CN102492043B - Anti-tumor and thrombolytic dual-effect chimeric protein with low immunogenicity, its preparation method and application - Google Patents

Abstract

The invention relates to an anti-tumor and thrombolytic dual-effect chimeric protein with low immunogenicity, its preparation method and application. The amino acid sequence of the chimeric protein is as shown in SEQ ID NO.11 or SEQ ID NO.12. The chimeric protein of the invention makes staphylokinase delta Sak with 10 amino acids deleted on the N end and staphylococcal enterotoxins delta SEC2 with 10 and 132 amino acids deleted respectively on the N and the C end jogged together, and keeps no connecting peptide between the two. The constructed chimeric proteins delta SEC2-delta Sak and delta Sak-delta SEC2 have substantially reduced molecular weights, and amino acid sequences with high immunogenicity are removed, so that the problems of large molecular weight and high immunogenicity of chimeric proteins are solved. Meanwhile, the anti-tumor and thrombolytic functions are retained.

Description

Antitumor thrombolytic double-effect chimeric protein of reduced immunogenicity and its preparation method and application

Technical field

The present invention relates to antitumor thrombolytic double-effect chimeric protein of reduced immunogenicity and its preparation method and application, belong to microbiology, molecular biology and genetically engineered field.

Background technology

2009, U.S. chairman ASCO mentioned when introducing tumour progression, and first cause of death of cancer is cancer itself and cancer return, and the second cause of the death is exactly thrombus.The relation of cancer and thrombus is very close, and prevention and the treatment of paying attention to the cancer thrombus have become one of emphasis of clinician's following work.

1865, Charles Robert Richet Armand Trousseau reported first patients with gastric cancer easily form phlebothrombosis, soon, Billroth has found to exist in the thrombus again cancer cells in 1878, further propose canceration and can cause Coagulation Dysfunction.Clinically the cancer-related thrombus is called the Trousseau syndromes.A large amount of pathology, experiment and clinical research confirmation this phenomenon, but also find, nearly all cancer patients exists part or whole hypercoagulative states.

Cancer patients's thrombotic diseases comprises deep venous thrombosis (deep venous thlmmbosis, DVT), pulmonary infarction (pulmonary embolism, PE), disseminated inravascular coagulation (disseminated intravascular coagulation, DIC), Portal Vein Thrombosis (portal vein thmrnbosis, PVT) and arterial thromboembolism (arterial thromboembolism, AT), the shallow table thrombophlebitis of migration (migratory thrombophlebitis superficial) etc., wherein lower limb DVT is the most common.

The pathogeny very complex that cancer patients's thrombus forms, not yet fully clear and definite so far.Many factors has caused cancer patients's thrombus easily to suffer from tendency by different approaches jointly, mainly comprises representation and the factors such as releaser and oncogene of anticancer therapy, tumour cell.

Anticancer therapy causes thrombosis, mainly is to form thrombus during the operative treatment, and chemotherapeutics and radiotherapy bring out thrombosis and patient during accepting the central venous puncture catheterization, manage all fibrin sheath and cause thrombosis.

The material of the thrombotic diseases that the cancer patients suffers from and tumor cells expression and release is in close relations.Tumour cell can activate the coagulation cascade reaction directly or indirectly by discharging the short solidifying factor or inflammatory factor, and on the other hand, inflammatory factor also can stimulate tumour cell to discharge the more short solidifying factor and the hypercoagulative state of aggravating blood.Three kinds of main coagulants of tumor cells expression and release are: tissue factor (TF), cancer coagulant (cancer procoagulant CP) and serine protease (hepsin).

Studies show that recently, some oncogene also can cause Coagulation Dysfunction when promoting malignant change of cell.Such as confirmations such as Yu J, cause some gene pathology that cancer produces such as Ras gene activation or p53 gene inactivation can cause the overexpression of tissue factor (TF), bring out the blood coagulation pathology.

Nearest research also shows, thrombosis has participated in the mechanism such as progress, vasculogenesis and transfer of tumour.Can be considered the extraneous factor except cancer therapy forms thrombus, all the other two kinds of formed thrombus of factor all answer the body canceration to occur.The research discovery, the mechanism that is beneficial to tumour generation and development that is formed with of thrombus may be that fibrin deposition provides 2 conveniences for tumor growth around tumour cell: provide matrix for tumour cell adheres on the one hand, be beneficial to its local diffusion and spread; On the other hand, scleroproein sludged blood and relevant thrombin can raise the expression (VEGF) of vascular endothelial growth factor, thereby have created condition for new vessel.

Staphylococcus aureus enterotoxin C 2 (staphylococcal enterotoxins, SEC2) be a bacterioid superantigen, they need not antigen presenting cell (antigen presenting cells, APCs) processing, be not subjected to the restriction of histocompatibility complex (MHC) yet, outside APC with φt cell receptor V β chain formation MHCII-SE-TCRV beta composite, when extremely low concentration, just can stimulate major part that the T cell proliferation of TCRV β sequence is arranged, make it to discharge cytokine profiles, produce extremely strong effect of immune response.SEC2 can produce the cytotoxicity (Sag-dependent cell-mediated T-cell-derived cytotoxicity, SDCC) that superantigen relies on to tumour cell, thus the antitumor reaction of the system of generation.But as potential stomach and intestine toxin, SEC2 can cause food poisoning, causes angina abdominis and diarrhoea, and as foreign protein, the SEC2 molecular weight is large, easily causes allergy.The Δ SEC2 molecular weight of 17 amino acid of N end and 132 aminoacid deletion of C end is little, and toxic side effect reduces, and its anti-tumor activity is kept.

Staphylokinase (staphylokinase, Sak) is that lysogenic streptococcus aureus produced in later stage exponential phase, a kind of extracellular protein of secretion by having.The thrombolytic effect mechanism of staphylokinase is to swash in vivo thrombus dissolving system, namely be transformed into plasmin (Pli) by plasminogen activation (Plg), and play thrombolytic effect, it is typical third generation thrombolytic drug, thrombolytic drug with other compares, it is energetic that staphylokinase has thrombolysis, thrombolytic effect is single-minded, low and the anaphylaxis of immunogenicity waits various features less, and after thrombus is dissolved, staphylokinase can be subject to the inhibition of α 2 antiplasmins in the human body to the activation of Profibrinolysin, avoided hemorrhage reaction.And the antithrombotic acitivity of 10 amino acid whose Δ SAK of N end disappearance is active similar to SAK's.

