CN100480270C - Fusion protein of chemoattracting small peptide and dual specific antibodies - Google Patents

Abstract

This invention relates to a fusion protein of chenotactic peptide and bispecific antibody against ovarian carcinoma and CD3 molecules. The fusion protein is constructed by sequentially connecting N-terminated 18-peptides of secondary lymphoid tissue chemokine, (GGGGS)2 linking peptide, and the bispecific antibody against ovarian carcinoma and CD3 molecules. C-myc label and His6 label are connected at the C-terminal. This invention also relates to the method for constructing, expressing and purifying the fusion protein, its coding sequence, its relevant expression vector, and the host cells containing the expression vector.

Description

The fusion rotein of a kind of chemoattracting small peptide and bispecific antibody

Technical field

The present invention relates to the genetic engineering antibody field, more specifically, relate to N-terminal ten octapeptides of a kind of chemokine SLC (Secondary lymphoid tissue chemokine, secondary lymphoid-tissue chemokines) and the fusion rotein of ovarian cancer resistance * anti-CD3 bispecific antibody; The structure of this fusion rotein, expression and purification process; The carrier and the e. coli host bacteria that contain this fusion rotein.

Background technology

Along with the development of antibody engineering, immunotherapy is after operation, radiotherapy, chemotherapy, the important method of another treatment tumour.The ideal treatment plan should be that the specific efficient at tumour cell kills and wounds, and can be the comparatively ideal candidate of this scheme in conjunction with the effector cell's of high cell toxicity bispecific antibody again in conjunction with the target tumour cell.It is on the one hand at tumour, realize combining with the specificity of target tumour cell, the effector cell is enriched in around the tumour cell on the other hand, the effect of simulation native ligand, combine with effector cell's trigger molecule at tumor locus, the activation effect cell is realized specific killing and cracking (reference 1-10) to tumour.But effector cell's the quantity that is enriched in tumor locus is limited after all, makes this effect be difficult to obtain maximum performance.

The discovery of chemokine makes the solution of this problem new dawn occur.Chemokine (abbreviation of chemotactic cytokine) is a complicated superfamily of being made up of the alkaline secretory protein of small molecules (8-10KD), and various white corpuscles are had chemotactic effect (reference 11-15).According to the quantity and the position of N-terminal halfcystine, chemokine is divided into four subfamily: CC, CXC, CX ₃C and C.Wherein SLC is member's (reference 16) of CC family, mainly expresses at secondary lymphoid tissue (in spleen, lymphoglandula, Pai Shi assembly, lymph endothelium), and T lymphocyte, bone-marrow-derived lymphocyte, sophisticated dendritic cell are had chemotaxis.

Recently, the three-dimensional structure of some chemokines has obtained resolving (reference 17-22), see that from existing data the three-dimensional structure of chemokine is a high conservative, they mostly have the N-terminal of a flexible disordered, it then is the N-terminal annular zone of corner in the shape of a spiral, then being three antiparallel βZhe Dies, is C-terminal α spiral at last, and N-terminal and N-terminal district are connected on the βZhe Die by two disulfide linkage.People carry out L-Ala scanning by brachymemma N-terminal district or to N-terminal district and N-terminal annular zone, have proved that these two zones are necessary (reference 23-26) in part combination and activated receptor.People such as Pius Loetscher confirm that the N-terminal district of SDF-1 and N-terminal annular zone have enough combinations active to its receptor CXCR 4, have certain chemotactic activity (reference 27) to the lymphocyte of expressing this receptor.This laboratory also confirms very high in conjunction with activity to acceptor CCR7 of the N-terminal district of CCL21 and N-terminal annular zone, and dendritic cell and T lymphocyte are had suitable chemotactic activity.

Characteristics in conjunction with bispecific antibody and chemokine, the inventor has successfully made up N-terminal ten octapeptides of chemokine SLC and the fusion rotein of ovarian cancer resistance * anti-CD3 bispecific antibody, and proved that this fusion rotein promptly has expressing the cell combination and the chemotactic activity of CCR7 acceptor, has combination and the killing activity of bispecific antibody to corresponding tumour cell again.This fusion rotein arrives tumor locus under the guide effect of bispecific antibody like this, then brings into play chemotaxis the dendritic cell and the T lymphocyte of q.s is attracted to tumor locus, and killing tumor cells can be treated tumour so efficiently.

