTCTAGATACTTC) and pUHR (TCAGAGACCGAGAGCAGCCTG) it is respectively upstream and downstream primer, expression plasmid pPICZa::UTI-rHSA, pPICZa::UTI- L-rHSA, pPICZa::UTI-rHSA-PDI and pPICZa::UTI-L-rHSA-PDI conversion bacterial strain should be able to be amplified about 2100bp DNA fragmentation.Using pUTIF and pUTIR as upstream and downstream primer, pPICZa::UTI conversion bacterial strain should be able to be amplified About 530bp DNA fragmentation.Each recombinant bacterial strain 36 monoclonals of random picking, are inoculated in the big examination containing 3mL BMGY respectively Guan Zhong, in 28 DEG C, 220rpm is cultivated 18 hours.Taking culture, 3000g is centrifuged 1 minute at room temperature, collects thalline, and use 2mL BMMY suspension thallines again, in 28 DEG C, 220rpm continues to cultivate.100% methanol was added into culture medium every 24 hours to end Concentration 1%.After culture 3 days, fermented liquid supernatant is collected by centrifugation.By the expression of PAGE glue detection fusion albumen, finally obtain Obtained the of a relatively high transformant of expression quantity, expression plasmid pPICZa::UTI-rHSA, pPICZa::UTI-L-rHSA, pPICZa::UTI-rHSA-PDI, pPICZa::UTI-L-rHSA-PDI and pPICZa::UTI conversion Pichia pastoris GS115s are obtained The high recombinant bacterial strain of expression quantity be respectively designated as ppBUH-UH, ppBUH-ULH, ppBUH-UHP and ppBUH-ULHP, ppUTI.

Have PDI be co-expressed bacterial strain ppBUH-UHP, ppBUH-ULHP less PDI coexpression bacterial strain ppBUH-UH, The expression quantity of ppBUH-ULH UTI fusion proteins is higher, as shown in Figure 5.

The shake flask fermentation and fusion protein purification of 5.UTI and UTI fusion protein recombinant bacterial strains

By expression the bacterial strain ppBUH-UHP and ppBUH-ULHP of fusion protein, the Zeocin containing 100 μ g/mL is applied respectively YPD flat boards on, be inoculated into from flat board picking monoclonal in 5ml YPD test tubes, 30 DEG C, 250rpm overnight incubations, after 18h OD600 should be inoculated in 100ml BMGY shaking flasks 15 or so, then by by 1 ‰ inoculum concentrations, 30 DEG C, 250rpm cultivate 24 hours, 3000g collected after centrifugation thalline, thalline is suspended from 110ml BMMY, the methanol of addition final concentration of 0.5%, 30 DEG C, 250rpm continues to cultivate, and adds a methanol per 24h and measures OD600, and culture terminates culture after 110 hours.5000g collects bacterium Liquid supernatant.Fermentation supernatant about 100ml is respectively taken, with 10Mm Tris pH:8.5 buffer solutions are diluted to 250ml, will be dilute with solid Tris Fermentation supernatant pH value after releasing is adjusted to 8.5, upper anion-exchange chromatography QFF.With the 10Mm Tris of the NaCl containing 0.1M PH8.5 wash buffers, continuous gradient elution is made with 0.1M containing NaCl to 1.0M 10Mm Tris pH8.5 buffer solutions, is collected To 2 protein peaks, detected with SDS-PAGE, without mesh in the flushing peak for flowing through peak and 0.1M NaCl during sample loading Albumen is marked, and target protein merges eluting peak, buffer solution is changed by the ultra-filtration centrifuge tube for retaining 10k substantially in 2 eluting peaks For in 20mM pH7.2 PB buffer solutions.Because containing human albumin in fusion protein, the Blue Sepharose of GE companies can be used HP affinity chromatographies, will change Blue Sepharose HP chromatographic columns on the protein liquid after liquid, with the pH7.2's of the 0-1.5M containing NaCl PB buffer solutions are cooked continuous gradient elution, collect protein peak.Followed by hydrophobic chromatography, sample electric conductivity value is adjusted with 5M NaCl To 155ms/cm or so, the highly dense hydrophobic chromatography of the phenyl of GE companies on protein sample, buffer solution is Buffer A:10mM Tris pH:7.52.5M NaCl, Buffer B:10mM Tris pH:7.5.Washing condition is 100%Buffer A, and elution requirement is 0-100%Buffer B, collect albumen wash-out peak, and buffer solution is changed into 20mM pH7.2's with retention 10k ultra-filtration centrifuge tube It is standby in -20 DEG C of refrigerators with as refined solution after 0.22um bacterial filter filtering in PB buffer solutions.