At present, the medicine for the treatment of tumour and thrombus all is the medicine of simple function, there is no specifics for the tumor complicated thrombus.Therefore, construct the NIF-Hirudin Hybrid of a kind of not only antitumor but also thrombolysis, significant for the case of the concurrent thrombus for the treatment of cancer.Although the not only chimeric protein of antitumor but also thrombolysis is also arranged, its molecular weight is up to 44kD, and has higher immunogenicity, therefore, reduces molecular weight, reduces immunogenicity and become current problem demanding prompt solution.

Summary of the invention

In order to address the above problem, the objective of the invention is to make up a kind of molecular weight low, the antitumor thrombolytic double-effect chimeric protein that immunogenicity is low.

To achieve these goals, the present invention takes following technical scheme: the antitumor thrombolytic double-effect chimeric protein of a kind of reduced immunogenicity, consisted of by the Δ SEC2 aminoacid sequence shown in the Δ Sak aminoacid sequence shown in the sequence table SEQ ID NO.3 and the sequence table SEQ ID NO.4, Δ SEC2 and Δ Sak linked together obtains the chimeric protein Δ Sak-Δ SEC2 of aminoacid sequence shown in SEQ ID NO.11, or the chimeric protein Δ SEC2 – Δ Sak of aminoacid sequence shown in SEQ ID NO.12.

Utilize escherichia expression system to produce the method for the antitumor thrombolytic double-effect chimeric protein Δ of above-mentioned reduced immunogenicity Sak-Δ SEC2 as follows:

1) carrier construction pET 28a- Δ sak-linker-Δ sec2, Δ sak-linker-Δ sec2Dna sequence dna shown in SEQ ID NO.5;

2) with the carrier pET 28a-that obtains Δ sak-linker-Δ sec2Remove through PCR Δ sakWith Δ sec2Between linker, construct the plasmid pET28a-that expresses chimeric protein Δ Sak-Δ SEC2 Δ sak-Δ sec2, Δ sak-Δ sec2Dna sequence dna shown in SEQ ID NO.7;

3) with plasmid pET28a- Δ sak-Δ sec2Change in the e. coli bl21 competent cell, obtain recombination bacillus coli;

4) recombination bacillus coli is inoculated in carries out shake-flask culture in the LB substratum, when bacterium liquid absorbance reaches 0.6, under 30 ℃ of conditions, induced 4～5 hours with 1 mM IPTG, afterwards centrifugal collection somatic cells;

5) use the ultrasonic disruption somatic cells, power: 200-300 W; Treatment time: 6 * 9 sec, 9 sec intervals; After the processing, centrifugal 10 min of 6000 rpm remove insoluble cell debris, collect supernatant liquor;

6) supernatant liquor is crossed the Ni-NTA affinity column, through gradient elution and concentrated desalination, obtained chimeric protein His-Δ Sak-Δ SEC2, its aminoacid sequence is shown in SEQ ID NO.9;

7) chimeric protein His-Δ Sak-Δ SEC2 crosses the Ni-NTA affinity column again through the enteropeptidase cutting, obtains chimeric protein Δ Sak-Δ SEC2, and its aminoacid sequence is shown in SEQ ID NO.11.

Utilize the method for the above-mentioned antitumor thrombolytic double-effect chimeric protein Δ of the reduced immunogenicity SEC2-Δ Sak of escherichia expression system production as follows:

1) carrier construction pET 28a- Δ sec2-linker-Δ sak, Δ sec2-linker-Δ sakDna sequence dna shown in SEQ ID NO.6;

2) with the carrier pET 28a-that obtains Δ sec2-linker-Δ sakRemove through PCR Δ sec2With Δ sakBetween linker, construct the plasmid pET28a-that expresses chimeric protein Δ SEC2-Δ Sak Δ sec2-Δ sak, Δ sec2-Δ sakDna sequence dna shown in SEQ ID NO.8;

3) with plasmid pET28a- Δ sec2-Δ sakChange in the e. coli bl21 competent cell, obtain recombination bacillus coli;

4) recombination bacillus coli is inoculated in carries out shake-flask culture in the LB substratum, when bacterium liquid absorbance reaches 0.6, under 30 ℃ of conditions, induced 4～5 hours with 1 mM IPTG, afterwards centrifugal collection somatic cells;

5) use the ultrasonic disruption somatic cells, power: 200-300 W; Treatment time: 6 * 9 sec, 9 sec intervals; After the processing, centrifugal 10 min of 6000 rpm remove insoluble cell debris, collect supernatant liquor;

6) supernatant liquor is crossed the Ni-NTA affinity column, through gradient elution and concentrated desalination, obtained chimeric protein His-Δ SEC2-Δ Sak, its aminoacid sequence is shown in SEQ ID NO.104;

7) chimeric protein His-Δ SEC2-Δ Sak crosses the Ni-NTA affinity column again through the enteropeptidase cutting, obtains chimeric protein Δ SEC2-Δ Sak, and its aminoacid sequence is shown in SEQ ID NO.12.

The application of chimeric protein of the present invention in the antitumor thrombolytic drug of preparation.

Take chimeric protein Δ Sak-Δ SEC2 of the present invention or chimeric protein Δ SEC2-Δ Sak as effective constituent, add pharmaceutically conventional auxiliary material, can prepare the various pharmaceutical preparations with antitumor thrombolytic double-effect.

The present invention, based on the antitumor activity of SEC2 and the thrombolysis activity of SAK, and for molecular weight and the immunogenicity that reduces bifunctional protein, with 10 amino acid whose staphylokinase Δ Sak of N end disappearance and N, the C end lacks respectively 10,132 amino acid whose enterotoxin Δ SEC2 are entrenched togather, and make between the two without any connection peptides, constructed chimeric protein Δ SEC2-Δ Sak and Δ Sak-Δ SEC2 molecular weight reduce greatly, removed the aminoacid sequence with high immunogenicity, solved the problem of the large and high immunogenicity of chimeric protein molecular weight, simultaneously antitumorly kept with function thrombolysis.