Summary of the invention

The present invention is with N-terminal ten octapeptides of the chemokine CC SLC of family, continuous successively N-terminal ten octapeptides of chemokine SLC (Secondary lymphoid tissuechemokine, secondary lymphoid-tissue chemokines) and the fusion rotein of ovarian cancer resistance * anti-CD3 bispecific antibody of making up of ovarian cancer resistance single-chain antibody * anti-CD3 single-chain antibody; This construction can be given full play to the advantage of the two.On the one hand, utilize the chemotaxis of N-terminal ten octapeptides of SLC, attract T lymphocyte and dendritic cell to corresponding site; On the other hand, utilize the bridging effect of ovarian cancer resistance * anti-CD3 bispecific antibody, specificity activates the T lymphocyte and kills and wounds ovarian cancer cell.This fusion rotein has bigger application prospect in the immunotherapy field of ovarian cancer.

Therefore, an object of the present invention is to provide the fusion rotein of a kind of chemoattracting small peptide and bispecific antibody.

In one embodiment of the invention, described chemoattracting small peptide is N-terminal ten octapeptides (SDGGAQDCCLKYSQRKIP) of the chemokine SLC of CC family (secondary lymphoid-tissue chemokines).

In another embodiment of the invention, described bispecific antibody is antitumor * anti-CD3 bispecific antibody.Preferred in the present invention described antitumor * anti-CD3 bispecific antibody is ovarian cancer resistance * anti-CD3 bispecific antibody.

In a specific embodiment of the present invention, described fusion rotein is N-terminal ten octapeptides, (GGGGS) by the chemokine SLC of CC family (secondary lymphoid-tissue chemokines) ₂Connection peptides, ovarian cancer resistance * anti-CD3 bispecific antibody are connected in sequence.Preferred described fusion rotein is characterized in that having the aminoacid sequence shown in the SEQ ID NO:2, and is coded by the nucleotide sequence shown in the SEQ ID NO:1, and its C-terminal can randomly have C-myc, and (aminoacid sequence is: EQKLISEEDLN) and His ₆Label.

Another one purpose of the present invention provides a kind of expression vector, it is characterized in that comprising the encoding gene of any one described fusion rotein among the claim 1-6.Preferred described carrier is p18TBHL.

The present invention also provides the host cell that contains above-mentioned carrier, preferred intestinal bacteria.

The present invention also provides the method for above-mentioned any fusion rotein of a kind of purifying on the other hand, it is characterized in that uniting adopting DEAE anionite-exchange resin and Ni affinity column to carry out purifying.

More specifically, according to an aspect of of the present present invention, N-terminal ten octapeptides of a kind of chemokine SLC that treats ovarian cancer and the fusion rotein of ovarian cancer resistance * anti-CD3 bispecific antibody are provided.

The method of the fusion rotein of a kind of N-terminal ten octapeptides that make up chemokine SLC and ovarian cancer resistance * anti-CD3 bispecific antibody is provided in another aspect of this invention.

The aminoacid sequence of preferred fusion rotein of the present invention is shown in following SEQ ID NO:2.

SDGGAQDCCLKYSQRKIP GGGGSGGGGS(bolded section is terminal ten octapeptides of the N of chemotactic factor (CF) SLC to LELEDVQLLESGPEAKKPGETVRISCKASGYTFTTAGMQWVQKMPGKGLKWLGWIN TNSEVPKYAEDFRGRFAFSLETSASTAYLQISNLKNEDTATFFCARSFTWGTMDYW GQGTPVTVSSTSGGGGSGGGGSGGGGSSRDVVMTQTPLSLPVSLGDQASISCRSSQ TLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV EAEDLGVYFCSQSTHVPYTFGGGTKLELKEFQNALLVRYTKKVPQVSTPTLVEVSE LDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLH SGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLELKRATS GGGGSGGGGSGGGGSSREVKLVESGPELVKPGASMKISCKASGYSFTGYTMNWVKQ SHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYY CARSGYYGDSDWYFDVWGAGTSVTVSS, and underscore partly is (GGGGS)₂Connection peptides is ovarian cancer resistance * anti-CD3 bispecific antibody with the lower section)

More preferably encode the nucleotide sequence of fusion rotein of the present invention shown in following SEQ ID NO:1.

agcgatggtggtgcacaggattgctgcctgaaatatagccagcgtaaaattccgggtggtggtggttctggtggtggtggttctctcgagctcgaggatgtgcagctgctggagtctggacctgaggcgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtataccttcacaactgctggaatgcagtgggtgcaaaagatgccaggaaagggtttgaagtggcttggctggataaacaccaactctgaagttccaaaatatgcagaagacttcaggggacggtttgccttctctttggagacctctgccagcactgcatatttacagataagcaacctcaaaaatgaggacacggctacgtttttctgtgcgagatcttttacttgggggactatggactattggggccaagggaccccggtcaccgtctcctcaactagtggtggtggtggttctggtggtggtggttctggtggtggtggttcttctagagatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagacccttgtacacagtaatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaaactcctgatctacaaggtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttccgtacacgttcggaggggggaccaagctggagctcaaagaattccagaatgcgctgttagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcagagctcgacatccagatgacccagaccacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagaaattatttaaactggtatcaacagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaagttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaggatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcgctggaggcaccaaactggaactgaagcgcgctactagtggtggtggtggttctggtggtggtggttctggtggtggtggttcttctagagaggtgaagctggtggagtctggacctgagctggtgaagcctggagcttcaatgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagtcatggaaagaaccttgagtggatgggacttattaatccttacaaaggtgttagtacctacaaccagaagttcaaggacaaggccacattaactgtagacaagtcatccagcacagcctacatggaactcctcagtctgacatctgaggactctgcagtctattactgtgcaagatcggggtactacggtgatagtgactggtacttcgatgtctggggcgcaggaacctcagtcactgtctcctca