Express bacterial strain ppUTI to apply on the YPD flat boards of the Zeocin containing 100 μ g/mL, be inoculated into from flat board picking monoclonal In 5ml YPD test tubes, 30 DEG C, 250rpm overnight incubations, then be inoculated in by by 1 ‰ inoculum concentrations in 100ml BMGY shaking flasks, 30 DEG C, 250rpm is cultivated 24 hours, and thalline is suspended from 110ml BMMY by 3000g collected after centrifugation thalline, adds final concentration of 0.5% Methanol, 30 DEG C, 250rpm continue to cultivate, add a methanol and measure OD600 per 24h, culture terminates training after 110 hours Support.5000g collects bacterium solution supernatant.Fermentation supernatant about 100ml is respectively taken, with 10Mm Tris pH:8.5 buffer solutions are diluted to 250ml, The fermentation supernatant pH value after dilution is adjusted to 8.5 with solid Tris, upper anion-exchange chromatography QFF.With the NaCl's containing 0.1M 10mM Tris pH8.5 wash buffers, continuous ladder is made with 0.1M containing NaCl to 1.0M 10Mm Tris pH8.5 buffer solutions Degree elution, collects protein peak, is detected with SDS-PAGE, merges target protein peak, will be buffered by the ultra-filtration centrifuge tube for retaining 10k Liquid is changed in 20mM pH7.2 PB buffer solutions.Sample electric conductivity value is adjusted on 160ms/cm or so, protein sample with 5M NaCl The highly dense hydrophobic chromatography of phenyl of GE companies, buffer solution is Buffer A:10mM Tris pH:7.5 2.5M NaCl, Buffer B:10mM Tris pH:7.5.Washing condition is 100%Buffer A, and elution requirement is 0-100%Buffer B, collects albumen Buffer solution, is changed in 20mM pH7.2 PB buffer solutions by eluting peak with retention 10k ultra-filtration centrifuge tube, degerming with 0.22um It is that refined solution is standby in -20 DEG C of refrigerators after filter filtering.PpBUH-UHP and ppBUH-ULHP fermentation supernatants and purifying UTI albumen, fusion protein UTI-rHSA, UTI-L-rHSA electrophoresis result as shown in fig. 6, UTI, UTI-rHSA after purification, UTI-L-rHSA purity is more than 95%.

6. the detection of bioactivity

According to《Chinese Pharmacopoeia》Two P of version in 2015_83-84Middle UTI solution titration method is carried out, with human urine The UTI of extraction is compared, as a result as shown in the table, and the potency of the UTI extracted in human urine is 3600U/mg, restructuring Expression UTI activity be 3700U/mg, and fusion protein UTI-rHSA and UTI-L-rHSA ratio live by molar concentration meter calculate with UTI is suitable, between the U/nmol of 4.4-5.0 ten thousand.

The bioactivity result of table 1

7. mouse abnormal toxicity test and serum level detection

It is experimental group to take UTI and UTI-rHSA after purification, respectively in intraperitoneal administration mode, is injected by 5mg/kg dosage Babl/c mouse (body weight 18-22g), using physiological saline as negative control group, every group of mouse 5 observes the abnormal conditions of each group, Observation 7 days, detects changes of weight.As a result as shown in the table, the increase of each group mouse weight is without exception.

The mouse undue toxicity result of table 2

It is experimental group to take UTI and UTI-rHSA after purification, to be injected intravenously administering mode, is noted by 1.0mg/kg/ dosage SD rats (body weight 230-250g) are penetrated, using physiological saline as negative control group, every group of rat 3 takes blood, when taking blood from tail vein Between point be 0,5min, 20min, 60min, 120min, (being resisted with UTI in ELISA method detection blood coating rabbit-anti UTI overnight, closing more Afterwards plus more than 100 times of blood serum sample of dilution, plus anti-UTI mouse source monoclonal antibody and sheep anti-Mouse enzyme labelled antibody, TMB develops the color, 2M sulfuric acid Terminating reaction, 450nm colorimetrics) the OD values of ELISA detections are determined as the positive more than 0.15, every part of serum dilutes from 100 times, after Doubling dilution, maximum dilution multiple when calculating every part of Virus monitory result for the positive is calculated as titre, the titre of every group of 3 rats As a result geometrical mean is taken, negative findings is calculated as N, and the result of each group is as shown in the table, and the titre of UTI groups was to the maximum at 5 minutes 102400, later rapid decrease, half-life period is less than 20 minutes, and at 120 minutes, titre was only 4031；Fusion protein UTI-rHSA The titre of group is also in 5 minutes maximums；Because fusion protein and the UTI molecular weight identical in quality but fusion protein being administered are 6.6 times of UTI molecular weight, so the serum titer of detection also differs 4 times or so, but the serum half-life of fusion protein compares UTI Group should be obviously prolonged, and fusion protein group half-life period should be more than 20 minutes, and at 120 minutes, titre still had 5080.Fusion protein Serum half-life compared with UTI is obviously prolonged.