The invention has the beneficial effects as follows: the constructed antitumor thrombolytic double-effect albumen of the present invention had not only kept the thrombolysis activity of Sak but also had not subtracted the anti-tumor function of SEC2, and molecular weight is little, only has 28kD, be easy to intravenous administration, having practice significance for reducing toxic side effect, is the desirable anticancer double effect biological preparation of thrombolysis.The present invention adopts gene engineering method, by escherichia coli prokaryotic expression system and Ni affinitive layer purification method, successfully obtained the chimeric protein of high protein purity, and experimental results show that through external thrombolytic experiment and external tumor suppression, chimeric protein of the present invention has kept respectively the thrombus dissolving activity of Sak and the anti-tumor activity of SEC2, has successfully realized the structure of the anticancer double effect biological preparation of thrombolysis of reduced immunogenicity.

Embodiment

Embodiment 1The preparation of the antitumor thrombolytic double-effect chimeric protein Δ of reduced immunogenicity Sak-Δ SEC2

(1) with mosaic gene Δ sak-Δ sec2Expression plasmid make up

1) Δ sakPcr amplification, Δ sakGene order shown in SEQ ID NO.1, Δ sakBy EcoRI/ XhoI is connected on the pET28a, makes up plasmid pET28a- Δ sak, with plasmid pET28a- Δ sakBe template, by designing specific primer:

Upstream: 5 '-ATTAATCATATGGACGACGACGACAAAAAAGGCGATGACGCGAGT-3 '

Downstream: 5 '-CGGGCCGAATTCTTTCTTTTCTATAACAAC-3 '

Utilize the PCR method amplification Δ sak, and make Δ sakThe coded amino acid upstream has enteropeptidase and is identified as a little, Δ sakTwo ends are respectively with restriction enzyme site NdeI and EcoRI.

2) pET28a- Δ sak-linker-Δ sec2Structure: Δ sec2Gene order shown in SEQ ID NO.2, Δ sec2By EcoRI/ XhoI is connected on the pET 28a, makes up plasmid pET28a- Δ sec2, Δ sakWarp NdeI and EcoInsert pET28a-behind the RI double digestion Δ sec2In Δ sec2The upstream NdeI and EcoThe RI site, 16 ℃ of connections of T4 ligase enzyme are spent the night, and obtain ligation liquid.Ligation liquid is joined in advance in the e. coli bl21 competent cell by the Calcium Chloride Method preparation, transform (42 ℃ of water-bath heat shocks) through chemical process, afterwards, bacterium liquid is coated on the LB flat board that contains the Kan resistance, picking positive colony after 37 ℃ of overnight incubation extracts plasmid, warp after cultivating NdeI and EcoThe checking of RI double digestion is determined Δ sakWhether be inserted into pET 28a- Δ sec2In, whether make up pET 28a- Δ sak-linker-Δ sec2(because Δ sakWith Δ sec2Be by EcoThe RI restriction enzyme site connects, so Δ sakWith Δ sec2Between have a sequence be GAATTC(namely EcoThe RI restriction enzyme site) linker).

3) pET28a- Δ sak-Δ sec2Structure, with pET28a- Δ sak-linker-Δ sec2Remove through PCR Δ sakWith Δ sec2Between linker(GAATTC), the PCR product is carried out terminal smoothing and 5 ' phosphatizing treatment, efficient ligase enzyme carries out self and connects (cyclization), then chemical conversion is to the e. coli bl21 competent cell, bacterium liquid is coated on the LB flat board that contains the Kan resistance, picking positive colony after 37 ℃ of overnight incubation extracts the plasmid warp after cultivating EcoThe checking of RI single endonuclease digestion determines whether linker is removed.Give birth to worker company (Sangon, China) to the recombinant plasmid that the obtains affirmation of checking order by Shanghai again.

Test-results:

Cut checking and sequencing analysis comprehensively shows through enzyme, express the Prokaryotic expression vector construction of chimeric protein His-Δ Sak-Δ SEC2 successfully.Acquisition is with recombinant plasmid pET28a- Δ sak-Δ sec2Recombination bacillus coli.

(2) acquisition of the abduction delivering purifying of His-Δ Sak-Δ SEC2 and Δ Sak-Δ SEC2

1, the abduction delivering of His-Δ Sak-Δ SEC2

Will be with recombinant plasmid pET28a- Δ sak-Δ sec2Recombination bacillus coli be inoculated in and contain the Kan(kantlex, Kanamycine, Kan) in the 10 ml LB substratum of (40 μ g/ml), cultivated 12 ~ 16 hours, press 2% inoculum size next day, cultured bacterium liquid is inoculated into contains Kan(40 μ g/ml) 200 ml LB substratum in cultivate, when bacterium liquid absorbance reaches 0.6, induce with 1 mM IPTG.For fear of in the expression process, inclusion body occurring, the present invention be chosen in thalli growth be not very active 30 ℃ as inducing temperature, induce 4-5 h.

2, the purifying of His-Δ Sak-Δ SEC2

Behind 4 hours abduction delivering, collected somatic cells in centrifugal 5 minutes by 8000 rpm.Subzero 20 ℃ of frozen thalline that spend the night, take out next day, and room temperature was placed 15 minutes.

Use the ultrasonic disruption somatic cells, power: 200-300 W; Treatment time: 6 * 9sec, the 9sec interval.After the processing, centrifugal 10 min of 6000 rpm remove insoluble cell debris, collect supernatant liquor.

Supernatant liquor low speed is splined on the Ni-NTA affinity column, level pad (30 mM Tris-HCl, 300 mM NaCl pH7.6) balance is to baseline, elution buffer (30 mM Tris-HCl, 300 mM NaCl, 50-300 mM imidazoles pH7.6) gradient elution, collect elution peak, concentrated desalination, the SDS-PAGE purity assay.Obtain with histidine-tagged restructuring His-Δ Sak-Δ SEC2.

Among the present invention, vector plasmid pET28a (+) three labels that are used for purifying of having encoded, C hold 6 * Histag, t7tag and N end 6 * Histag, the present invention has selected N to hold 6 * Histag.Utilize the characteristics of Ni ion specificity chelating His, realize containing the Δ Sak-Δ SEC2 of 6 * Histag and separating of foreign protein, realize again the wash-out of His-Δ Sak-Δ SEC2 by the combination of imidazoles competition 6 * Histag and Ni ion.

3, the preparation of Δ Sak-Δ SEC2

Mixture after His-Δ Sak-Δ SEC2 cuts through recombination ox intestine kinase is crossed the Ni-NTA affinity column again, obtains chimeric protein Δ Sak-Δ SEC2.