Detailed Description Of The Invention

In the context of the present invention, employed term generally has the implication of those of ordinary skill in the art's common sense unless otherwise indicated.

N-terminal ten octapeptides of the chemokine SLC that the present invention is constructed and the fusion rotein of ovarian cancer resistance * anti-CD3 bispecific antibody are to make up by the method that genetically engineered is recombinated, promptly have the ability that attracts T lymphocyte and dendritic cell, have the function that activates T lymphocyte killing tumor cells again.Particularly, the fusion rotein of N-terminal ten octapeptides of chemokine SLC of the present invention and ovarian cancer bispecific antibody is to have the N-terminal ten octapeptides warps (GGGGS) of the chemokine SLC of CC family of chemotactic ability ₂The molecule that connection peptides and ovarian cancer resistance * anti-CD3 bispecific antibody is in series.The present invention selects N-terminal ten octapeptides of the chemokine SLC of CC family with chemotactic ability for use, and not with the full molecule of SLC, and this is just avoided causing because of molecule is excessive the follow-up purifying and the difficulty of medication.Between ten octapeptides and bispecific antibody, add connection peptides, can guarantee that two portions can both correctly fold, and exercise normal function.Add C-myc label and histidine-tagged (reference 28,29) at the C of bispecific antibody end, be convenient to active detection and purifying.This fusion rotein has the following advantages:

1. utilize the chemotactic ability of N-terminal ten octapeptides of the chemokine SLC of CC family can attract a large amount of T lymphocytes and dendritic cell to corresponding position;

2. the function of performance ovarian cancer bispecific antibody activates the T lymphocyte and kills and wounds ovarian cancer cell.

Purifying of the present invention, be to adopt DEAE anionite-exchange resin and Ni affinity column to be coupled, at first with soluble-expression product in the born of the same parents, through the DEAE anion-exchange chromatography, most of foreign protein is attracted on the DEAE post, and the fusion rotein of N-terminal ten octapeptides of chemokine SLC and bispecific antibody is worn liquid with stream and is flowed out, and then stream is worn liquid and crossed the Ni affinity column and further remove residual foreign protein, and purity can reach 90%.

Technical scheme of the present invention is as follows:

At first carrier pTMF enzyme is cut processing, linking to each other with fragment c-myc-his6 forms pTMFMH.Then carrier pTMFMH double digestion being linked to each other with the BHL-1 of amplification makes up pTCMBO, then the pTCMBO enzyme is cut, mended flat, and enzyme is cut again; Two complementary strands of ten octapeptides form double-stranded fragment in proper temperature annealing, are connected with the carrier of handling well subsequently to make up p18TBHL.Plasmid p18TBHL transformed into escherichia coli BL21 (DE3) (available from American I nvitrogen company), carry out low temperature induction with IPTG (isopropylthio-) (available from precious biotechnology (Dalian) company limited) and express, the ultrasonic supernatant of expression product DEAE anion-exchange column (Pharmacia company) and Ni affinity column (Pharmacia company) purifying.The SDS-PAGE and the immune marking detect the expression and purification situation.Detect the chemotaxis of purified product with chemotactic experiment to the peripheral blood lymphocyte that stimulated.Enzyme-linked immunosorbent assay (ELISA) detects the antigen-binding activity of purified product, and monochromatic flow cytometry (FACS) detects it to tumour cell and lymphocytic in conjunction with active.Mtt assay (tetramethyl-azo azoles blue laws) detects the effect that purified product mediation T lymphocyte kills and wounds ovarian cancer cell.Double-colored FACS method detects purified product mediation T lymphocyte killing and wounding ovarian cancer cell.

Should be pointed out that the context at this specification sheets, especially on the basis of the disclosure of " embodiment " part, other aspects and advantages of the present invention are conspicuous to those skilled in the art.