The UTI of table 3 and UTI-rHSA the content ELISA testing results in rat blood serum

Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent The present invention is described in detail with reference to foregoing embodiments for pipe, but it will be understood by those within the art that：Its The technical scheme described in foregoing embodiments can still be modified, or to which part or all technical characteristic Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill The scope of art scheme.

SEQUENCE LISTING

<110>Remittance bio tech ltd of the China of Beijing hundred hundred

<120>UTI fusion protein and its preparation and application

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tgtagaactg ttgctgcttg taacttgcca atcgttagag gtccatgtag agctttcatc 180

caattgtggg ctttcgacgc tgttaagggt aagtgtgttt tgttcccata cggtggttgt 240

caaggtaacg gtaacaagtt ctactctgaa aaggaatgta gagaatactg tggtgttcca 300

ggtgacggtg acgaagaatt gttg 324

<210> 5

<211> 1758

<212> DNA

<213>Artificial sequence

<400> 5

gacgctcaca agtctgaagt tgctcacaga ttcaaggact tgggtgaaga aaacttcaag 60

gctttggttt tgatcgcttt cgctcaatac ttgcaacaat gtccattcga agaccacgtt 120

aagttggtta acgaagttac tgaattcgct aagacttgtg ttgctgacga atctgctgaa 180

aactgtgaca agtctttgca cactttgttc ggtgacaagt tgtgtactgt tgctactttg 240

agagaaactt acggtgaaat ggctgactgt tgtgctaagc aagaaccaga aagaaacgaa 300

tgtttcttgc aacacaagga cgacaaccca aacttgccaa gattggttag accagaagtt 360

gacgttatgt gtactgcttt ccacgacaac gaagaaactt tcttgaagaa gtacttgtac 420

gaaatcgcta gaagacaccc atacttctac gctccagaat tgttgttctt cgctaagaga 480

tacaaggctg ctttcactga atgttgtcaa gctgctgaca aggctgcgtg cctgttgcca 540

aagttggacg aattgagaga cgaaggtaag gcttcttctg ctaagcaaag attgaagtgc 600

gcgagcttgc aaaagttcgg cgaaagagct ttcaaggctt gggctgttgc tagattgtct 660

caaagattcc caaaggctga attcgctgaa gtttctaagt tggttactga cttgactaag 720

gttcacactg aatgttgtca cggtgacttg ttggaatgtg ctgacgacag agctgacttg 780

gctaagtaca tctgtgaaaa ccaagactct atctcttcta agttgaagga atgttgtgaa 840

aagccattgt tggaaaagtc tcactgtatc gctgaagttg aaaacgacga aatgccagct 900

gacctcccaa gcttggctgc tgactttgtt gaatctaagg acgtttgtaa gaactacgct 960

gaagctaagg acgttttctt gggtatgttc ttgtacgaat acgctagaag acacccagac 1020

tactcggttg tactattgtt gagattggct aagacttacg aaactacttt ggaaaagtgt 1080

tgtgctgctg ctgacccaca cgaatgttac gctaaggttt tcgacgaatt caagccattg 1140

gttgaagaac cacaaaactt gatcaagcaa aactgtgaat tgttcgaaca attgggtgaa 1200

tacaagttcc aaaacgcttt gttggttaga tacactaaga aggttccaca agtttctact 1260

cccactttgg ttgaagtaag cagaaacttg ggtaaggttg gttctaagtg ttgtaagcac 1320

ccagaagcta agagaatgcc atgtgctgaa gactacttgt ctgttgtttt gaaccaattg 1380

tgtgttttgc acgaaaagac tccagtttct gacagagtta ctaagtgttg tactgaatct 1440

ttggttaaca gaagaccatg tttctctgct ttggaagttg acgaaactta cgttccaaag 1500

gaattcaacg ctgaaacttt cactttccac gctgacatct gtactttgtc tgaaaaggaa 1560

agacaaatca agaagcaaac tgctttggtt gaattggtta agcacaagcc aaaggctact 1620

aaggaacaat tgaaggctgt tatggacgac ttcgctgctt tcgttgaaaa gtgttgtaag 1680

gctgacgaca aggagacatg cttcgcagaa gaaggtaaga agttggttgc tgctagtcag 1740

gctgctctcg gtctctga 1758

<210> 6

<211> 45

<212> DNA

<213>Artificial sequence

<400> 6

ggtggtggtg gtagcggtgg tggtggtagc ggtggtggtg gtagc 45