(3) SDS-PAGE of Δ Sak-Δ SEC2 checking and western blot analyze

Δ Sak-Δ SEC2 is behind SDS-PAGE, and electricity is transferred on the NC film, through 5% BSA sealing, after the TBST rinsing, adds the anti-SEC2 antibody of rabbit, 37 ℃ of reaction 1 h; The TBST rinsing, the goat anti-rabbit igg of adding HRP mark, 37 ℃ of 1 h, TBST rinsing 3 times, DAB dyeing.

The result shows: verify through SDS-PAGE, success obtains chimeric protein Δ Sak-Δ SEC2, Δ Sak-Δ SEC2 albumen can with the anti-SEC antibodies of rabbit, at the about visible specific reaction band in 28kD place of relative molecular mass, show that Δ Sak-Δ SEC2 albumen has good reactionogenicity, Δ SEC2 has kept the antigenic determinant relevant with the SEC2 superantigen.Δ Sak-Δ SEC2 aminoacid sequence is shown in SEQ ID NO.11.

(4) the external thrombolysis activity analysis of Δ Sak-Δ SEC2

The thrombolysis activity of Sak and Δ Sak-Δ SEC2 is compared the urokinase standard substance and is compared and record.Implementation is as follows:

Take by weighing 125 mg agaroses (electrophoresis level), add 23 ml physiological saline, boil dissolving, 60 ℃ of water-bath balances add zymoplasm 14 μ l(100 IU/ml), human plasminogen 280 μ l(0.5 mg/ml), human fibrinogen 2.2 ml(6 mg/ml), pour cooling in the 80 mm flat boards into, make the human fibrin flat board, place half hour with front in 4 ℃.Punching (diameter 2 mm) in the fibrin plate that forms, point sample 10 μ l in every hole are placed horizontally at 37 ℃ of incubator 24 h.

Standard substance urokinase (1280 IU) is diluted to 640 IU, 320 IU, 160 IU, 80 IU, 40 IU, 20 IU, 10 IU production standard curves.The point sample flat board is through removing in length and breadth the solusphere diameter for twice, take the logarithm (x) of each dilution activity as X-coordinate, take the logarithm of the mean number of solusphere diameter (four times measure numerical value) as ordinate zou (y), utilize the regression analysis in the statistics software to make typical curve, and try to achieve a and b and linear regression coefficient r value among the y=a+bx, solusphere diameter per sample can be tried to achieve the activity of sample.

Experimental result:

The thrombolysis activity that gets Sak according to urokinase typical curve side is 7.5 * 10 ³IU/mg, Δ Sak-Δ SEC2 is 7.5 * 10 ³IU/mg, Δ Sak-Δ SEC2 has kept the thrombolysis activity of Sak.

(5) Δ Sak-Δ SEC2 mouse spleen lymphocyte proliferation test

Get the 4-6 mouse in age in week, male, body weight 18-22 g.Take off neck and put to death mouse, 75% alcohol-pickled sterilization 5 min.Cut mouse open under the aseptic condition, spleen is got in the abdominal cavity, grinds 100 eye mesh screens, DThe splenocyte suspension that separates is collected in the flushing of-Hank ' s liquid.Centrifugal 5 min of 1000 r/min normal temperature abandon supernatant; Add erythrocyte cracked liquid 10 ml, the mixing splenocyte, static 4-5 min makes red corpuscle fully broken.Centrifugal 5 min of 1000 r/min normal temperature abandon supernatant, remove red corpuscle. D-Hank ' s liquid is washed 2-3 time, and with 2 ml RPMI1640 complete culture solution re-suspended cells, with the blood counting chamber counting, adjusting cell concn with the RPMI1640 nutrient solution is 2 * 10 ⁶Individual/ml.With RPMI1640 nutrient solution dilution trial-product, every hole 100 μ l join in 96 orifice plates, with the positive contrast of PHA-P, with the negative control wells of BSA, take the RPMI1640 nutrient solution as the blank hole, if 6 multiple holes add respectively 100 μ l splenic lymphocyte suspensions in each hole.After splenic lymphocyte is adherent, application of sample.Each hole adds 100 μ l samples.To add excellent 96 orifice plates and put 37 ^oC, 5% CO ₂Cultivate 72 h in the incubator.Stop cultivating, centrifugal 10 min of 3000 rpm discard nutrient solution in the hole.Every hole adds 120 μ l dimethyl sulfoxide (DMSO) (DMSO), and 10 min that vibrate are fully dissolving crystallized, and putting and measuring each hole on the microplate reader is the light absorption value at 570 nm places at wavelength.

Experimental result:

Can find out from the propagation situation of mouse splenic lymphocyte, the proliferation rate of Δ Sak-Δ SEC2 and enterotoxin SEC2 is similar, and the promoting lymphocyte proliferation vigor of Δ Sak-Δ SEC2 and SEC2 is effect relation with concentration within the specific limits.The results are shown in Table 1.

Table 1

(6) external antitumor activity analysis

Implementation is as follows:

Get the 4-6 mouse in age in week, male, body weight 18-22 g.Take off neck and put to death mouse, 75% alcohol-pickled sterilization 5 min.Cut mouse open under the aseptic condition, spleen is got in the abdominal cavity, grinds 100 eye mesh screens, DThe splenocyte suspension that separates is collected in the flushing of-Hank ' s liquid.Centrifugal 5 min of 1000 r/min normal temperature abandon supernatant; Add erythrocyte cracked liquid 10 ml, the mixing splenocyte, static 4-5 min makes red corpuscle fully broken.Centrifugal 5 min of 1000 r/min normal temperature abandon supernatant, remove red corpuscle. D-Hank ' s liquid is washed 2-3 time, and with 2 ml RPMI1640 complete culture solution re-suspended cells, with the blood counting chamber counting, adjusting cell concn with the RPMI1640 nutrient solution is 2 * 10 ⁶Individual/ml.With lymphocyte with 2 * 10 ⁵The amount in individual/hole joins in 96 orifice plates; Outwell old substratum in the Hella Tissue Culture Flask, add the residual substratum of 2-3 ml D-Hanks liquid flush away, or rinse with a small amount of pancreatin and to wash once, add 1-2 ml pancreatin, under inverted microscope, observe after building bottle cap, regain projection when cell and become the bowlder culturing bottle that overturns immediately, make the cell detachment pancreatin, pancreatin is outwelled, add the RPMI1640 substratum that contains on a small quantity 10% serum, the cell that piping and druming digestion is good repeatedly makes it take off wall and disperses counting.With the Hella cell with 1 * 10 ⁴The amount in individual/hole joins in 96 orifice plates, with the RPMI1640 substratum that contains 10% serum testing sample is diluted to 20 ng/ml and two concentration of 200 ng/ml, join in 96 orifice plates, only add the Hella cell as the tumour cell control wells, only add with the RPMI1640 of experimental port equivalent and testing sample as the background release aperture, 3 multiple holes of every sample, 5% CO2 cultivates 36 h for 37 ℃; Take out 96 orifice plates, add 50 μ l/ hole MTT, 5% CO2 cultivates 4 h for 37 ℃; Centrifugal 10 min of 3000 rpm add DMSO 120 μ l/ holes, 37 ℃ of dissolving 20 min, and 570 nm measure the light absorption value in each hole on the microplate reader.And calculate tumour inhibiting rate according to following formula:

Experimental result:

From tumor killing effect, 100 ng, 1 μ g, 10 μ gUnder three kinds of different concns, Δ Sak – Δ SEC2 shows the vigor similar with enterotoxin SEC2.The results are shown in Table 2.

Table 2

(7) Δ Sak-Δ SEC2 Evaluation of Immunogenicity

Mouse immune SEC2 and Δ Sak-Δ SEC2 albumen respectively with equivalent Freund's complete adjuvant mixing, be prepared into the suspension of 250 μ g/ ml, get 200 μ l/ mouse and make subcutaneous inoculation, establish simultaneously the PBS control group.Distinguish booster immunization 1 time in 11d and 27d.After the last immunity the 9th day, blood was got in docking, separation of serum, and on the same group behind the serum equivalent mixing ,-80 ℃ of preservations.(0.05 mol/ L yellow soda ash-sodium bicarbonate buffer liquid, pH9.6 are diluted to 5 μ g/ ml, and every antigen hole adds 100 μ l, 4 ℃ of coated spending the night with coating buffer with SEC2.Discard liquid in the hole next day, with 37 ℃ of sealings of confining liquid (10 % calf serum PBS) 2h.Add respectively different dilution various antiserum(antisera) 100 μ l after the washing, hatch 1h for 37 ℃.Add the goat anti-mouse igg 100 μ l of the horseradish peroxidase-labeled of working concentration after the washing, hatch 1h for 37 ℃.Washing also adds substrate solution OPD-H ₂O ₂100 μ l, 37 ℃ of lucifuge colour developing 15～20min, every hole adds stop buffer 50 μ l termination reactions, detects the OD value with the 492nm wavelength.

ELISA result shows, the mouse of SEC2 and two kinds of protein immunizations of Δ Sak-Δ SEC2, can both produce a certain amount of antibody, and it is much bigger that the antibody horizontal that SEC2 produces produces antibody horizontal than Δ Sak-Δ SEC2, and PBS control group nonreactive SEC2 antibody produces, and proves that Δ Sak-Δ SEC2 is lower than the immunogenicity of SEC2.

Embodiment 2The preparation of antitumor thrombolytic double-effect chimeric protein Δ SEC2-Δ Sak

(1) with mosaic gene Δ sec2-Δ sakExpression plasmid make up

1) Δ sec2Pcr amplification, Δ sec2Gene order shown in SEQ ID NO.2, Δ sec2By EcoRI/ XhoI is connected on the pET28a, makes up plasmid pET28a- Δ sec2, with plasmid pET28a- Δ sec2Be template, by designing specific primer:

Upstream: 5 '-TTATCGCATATGGACGACGACGACAAAACTGGTACGATGGGTAATAT-3 '

Downstream: 5 '-CGCGAATTCACCACCTGTAACTTTAC-3 '

Utilize the PCR method amplification Δ sec2, and make Δ sec2The coded amino acid upstream has enteropeptidase and is identified as a little, Δ sec2Two ends are respectively with restriction enzyme site NdeI and EcoRI.

2) pET28a- Δ sec2-linker-Δ sakStructure, Δ sakGene order is shown in SEQ ID NO.1 , Δ sakBy EcoRI/ XhoI is connected on the pET28a, makes up plasmid pET28a- Δ sak, Δ sec2Warp NdeI and EcoInsert pET28a-behind the RI double digestion Δ sakIn Δ sakThe upstream NdeI and EcoThe RI site, 16 ℃ of connections of T4 ligase enzyme are spent the night, and obtain ligation liquid.Ligation liquid is joined in advance in the e. coli bl21 competent cell by the Calcium Chloride Method preparation, transform (42 ℃ of water-bath heat shocks) through chemical process, afterwards, bacterium liquid is coated on the LB flat board that contains the Kan resistance, picking positive colony after 37 ℃ of overnight incubation extracts the plasmid warp after cultivating NdeI and EcoThe checking of RI double digestion is determined Δ sec2Whether be inserted into pET 28a- Δ sakIn, whether make up pET 28a- Δ sec2-linker-Δ sak(because Δ sec2With Δ sakBe by EcoRThe I restriction enzyme site connects, so Δ sec2With Δ sakBetween have a sequence be GAATTC(namely EcoRThe I restriction enzyme site) linker).

3) pET 28a- Δ sec2 – Δ sakStructure, with pET 28a- Δ sec2-linker-Δ sakRemove through PCR Δ sec2With Δ sakBetween linker(GAATTC), the PCR product is carried out terminal smoothing and 5 ' phosphatizing treatment, efficient ligase enzyme carries out self and connects (cyclization), then chemical conversion is to transforming the e. coli bl21 competent cell, bacterium liquid is coated on the LB flat board that contains the Kan resistance, picking positive colony after 37 ℃ of overnight incubation extracts the plasmid warp after cultivating EcoThe checking of RI single endonuclease digestion determines whether linker is removed.Give birth to worker company (Sangon, China) to the recombinant plasmid that the obtains affirmation of checking order by Shanghai again.

Test-results:

Cut checking and sequencing analysis comprehensively shows through enzyme, express the Prokaryotic expression vector construction of chimeric protein His-Δ SEC2-Δ Sak successfully.Acquisition is with recombinant plasmid pET28a- Δ sec2 – Δ sakRecombination bacillus coli.