Description of drawings

Fig. 1. make up N-terminal 18 peptides of SLC and the plasmid figure of ovarian cancer bispecific antibody fusion rotein.N-terminal 18 peptides of SLC are building up to the N-terminal of ovarian cancer bispecific antibody BHL with NcoI and XhoI, at the C-terminal of fusion rotein detection label C-myc and purification tag 6His are arranged.

Fig. 2. expressing fusion protein purifying figure.First swimming lane, the ultrasonic supernatant of expression product; Second swimming lane, DEAE stream is worn; The 3rd swimming lane, the DEAE wash-out; The 4th swimming lane, all product on the Ni post; The 5th swimming lane, Ni post stream is worn; The 6th swimming lane, the washing of Ni post; The 7th swimming lane, Ni post wash-out; The 8th swimming lane, low molecular weight protein (LMWP) standard (Shanghai biochemical research institute).

Fig. 3. detect the fusion rotein of purifying.First swimming lane, the fusion rotein of purifying; Second swimming lane dyes the molecular weight of albumen standard in advance.

Fig. 4. chemotactic test-results figure.SLC, 18T-BHL, 18T, BHL, BSA be 0.1,1,10,100,1000, during 10000nM to the chemotaxis of peripheral blood mononuclear cell PBMC.

Fig. 5 .BHL and 18T-BHL detect SKOV3 membrane antigen bonded ELISA.The concentration of two kinds of samples is respectively 20,10,5,2.5,1.25,0.625 and 0.3125 μ g/ml, curve A g among the figure ^-18T-BHL and Ag ^-BHL represents not wrap the membrane antigen by SKOV3, adds the result of a certain amount of 18T-BHL and BHL respectively.Curve A g among the figure ⁺18T-BHL and Ag ⁺When BHL represents bag by the SKOV3 membrane antigen, add the result of a certain amount of 18T-BHL and BHL.

Fig. 6 .BHL and 18T-BHL detect PBL membrane antigen bonded ELISA.The concentration of two kinds of samples is respectively 20,10,5,2.5,1.25,0.625,0.3125 μ g/ml, curve A g among the figure ^-18T-BHL and Ag ^-BHL represents not wrap the membrane antigen by PBL, adds the result of a certain amount of 18T-BHL and BHL respectively.Curve A g among the figure ⁺18T-BHL and Ag ⁺When BHL represents bag by the PBL membrane antigen, add the result of a certain amount of 18T-BHL and BHL.

Fig. 7 .BHL and 18T-BHL detect Jurkat membrane antigen bonded.The concentration of two kinds of samples is respectively 20,10,5,2.5,1.25,0.625,0.3125 μ g/ml, curve A g among the figure ^-18T-BHL and Ag ^-BHL represents not wrap the membrane antigen by Jurkat, adds the result of a certain amount of 18T-BHL and BHL respectively.Curve A g among the figure ⁺18T-BHL and Ag ⁺When BHL represents bag by the Jurkat membrane antigen, add the result of a certain amount of 18T-BHL and BHL.

Fig. 8 .18T-BHL and BHL identify that to SKOV3 cell bonded FACS the peak of band shade is the control sample measurement result that does not add sample 18T-BHL and BHL among the figure, and the peak with shade is not the sample determination result who adds 18T-BHL and BHL.Wherein the figure on the left side is the measurement result of BHL, and the figure on the right is the measurement result of 18T-BHL.

Fig. 9 .18T-BHL and BHL identify that to Jurkat cell bonded the peak of band shade is the control sample measurement result that does not add sample 18T-BHL and BHL among the figure, and the peak with shade is not the sample determination result who adds 18T-BHL and BHL.Wherein the figure on the left side is the measurement result of BHL, and the figure on the right is the measurement result of 18T-BHL.

Figure 10 .18T-BHL and BHL identify that to PBL cell bonded the peak of band shade is the control sample measurement result that does not add sample 18T-BHL and BHL among the figure, and the peak with shade is not the sample determination result who adds 18T-BHL and BHL.Wherein the figure on the left side is the measurement result of BHL, and the figure on the right is the measurement result of 18T-BHL.

Figure 11 .MTT detects the result of the 18T-BHL and the T lymphocyte killing tumor cell that BHL mediates of different concns.When the ratio of effector cell and target cell was 10:1, concentration was respectively 80,40, and 20,10,5,2.5, the T lymphocyte of the 18T-BHL of 1.25,0.625 μ g/ml and BHL mediation is to the Mortaility results of target cell SKOV3.

The FACS detected result of the tumour cell (SKOV3) that Figure 12 .PKH26 and AnnexinV-FITC double-tagging are killed and wounded.When the ratio of effector cell and target cell was 10:1, the concentration of 18T-BHL and BHL was respectively 20,10, mediation T lymphocyte killing and wounding tumour cell during 1,0 μ g/ml.Upper left district is the SKOV3 cell of the painted not apoptosis of PKH26, and upper right district is PKH26 and amphophilic apoptotic cell.