<210> 7

<211> 2237

<212> DNA

<213> Pichia pastoris

<400> 7

aagcttaaag aactgcccga tgaacgaact ttacgtaaga aggagtggct gaagagaaga 60

ccccgaacac tccctgttga atagaacacg aacactgtaa atagaataaa agaaaacttg 120

gatagtagaa cttcaatgta gtgtttctat tgtcttacgc ggctctttag attgcaatcc 180

ccagaatgga atcgtccatc tttctcaacc cactcaaaga taatctacca gacataccta 240

cgccctccat cccagcacca cgtcgcgatc acccctaaaa cttcaataat tgaacacgta 300

ctgatttcca aaccttcttc ttcttcctat ctataagaat gcaattcaac tggaatatta 360

aaactgtggc aagtattttg tccgctctca cactagcaca agcaagtgat caggaggcta 420

ttgctccaga ggactctcat gtcgtcaaat tgactgaagc cacttttgag tctttcatca 480

ccagtaatcc tcacgttttg gcagagtttt ttgccccttg gtgtggtcac tgtaagaagt 540

tgggccctga acttgtttct gctgccgaga tcttaaagga caatgagcag gttaagattg 600

ctcaaattga ttgtacggag gagaaggaat tatgtcaagg ctacgaaatt aaagggtatc 660

ctactttgaa ggtgttccat ggtgaggttg gggtcccaag tgactatcaa ggtcaaagac 720

agagccaaag cattgtcagc tatatgctaa agcagagttt accccctgtc agtgaaatca 780

atgcaaccaa agatttagac gacacaatcg ccgaggcaaa agagcccgtg attgtgcaag 840

tactaccgga agatgcatcc aacttggaat ctaacaccac attttacgga gttgccggta 900

ctctcagaga gaaattcact tttgtctcca ctaagtctac tgattatgcc aaaaaataca 960

ctaacgactc gactcctgcc tatttgcttg tcagacctgg cgaggaacct agtgtttact 1020

ctggtgagga gttagatgag actcatttgg tgcactggat tgatattgag tccaaacctc 1080

tatttggaga cattgacgga tccaccttca aatcatatgc tgaagctaac atccctttag 1140

cctactattt ctatgagaac gaagaacaac gtgctgctgc tgccgatatt attaaacctc 1200

ttgctaaaga gcaacgtggc aaaattaact ttgttggctt agaagccgtt aaattcggta 1260

agcatgccaa gaacttaaac atggatgaag agaaactccc tctatttgtc attcatgatt 1320

tggtgagcaa caagaagttt ggagttcctc aagaccaaga attgacgaac aaagatgtga 1380

ccgagctgat tgagaaattc atcgcaggag aggcagaacc aattgtgaaa tcagagccaa 1440

ttccagaaat tcaagaagag aaagtcttca agctagtcgg aaaggcccac gatgaagttg 1500

tcttcgatga atctaaagat gttctagtca agtactacgc cccttggtgt ggtcactgta 1560

agagaatggc tcctgcttat gaggaattgg ctactcttta cgccaatgat gaggatgcct 1620

cttcaaaggt tgtgattgca aaacttgatc acactttgaa cgatgtcgac aacgttgata 1680

ttcaaggtta tcctactttg atcctttatc cagctggtga taaatccaat cctcaactgt 1740

atgatggatc tcgtgaccta gaatcattgg ctgagtttgt aaaggagaga ggaacccaca 1800

aagtggatgc cctagcactc agaccagtcg aggaagaaaa ggaagctgaa gaagaagctg 1860

aaagtgaggc agacgctcac gacgagcttt aagaatgtcc ggggtttcta tatttactgt 1920

aactaggttt tattcttttg tatagtccaa caaaccatat ggattagcat cacttcgcgc 1980

aatacgttct agttgcccac ttgctaaaac caatttagca atgattcaac agcaattatt 2040

atcaccatga ctaactggaa agcgatattg actcccgctc aataccaagt cctccgtttg 2100

ggcggaacag aaagaccgta taccggacag tatgtgaact tcaagaaaaa tggaacctac 2160

ttgtgtagtg ggtgtcaaac tccgctttac aaaagtggca caaaatttga ttcatcttgt 2220

ggttggcctg cggccgc 2237

<210> 8

<211> 508

<212> DNA

<213> Pichia pastoris

<400> 8

agatctatgt cttggtgtcc tcgtccaatc aggtagccat ctctgaaata tctggctccg 60

ttgcaactcc gaacgacctg ctggcaacgt aaaattctcc ggggtaaaac ttaaatgtgg 120

agtaatggaa ccagaaacgt ctcttccctt ctctctcctt ccaccgcccg ttaccgtccc 180

taggaaattt tactctgctg gagagcttct tctacggccc ccttgcagca atgctcttcc 240

cagcattacg ttgcgggtaa aacggaggtc gtgtacccga cctagcagcc cagggatgga 300

aaagtcccgg ccgtcgctgg caataatagc gggcggacgc atgtcatgag attattggaa 360