(2) acquisition of the abduction delivering purifying of His-Δ SEC2-Δ Sak and Δ SEC2-Δ Sak

1, the abduction delivering of His-Δ SEC2-Δ Sak

Will be with recombinant plasmid pET28a- Δ sec2 – Δ sakRecombination bacillus coli be inoculated in and contain the Kan(kantlex, Kanamycine, Kan) in the 10 ml LB substratum of (40 μ g/ml), cultivated 12 ~ 16 hours, press 2% inoculum size next day, cultured bacterium liquid is inoculated into contains Kan(40 μ g/ml) 200 ml LB substratum in cultivate, when bacterium liquid absorbance reaches 0.6, induce with 1 mM IPTG.For fear of in the expression process, inclusion body occurring, the present invention be chosen in thalli growth be not very active 30 ℃ as inducing temperature, induce 4-5 h.

2, the purifying of His-Δ SEC2-Δ Sak

Behind 4 hours abduction delivering, collected somatic cells in centrifugal 5 minutes by 8000 rpm.Subzero 20 ℃ of frozen thalline that spend the night, take out next day, and room temperature was placed 15 minutes.

Use the ultrasonic disruption somatic cells, power: 200-300 W; Treatment time: 6 * 9sec, the 9sec interval.After the processing, centrifugal 10 min of 6000 rpm remove insoluble cell debris, collect supernatant liquor.

Supernatant liquor low speed is splined on the Ni-NTA affinity column, level pad (30 mM Tris-HCl, 300 mM NaCl pH7.6) balance is to baseline, elution buffer (30 mM Tris-HCl, 300 mM NaCl, 50-300 mM imidazoles pH7.6) gradient elution, collect elution peak, concentrated desalination, the SDS-PAGE purity assay.Obtain with histidine-tagged restructuring His-Δ SEC2-Δ Sak.

Among the present invention, vector plasmid pET28a (+) three labels that are used for purifying of having encoded, C hold 6 * Histag, t7tag and N end 6 * Histag, the present invention has selected N to hold 6 * Histag.Utilize the characteristics of Ni ion specificity chelating His, realize containing the His-Δ SEC2-Δ Sak of 6 * Histag and separating of foreign protein, realize again the wash-out of His-Δ SEC2-Δ Sak by the combination of imidazoles competition 6 * Histag and Ni ion.

3, the preparation of Δ SEC2-Δ Sak

Mixture after His-Δ SEC2-Δ Sak cuts through recombination ox intestine kinase is crossed the Ni-NTA affinity column again, obtains chimeric protein Δ SEC2-Δ Sak.

(3) SDS-PAGE of Δ SEC2-Δ Sak checking and western blot analyze

Δ SEC2-Δ Sak is behind SDS-PAGE, and electricity is transferred on the NC film, through the 5%BSA sealing, after the TBST rinsing, adds the anti-SEC2 antibody of rabbit, 37 ℃ of reaction 1h; The TBST rinsing, the goat anti-rabbit igg of adding HRP mark, 37 ℃ of 1 h, TBST rinsing 3 times, DAB dyeing.

The result shows: verify through SDS-PAGE, success obtains chimeric protein Δ SEC2-Δ Sak, Δ SEC2-Δ Sak albumen can with the anti-SEC antibodies of rabbit, at the about visible specific reaction band in 28kD place of relative molecular mass, show that Δ SEC2-Δ Sak albumen has good reactionogenicity, Δ SEC2-Δ Sak has kept the antigenic determinant relevant with the SEC2 superantigen.Δ SEC2-Δ Sak aminoacid sequence is shown in SEQ ID NO.12.

(4) the external thrombolysis activity analysis of Δ SEC2-Δ Sak

Implementation: with embodiment 1

Experimental result:

The thrombolysis activity that gets Sak according to urokinase typical curve side is 7.5 * 10 ³IU/mg, Δ SEC2-Δ Sak is 7.5 * 10 ³IU/mg, Δ SEC2-Δ Sak has kept the thrombolysis activity of Sak.

(5) lymphocytes in vitro (PBMC) proliferation activity analysis

Implementation: with embodiment 1

Experimental result:

No matter can find out from the propagation situation of mouse splenic lymphocyte, be 20 ng of low concentration or 200 ng of higher concentration, and the proliferation rate of Δ SEC2-Δ Sak and enterotoxin SEC2 is identical.The results are shown in Table 3.

Table 3

(6) external antitumor activity analysis

Implementation: with embodiment 1

Experimental result:

From tumor killing effect, under 20 ng and two kinds of different concns of 200 ng, Δ SEC2-Δ Sak shows the vigor identical with enterotoxin SEC2.The results are shown in Table 4.

The external tumor suppression experiment of table 4

(7) Δ SEC2-Δ Sak Evaluation of Immunogenicity

Mouse immune SEC2 and Δ SEC2-Δ Sak albumen respectively with equivalent Freund's complete adjuvant mixing, be prepared into the suspension of 250 μ g/ ml, get 200 μ l/ mouse and make subcutaneous inoculation, establish simultaneously the PBS control group.Distinguish booster immunization 1 time in 11d and 27d.After the last immunity the 9th day, blood was got in docking, separation of serum, and on the same group behind the serum equivalent mixing ,-80 ℃ of preservations.(0.05 mol/ L yellow soda ash-sodium bicarbonate buffer liquid, pH9.6 are diluted to 5 μ g/ ml, and every antigen hole adds 100 μ l, 4 ℃ of coated spending the night with coating buffer with SEC2.Discard liquid in the hole next day, with 37 ℃ of sealings of confining liquid (10 % calf serum PBS) 2h.Add respectively different dilution various antiserum(antisera) 100 μ l after the washing, hatch 1h for 37 ℃.Add the goat anti-mouse igg 100 μ l of the horseradish peroxidase-labeled of working concentration after the washing, hatch 1h for 37 ℃.Washing also adds substrate solution OPD-H ₂O ₂100 μ l, 37 ℃ of lucifuge colour developing 15～20min, every hole adds stop buffer 50 μ l termination reactions, detects the OD value with the 492nm wavelength.

ELISA result shows, the mouse of SEC2 and two kinds of protein immunizations of Δ SEC2-Δ Sak, can both produce a certain amount of antibody, and it is much bigger that the antibody horizontal that SEC2 produces produces antibody horizontal than Δ SEC2-Δ Sak, and PBS control group nonreactive SEC2 antibody produces, and proves that Δ SEC2-Δ Sak is lower than the immunogenicity of SEC2.