The statistical graph of the FACS detected result of the tumour cell (SKOV3) that Figure 13 .PKH26 and AnnexinV-FITC double-tagging are killed and wounded.When BHL concentration was 20,10,1,0 μ g/ml, apoptotic cells was respectively 45.58%, 43.54%, and 28.37%, 26.02%.When 18T-BHL concentration was 20,10,1,0 μ g/ml, apoptotic cell was respectively 52.33% 57.33% 47.04% 24.68%.

Embodiment

Below in conjunction with specific embodiment, and the present invention is described in further detail with reference to accompanying drawing.Should be understood that the embodiment in this specification sheets is in order to demonstrate the invention, but not limit the scope of the invention by any way.

Embodiment 1: the structure of carrier pTCMBO.

(1) processing of carrier pTMF.

The structure flow process of PTMFMH.At first (carrier pTMF derives from carrier pET28a (+) (Novagen company) with carrier pTMF, by our company the multiple clone site structure of synthetic one section multiple clone site replacement vector pET28a (+) is formed, document 30 sees reference) usefulness restriction enzyme BamHI (precious biotechnology (Dalian) company limited, down together) enzyme is cut, specific as follows: as to get carrier pTMF18 μ l, BamHI 1.5 μ l, K damping fluid 3 μ l use ddH ₂O supplies volume to 30 μ l, and 37 ℃ of water enzyme digestions reacted 4 hours.Reclaim test kit (magnificent Shun Bioisystech Co., Ltd product, down with) according to a small amount of glue the DNA after purifying recovery enzyme is cut be described.Reclaim product and cut with the Bpu1102I enzyme, specific as follows: pTMF 16 μ l, Bpu1102I 1.5 μ l (available from precious biotechnology (Dalian) company limited, down together), basic damping fluid 3 μ l use ddH ₂O supplies volume to 30 μ l, and 37 ℃ of water enzyme digestions reacted 4 hours.After 1% agarose gel electrophoresis detects digestion fully, reclaim required band at long wavelength ultraviolet lamp incision glue, the test kit explanation is reclaimed in reference glue in a small amount, reclaims and the required endonuclease bamhi of purifying with post.

(2) preparation of fragment c-myc-his

According to the sequence of c-myc and his, synthetic two complementary strands of design

Upstream 5 ' aacggatccgaacaaaaactcatctcagaagaggatctgaatggggccgcacatca t3 '

Downstream 5 ' gacgcttagcttattgctcgtgatggtgatgatgatgtgcggccccattcagatcc tctt 3 '

Article two, the complementary strand pcr amplification obtains containing the complementary double-stranded of restriction enzyme site BamHI and Bpu1102I.Concrete operations are as follows: upstream primer 2 μ l, downstream primer 2 μ l, PCR damping fluid 2 μ l are (available from precious biotechnology (Dalian) company limited, down together), dNTP 2 μ l (Shanghai Bo Ya Bioisystech Co., Ltd, down together), pfu enzyme 1 μ l is (available from precious biotechnology (Dalian) company limited, down together), H ₂O 11 μ l, 95 ℃ of sex change 5min of response procedures; 25 circulations of 72 ℃ of 1min reactions of 53 ℃ of 1min of 94 ℃ of 1min, 72 ℃ of 5min.The PCR product carries out 1% agarose gel electrophoresis, and utilizing in a small amount, glue reclaims the required fragment of test kit purifying recovery.The fragment that reclaims carries out BamHI respectively and the Bpu1102I enzyme is cut, and concrete operations are with reference to (1), and glue reclaims the test kit purifying and reclaims the fragment that enzyme is cut in a small amount.

(3) carrier segments pTMF and fragment c-myc-his's is connected

Fetch the about 40ng of double digestion pTMF of receipts, the about 20ng of c-myc-his fragment adds 2 μ l, 10 * T ₄(available from precious biotechnology (Dalian) company limited, component is as follows: 660mM Tris-HCl (pH7.6), 66mM MgCl for the dna ligase damping fluid ₂, 100mM DTT, 1mM ATP, down together), 1 μ l T ₄Dna ligase (available from precious biotechnology (Dalian) company limited, down together) adds ddH ₂O is to cumulative volume 20 μ l, mixing, 16 ℃ of connections spend the night (about 16 hours).