accaccagaa tcgaatataa aaggcgaaca cctttcccaa ttttggtttc tcctgaccca 420

aagactttaa atttaattta tttgtcccta tttcaatcaa ttgaacaact atcaaaacac 480

aatggctatc actgtcggta ttaagctt 508

Claims

1. a kind of UTI fusion protein, its structure is shown in UTI-rHSA；

Wherein, UTI is UTI, or part thereof fragment, mutant and deletant；

RHSA is human serum albumin, or part thereof fragment, mutant and deletant.

2. UTI fusion protein according to claim 1, it is characterised in that the amino acid sequence of the UTI such as SEQ ID NO：Shown in 1；

It is preferred that, the amino acid sequence such as SEQ ID NO of the rHSA：Shown in 2.

3. UTI fusion protein according to claim 1 or 2, it is characterised in that the UTI is also wrapped with the rHSA Include Linker；

It is preferred that, the amino acid number of the Linker is 1~30；

It is preferred that, it can be 1,2,3 that the sequence of the Linker, which is selected from (GGGGS) n, (GGGS) n, (GGS) n or Gn, wherein n, 4,5 or 6；

4. encode the nucleotide sequence of the UTI fusion protein described in any one of claims 1 to 3.

5. a kind of expression cassette, it contains the nucleotide sequence described in claim 4；

6. expression cassette according to claim 5, it is characterised in that include promoter between the rHSA and the PDI Element；

7. recombinant vector, it contains nucleotide sequence according to claim 4, or the expression described in claim 6 or 7 Box.

8. host cell, it is by the nucleotide sequence described in claim 4, or the expression cassette described in claim 6 or 7, or power Profit requires that the recombinant vector described in 7 is converted；

It is preferred that, the host cell is yeast.

9. a kind of method for preparing UTI fusion protein, it is characterised in that including：

In the medium with the host cell described in culture claim 8 under suitable condition of culture；

10. the UTI fusion protein described in claim 1 or 2 is being prepared for treatment and/or pre- preventing tumor, autoimmunity Application in property disease, nerve degenerative diseases, allergy and the medicine of infection；

It is preferred that, the tumour includes：Leukaemia, lymthoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell Lung cancer, nasopharyngeal carcinoma, cancer of the esophagus, stomach cancer, cancer of pancreas, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, oophoroma, cervical carcinoma, uterus Endometrial carcinomas, sarcoma of uterus, prostate cancer, carcinoma of urinary bladder, clear-cell carcinoma, melanoma；

It is preferred that, the autoimmune disease includes：Arthritis, rheumatic arthritis, psoriasis, multiple sclerosis, burst Ulcer colitis, Crohn disease, systemic loupus erythematosus, glomerulonephritis, dilated cardiomyopathy sample disease, Siogren are comprehensive Simulator sickness, allergic contact dermatitis, polymyositis, chorionitis, artery week property panarteritis, rheumatic fever, leucoderma, insulin are relied on Property diabetes, Behcet's syndrome and chronic thyroiditis；

It is preferred that, the nerve degenerative diseases include：Parkinson's disease, Huntington's disease, Machado-Joseph disease, flesh wither Contracting lateral schlerosis, Creutzfeldt-Jakob disease.