＜110〉Liaoning University

＜120〉antitumor thrombolytic double-effect chimeric protein of reduced immunogenicity and its preparation method and application

<160> 12

<210> 1

<211> 378

<212> DNA

<213> Δsak

<400> 1

AAAGGCGATGACGCGAGTTATTTTGAACCAACAGGCCCGTATTTGATGGTAAATGTGACTGGAGTTGATGGTAAAGGAAATGAATTGCTATCCCCTCATTATGTCGAGTTTCCTATTAAACCTGGGACTACACTTACAAAAGAAAAAATTGAATACTATGTCGAATGGGCATTAGATGCGACAGCATATAAAGAGTTTAGAGTAGTTGAATTAGATCCAAGCGCAAAGATCGAAGTCACTTATTATGATAAGAATAAGAAAAAAGAAGAAACGAAGTCTTTCCCTATAACAGAAAAAGGTTTTGTTGTCCCAGATTTATCAGAGCATATTAAAAACCCTGGATTCAACTTAATTACAAAGGTTGTTATAGAAAAGAAA

<210> 2

<211> 270

<212> DNA

<213> Δsec2

<400> 2

ACTGGTACGATGGGTAATATGAAATATTTATATGATGATCATTATGTATCAGCAACTAAAGTTATGTCTGTAGATAAATTTTTGGCACATGATTTAATTTATAACATTAGTGATAAAAAACTAAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGAAGATTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGTAAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGT

<210> 3

<211> 126

<212> PRT

<213> ΔSak

<400> 3

KGDDASYFEPTGPYLMVNVTGVDGKGNELLSPHYVEFPIKPGTTLTKEKIEYYVEWALDATAYKEFRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKK

<210> 4

<211> 90

<212> PRT

<213> ΔSEC2

<400> 4

TGTMGNMKYLYDDHYVSATKVMSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDEVVDVYGSNYYVNCYFSSKDNVGKVTGG

<210> 5

<211> 654

<212> DNA

<213> Δsak-linker-Δsec2

<400> 5

AAAGGCGATGACGCGAGTTATTTTGAACCAACAGGCCCGTATTTGATGGTAAATGTGACTGGAGTTGATGGTAAAGGAAATGAATTGCTATCCCCTCATTATGTCGAGTTTCCTATTAAACCTGGGACTACACTTACAAAAGAAAAAATTGAATACTATGTCGAATGGGCATTAGATGCGACAGCATATAAAGAGTTTAGAGTAGTTGAATTAGATCCAAGCGCAAAGATCGAAGTCACTTATTATGATAAGAATAAGAAAAAAGAAGAAACGAAGTCTTTCCCTATAACAGAAAAAGGTTTTGTTGTCCCAGATTTATCAGAGCATATTAAAAACCCTGGATTCAACTTAATTACAAAGGTTGTTATAGAAAAGAAA GAATTCACTGGTACGATGGGTAATATGAAATATTTATATGATGATCATTATGTATCAGCAACTAAAGTTATGTCTGTAGATAAATTTTTGGCACATGATTTAATTTATAACATTAGTGATAAAAAACTAAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGAAGATTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGTAAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGT

<210> 6

<211> 654

<212> DNA

<213> Δsec2-linker-Δsak

<400> 6

ACTGGTACGATGGGTAATATGAAATATTTATATGATGATCATTATGTATCAGCAACTAAAGTTATGTCTGTAGATAAATTTTTGGCACATGATTTAATTTATAACATTAGTGATAAAAAACTAAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGAAGATTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGTAAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGT GAATTCAAAGGCGATGACGCGAGTTATTTTGAACCAACAGGCCCGTATTTGATGGTAAATGTGACTGGAGTTGATGGTAAAGGAAATGAATTGCTATCCCCTCATTATGTCGAGTTTCCTATTAAACCTGGGACTACACTTACAAAAGAAAAAATTGAATACTATGTCGAATGGGCATTAGATGCGACAGCATATAAAGAGTTTAGAGTAGTTGAATTAGATCCAAGCGCAAAGATCGAAGTCACTTATTATGATAAGAATAAGAAAAAAGAAGAAACGAAGTCTTTCCCTATAACAGAAAAAGGTTTTGTTGTCCCAGATTTATCAGAGCATATTAAAAACCCTGGATTCAACTTAATTACAAAGGTTGTTATAGAAAAGAAA

<210> 7

<211> 648

<212> DNA

<213> Δsak-Δsec2

<400> 7

AAAGGCGATGACGCGAGTTATTTTGAACCAACAGGCCCGTATTTGATGGTAAATGTGACTGGAGTTGATGGTAAAGGAAATGAATTGCTATCCCCTCATTATGTCGAGTTTCCTATTAAACCTGGGACTACACTTACAAAAGAAAAAATTGAATACTATGTCGAATGGGCATTAGATGCGACAGCATATAAAGAGTTTAGAGTAGTTGAATTAGATCCAAGCGCAAAGATCGAAGTCACTTATTATGATAAGAATAAGAAAAAAGAAGAAACGAAGTCTTTCCCTATAACAGAAAAAGGTTTTGTTGTCCCAGATTTATCAGAGCATATTAAAAACCCTGGATTCAACTTAATTACAAAGGTTGTTATAGAAAAGAAAACTGGTACGATGGGTAATATGAAATATTTATATGATGATCATTATGTATCAGCAACTAAAGTTATGTCTGTAGATAAATTTTTGGCACATGATTTAATTTATAACATTAGTGATAAAAAACTAAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGAAGATTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGTAAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGT

<210> 8

<211> 648

<212> DNA

<213> Δsec2-Δsak

<400> 8

ACTGGTACGATGGGTAATATGAAATATTTATATGATGATCATTATGTATCAGCAACTAAAGTTATGTCTGTAGATAAATTTTTGGCACATGATTTAATTTATAACATTAGTGATAAAAAACTAAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGAAGATTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGTAAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGTAAAGGCGATGACGCGAGTTATTTTGAACCAACAGGCCCGTATTTGATGGTAAATGTGACTGGAGTTGATGGTAAAGGAAATGAATTGCTATCCCCTCATTATGTCGAGTTTCCTATTAAACCTGGGACTACACTTACAAAAGAAAAAATTGAATACTATGTCGAATGGGCATTAGATGCGACAGCATATAAAGAGTTTAGAGTAGTTGAATTAGATCCAAGCGCAAAGATCGAAGTCACTTATTATGATAAGAATAAGAAAAAAGAAGAAACGAAGTCTTTCCCTATAACAGAAAAAGGTTTTGTTGTCCCAGATTTATCAGAGCATATTAAAAACCCTGGATTCAACTTAATTACAAAGGTTGTTATAGAAAAGAAA