(4) preparation of competent escherichia coli cell

Prepare colibacillary competent cell with Calcium Chloride Method, method is as follows: picking list bacterium colony from wild coli strain Top10 (Invitrogen company) flat board, be inoculated in 3ml LB (10g/l Tryptones (GIBCO company), 5g/l yeast extract paste (GIBCO company), 5g/l NaCl, pH7.5) in the liquid nutrient medium, 37 ℃ of concussion overnight incubation.Transfer in the LB of 40ml liquid nutrient medium with 1% inoculum size, 37 ℃ of concussions are cultured to OD ₆₀₀When value is 0.3-0.4, bacterium liquid was placed ice bath 10 minutes, in 4 ℃, centrifugal 10 minutes of 4000rpm, supernatant discarded, precipitation is resuspended in the 0.1mol/L CaCl of 20ml precooling ₂In (Sigma company), ice bath was placed 30 minutes, and 4 ℃, centrifugal 10 minutes of 4000rpm, supernatant discarded, the 0.1mol/L CaCl of adding 2ml precooling ₂(containing 20% glycerine) re-suspended cell, standby with every pipe 200 μ l packing.Each Guan Yu-70 ℃ preservation of untimely use.

(5) conversion of recombinant DNA, the screening of positive colony and order-checking are identified

10 μ l connect product and join among the competent cell Top10 of 200 μ l, mixing gently, and ice bath was placed 30 minutes, 42 ℃ of water-baths 90 seconds placed ice bath 2-5 minute immediately, added the not liquid LB substratum of added with antibiotic of 800 μ l then, mixing gently, 37 ℃ of jogs are cultivated recovery 45-60 minute.Get 200 μ l nutrient solutions and coat on LB (the containing 50 μ g/ml Km) flat board, after the surface drying, be inverted overnight incubation for 37 ℃.Single bacterium colony of growing on the picking flat board adopts alkaline process to extract plasmid DNA in a small amount, and with BamHI and Bpu1102I double digestion, the fragment that can downcut about 87bp promptly tentatively is defined as positive colony; Simultaneously, carrying out PCR identifies.Primer sequence is as follows:

Upstream primer: 5 ' atagcgatggtggtgcacaggattgctg3 ',

Downstream primer 5 ' gacggatccttattgctcgtgatggtgatgatgatgtgcggccccattcagatcct ctt, the PCR reaction is with reference to the molecular cloning experiment guide (third edition, J. Sa nurse Brooker, D.W. Russell, cold spring harbor laboratory publishes), the PCR product carries out 1% agarose gel electrophoresis, obtains and expects big or small identical segments, has further identified the segmental plasmid of insertion external source.Plasmid after identifying is served the order-checking of Hai Boya Bioisystech Co., Ltd identify, identify the correct positive plasmid called after pTMFMH of back order-checking.

(6) primer of a pair of amplification of design BHL-I (sequence of coding ovarian cancer bispecific antibody) contains restriction enzyme site XhoI and BamHI respectively.Primer sequence is as follows:

Primer 15 ' ttctcgaggatgtgcagcctgctggagtc3 '

Primer 25 ' atggatcctgaggagacagtgactgagg3 '

With pBHL-I is template (reference 10), and PCR reacts with reference to molecular cloning, and the PCR product carries out 1% agarose gel electrophoresis, obtains the fragment of 1590bp size.And fragment carried out XhoI (available from precious biotechnology (Dalian) company limited, down with) and BamHI double digestion, glue reclaims the test kit recovery in a small amount.

(7) structure of pTCMBO

Plasmid pTMFMH is carried out XhoI and BamHI double digestion, and glue reclaims the test kit recovery in a small amount, is connected transformed into escherichia coli competent cell Top10, then screening positive clone with the fragment BHL-I that enzyme is cut.Advanced performing PCR is identified, amplifies the fragment of about 1600bp size, with XhoI and BamHI recombinant plasmid is carried out enzyme again and cuts evaluation, downcuts the fragment of 1600bp size.To identify that the male plasmid serves Hai Boya Bioisystech Co., Ltd order-checking and identify the positive plasmid called after pTCMBO that checks order correct.

Embodiment 2: the structure of carrier p18TBHL

The preparation of chemokine 18 peptides

According to the sequence of SLC, synthetic two complementary strands that produce N-terminal 18 peptides of design

etup 5’agcgatggtgcacaggattgctgcctgaaatatagccagcgtaaaattccg3’

Two complementary strand pcr amplifications of etdown 5 ' ctcgagagaaccaccaccaccagaaccaccaccacccggaattttacgctggctat a3 ' obtain 3 ' end and contain the complementary double-stranded of restriction enzyme site XhoI.Concrete operations are with reference to the molecular cloning guide.Reclaim test kit with a small amount of glue and reclaim the PCR product.The PCR product that reclaims carries out the XhoI single endonuclease digestion, and endonuclease reaction reclaims the test kit purifying with a small amount of glue and reclaims the fragment that enzyme is cut with reference to product description.