<210> 9

<211> 242

<212> PRT

<213> His-ΔSak-ΔSEC2

<400> 9

MGSSHHHHHHSSGLVPRGSHMDDDDKKGDDASYFEPTGPYLMVNVTGVDGKGNELLSPHYVEFPIKPGTTLTKEKIEYYVEWALDATAYKEFRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKKTGTMGNMKYLYDDHYVSATKVMSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDEVVDVYGSNYYVNCYFSSKDNVGKVTGG

<210> 10

<211> 242

<212> PRT

<213> His-ΔSEC2-ΔSak

<400> 10

MGSSHHHHHHSSGLVPRGSHMDDDDKTGTMGNMKYLYDDHYVSATKVMSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDEVVDVYGSNYYVNCYFSSKDNVGKVTGGKGDDASYFEPTGPYLMVNVTGVDGKGNELLSPHYVEFPIKPGTTLTKEKIEYYVEWALDATAYKEFRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKK

<210> 11

<211> 216

<212> PRT

<213> ΔSak-ΔSEC2

<400> 11

KGDDASYFEPTGPYLMVNVTGVDGKGNELLSPHYVEFPIKPGTTLTKEKIEYYVEWALDATAYKEFRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKKTGTMGNMKYLYDDHYVSATKVMSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDEVVDVYGSNYYVNCYFSSKDNVGKVTGG

<210> 12

<211> 216

<212> PRT

<213> ΔSEC2-ΔSak

<400> 12

TGTMGNMKYLYDDHYVSATKVMSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDEVVDVYGSNYYVNCYFSSKDNVGKVTGGKGDDASYFEPTGPYLMVNVTGVDGKGNELLSPHYVEFPIKPGTTLTKEKIEYYVEWALDATAYKEFRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKK

Claims (3)

1. the antitumor thrombolytic double-effect chimeric protein of reduced immunogenicity, it is characterized in that: described chimeric protein is the chimeric protein Δ Sak-Δ SEC2 of aminoacid sequence shown in SEQ ID NO.11, or the chimeric protein Δ SEC2 – Δ Sak of aminoacid sequence shown in SEQ ID NO.12.

2. method of utilizing escherichia expression system to produce the antitumor thrombolytic double-effect chimeric protein Δ of reduced immunogenicity claimed in claim 1 Sak-Δ SEC2 is characterized in that step is as follows:

1) carrier construction pET 28a- Δ sak-linker-Δ sec2, Δ sak-linker-Δ sec2Dna sequence dna shown in SEQ ID NO.5;

2) with the carrier pET 28a-that obtains Δ sak-linker-Δ sec2Remove through PCR Δ sakWith Δ sec2Between linker, construct the plasmid pET28a-that expresses chimeric protein Δ Sak-Δ SEC2 Δ sak-Δ sec2, Δ sak-Δ sec2Dna sequence dna shown in SEQ ID NO.7;

3) with plasmid pET28a- Δ sak-Δ sec2Change in the e. coli bl21 competent cell, obtain recombination bacillus coli;

4) recombination bacillus coli is inoculated in carries out shake-flask culture in the LB substratum, when bacterium liquid absorbance reaches 0.6, under 30 ℃ of conditions, induced 4～5 hours with 1 mM IPTG, afterwards centrifugal collection somatic cells;

5) use the ultrasonic disruption somatic cells, power: 200-300 W; Treatment time: 6 * 9 sec, 9 sec intervals; After the processing, centrifugal 10 min of 6000 rpm remove insoluble cell debris, collect supernatant liquor;

6) supernatant liquor is crossed the Ni-NTA affinity column, through gradient elution and concentrated desalination, obtained chimeric protein His-Δ Sak-Δ SEC2, its aminoacid sequence is shown in SEQ ID NO.9;

7) chimeric protein His-Δ Sak-Δ SEC2 crosses the Ni-NTA affinity column again through the enteropeptidase cutting, obtains chimeric protein Δ Sak-Δ SEC2, and its aminoacid sequence is shown in SEQ ID NO.11.

3. method of utilizing escherichia expression system to produce the antitumor thrombolytic double-effect chimeric protein Δ of reduced immunogenicity claimed in claim 1 SEC2 – Δ Sak is characterized in that step is as follows:

1) carrier construction pET 28a- Δ sec2-linker-Δ sak, Δ sec2-linker-Δ sakDna sequence dna shown in SEQ ID NO.6;

2) with the carrier pET 28a-that obtains Δ sec2-linker-Δ sakRemove through PCR Δ sec2With Δ sakBetween linker, construct the plasmid pET28a-that expresses chimeric protein Δ SEC2-Δ Sak Δ sec2-Δ sak, Δ sec2-Δ sakDna sequence dna shown in SEQ ID NO.8;

3) with plasmid pET28a- Δ sec2-Δ sakChange in the e. coli bl21 competent cell, obtain recombination bacillus coli;

4) recombination bacillus coli is inoculated in carries out shake-flask culture in the LB substratum, when bacterium liquid absorbance reaches 0.6, under 30 ℃ of conditions, induced 4～5 hours with 1 mM IPTG, afterwards centrifugal collection somatic cells;

5) use the ultrasonic disruption somatic cells, power: 200-300 W; Treatment time: 6 * 9 sec, 9 sec intervals; After the processing, centrifugal 10 min of 6000 rpm remove insoluble cell debris, collect supernatant liquor;

6) supernatant liquor is crossed the Ni-NTA affinity column, through gradient elution and concentrated desalination, obtained chimeric protein His-Δ SEC2-Δ Sak, its aminoacid sequence is shown in SEQ ID NO.10;

7) chimeric protein His-Δ SEC2-Δ Sak crosses the Ni-NTA affinity column again through the enteropeptidase cutting, obtains chimeric protein Δ SEC2-Δ Sak, and its aminoacid sequence is shown in SEQ ID NO.12.