The processing of carrier pTCMBO

Carrier pTCMBO cuts (available from precious biotechnology (Dalian) company limited, down together) with the NcoI enzyme earlier, mends and puts down, and cuts with the XhoI enzyme again.Concrete operations are as follows: the NcoI enzyme of carrier pTCMBO is cut, and endonuclease reaction reclaims the test kit purifying with a small amount of glue and reclaims the fragment that enzyme is cut with reference to product description.Enzyme is cut the benefit of carrier and is put down: DNA15 μ l PCR damping fluid 3 μ l pfu enzymes 1 μ l dNTP 3 μ lH ₂O 8 μ l, 72 ℃ of 20min of 94 ℃ of 10min reclaim the test kit purifying with a small amount of glue and reclaim the flat fragment of benefit.Mending the plain film section cuts with the XhoI enzyme: endonuclease reaction reclaims the test kit purifying with a small amount of glue and reclaims the fragment that enzyme is cut with reference to product description.

Carrier pTCMBO is connected with ten octapeptides

Fetch the enzyme of receipts and cut the about 40ng of the flat carrier pTCMBO of benefit, the about 20ng of ten octapeptide fragments adds 2 μ l10 * T ₄The dna ligase damping fluid, 1 μ l T ₄Dna ligase adds ddH ₂O is to cumulative volume 20 μ l, mixing, 16 ℃ of connections spend the night (about 16 hours).

(4) conversion of recombinant DNA, the screening of positive colony and order-checking are identified

Plasmid transforms (5) with reference to embodiment 1.Single bacterium colony of growing on the picking flat board adopts alkaline process to extract plasmid DNA in a small amount, carries out PCR and identifies.Primer sequence is as follows:

Upstream primer 5 ' agcgatggtgcacaggattgctgcctgaaatatagccagcgtaaaattccg3 '

Downstream primer 5 ' tgaggagacagtgactgaggttc 3 '

The PCR reaction is with reference to molecular cloning, the PCR product carries out 1% agarose gel electrophoresis, obtain and expect big or small identical segments, the plasmid after identifying is served the order-checking of Hai Boya Bioisystech Co., Ltd identify, identify the correct positive plasmid called after p18TBHL of back order-checking.

Soluble-expression in the low temperature induction born of the same parents of embodiment 3:p18TBHL

(1) with p18TBHL transformed into escherichia coli BL21 (DE3)

According to the method for preparing competent cell described in embodiment 1 (4), preparation e. coli bl21 (DE3) (Invitrogen company) competent cell.Specification sheets according to plasmid extraction kit (Shanghai Hua Shun company) extracts plasmid p18TBHL, carries out transformation experiment according to embodiment 1 (5).

(2) low temperature induction is expressed

The BL21 (DE3) that will contain p18TBHL is coated with the LB-K flat board, 37 ℃ of incubated overnight, and the picking mono-clonal is inoculated in the 5ml LB-K liquid nutrient medium then, in Boiling tube, 37 ℃ of shaking table overnight incubation (200 rev/mins).Get overnight culture next day and be transferred in the 250mlLB-K liquid nutrient medium, continue 37 ℃ of shaking tables and cultivate (200 rev/mins) to A in 1/100 ratio ₆₀₀=0.6, adding IPTG (available from precious biotechnology (Dalian) company limited) is 0.4mmol/ml to final concentration, and continuation was cultivated 4 hours at 30 ℃, carried out low temperature induction and expressed.Room temperature 12, centrifugal 10 minutes of 000rpm collects thalline, is suspended from the damping fluid of 1/5 volume of culture, carries out the thalline ultrasonication.Through further room temperature 12,000m is after centrifugal 10 minutes, and centrifugal supernatant component contains N-terminal ten octapeptides of the chemokine SLC of soluble-expression in the born of the same parents and the fusion rotein of ovarian cancer bispecific antibody.Because ultrasonic supernatant can be directly used in purifying and follow-up external activity experiment, saved the step of sex change and renaturation, save production cost greatly and the time.

Embodiment 4: adopt DEAE anion-exchange column and N-terminal ten octapeptides of Ni affinity column purifying chemokine SLC and the fusion rotein of bispecific antibody

With soluble-expression product room temperature 12 in the low temperature induction born of the same parents of above-mentioned 250ml, centrifugal 10 minutes of 000rpm, collect thalline and be suspended from level pad (the 10mmol/ml NaCl of the DEAE anionite-exchange resin (Pharmacia company) of 1/5 volume (50ml), 50mmol/ml Tris-HCl, pH9.0) in, carry out ultrasonication.Room temperature 12 then, centrifugal 10 minutes of 000rpm, and centrifugal supernatant is directly used in purifying.

20ml DEAE anionite-exchange resin is suspended in the 100ml level pad, adorns post (the magnificent company in Shanghai), the specification of post is: 16mm * 20cm; Use the level pad of 5 column volumes to carry out balance then, flow velocity is 1ml/ minute; With the direct upper prop of centrifugal supernatant of above-mentioned ultrasonic product, last sample flow velocity is 0.25ml/ minute, collects stream and wears component; After collect finishing, (500mmol/ml NaCl, 50mmol/ml Tris-HCl pH9.0) carry out wash-out, and flow velocity is 0.25ml/ minute to use the elution buffer of 2 column volumes (about 40ml); Use 2 column volumes (about 40ml) 0.5mol/L NaOH to clean the post material, 2 column volumes (40ml) 1mol/L NaCl carries out the regeneration of post, and flow velocity is 0.5ml/ minute; Re-use 2 column volumes (40ml) level pad balance columns material at last, flow velocity is to be used for purifying next time in 1ml/ minute.If the long period does not use, must use 20% above aqueous ethanolic solution of 4 column volumes to cross post after, be kept at 4 ℃, in order to avoid post material breed bacteria.

10ml Ni affinity column material (Pharmacia company) is suspended in 50ml level pad (50mMNaH ₂PO ₄150mM NaCl PH8.0) in, adorn post (the magnificent company in Shanghai), the specification of post is: 10mm * 8cm; Level pad with 5 column volumes carries out balance then, and flow velocity is 0.5ml/ minute; Above-mentioned DEAE stream is worn the liquid upper prop, and last sample flow velocity is 0.25ml/ minute, collects stream and wears component, is washed till baseline with balance liquid; Use washings (the 50mM NaH of 2 column volumes ₂PO ₄150mMNaCl PH8.0 20mM imidazoles) washing column is collected scrubbed component; Elutriant (50mM NaH with 2 column volumes ₂PO ₄150mM NaCl PH8.0250mM imidazoles) wash-out is collected elution fraction, uses 2 column volumes (about 20ml) 0.5mol/L NaOH to clean post material, final rinse water.If the long period does not use, must use 20% above aqueous ethanolic solution of 4 column volumes to cross post after, be kept at 4 ℃, in order to avoid post material breed bacteria.

The regeneration of Ni post is at first used 5 column volumes of 0.2M acetic acid (sigma company) washing, the water flushing; Again with 5 column volumes of 0.5M NaOH washing, water flushing; And then close down the Ni ion with 100mM EDTA (ethylenediamine tetraacetic acid (EDTA)) a flat iron plate for making cakes, with enough water flushings; Use 200mM NiSO then ₄3 column volumes of actifier column, final rinse water falls unconjugated Ni ion.

Each component of above-mentioned collection is directly reduced SDS-PAGE, the purification Identification effect.The result of SDS-PAGE shows: through DEAE anionresin and Ni affinity column purifying, can remove the most of foreign protein in the ultrasonic supernatant, use digital image analyzer (U.S. AlphaInnotech company, AlphaImage 2200 Documentation ﹠amp; Analysis system) purity of determining 18TBHL reaches about 90%, the results are shown in Figure 2.Method with immunoblotting detects purified product, and working method shows equally that with reference to molecular cloning degree of purity of production is very high, the results are shown in Figure 3.

Above-mentioned purification of samples need be to PBS (phosphate buffered saline buffer) (8 gram NaCl, 0.2 gram KCl, 1.44 gram Na ₂HPO ₄With 0.24 gram KH ₂PO ₄, transfer pH to 7.4 with HCl, be settled to 1 liter) dialysed overnight (4 ℃).The dialysis volume was 500ml, changed dialyzate one time every 6 hours.Adopt the Bradford method that dialyzed sample is carried out protein quantification, concrete operations according to " fine works molecular biology experiment guide " (Yan Ziying, Wang Hailin is translated, the Jin Dongyan school, 1999, Science Press) carry out.Sample is quantitatively added NaN ₃(Sigma company) is to final concentration 0.05% (W/V), as sanitas; Add trehalose (available from Institute of Microorganism, Academia Sinica) to final concentration 0.15mol/L, as stablizer.It is frozen in-80 ℃ to be packed as 1ml/ part then, stand-by.

Embodiment 5: the chemotactic test

(1) separation of peripheral blood mononuclear cell (PBMC)

The dense white blood that blood station, Red Cross center, Beijing is buied is with the RPMI1640 of 2 times of volumes (GIBCO company, down with) dilution.Add the 3ml lymphocyte separation medium to the 10ml centrifuge tube, then slowly add 6ml blood, keep layering not to be destroyed to same centrifuge tube.Layering appears in centrifugal 25 minutes of 2500rpm, and the upper strata is the blood plasma of dilution, and the middle level is a lymphocyte separation medium, and lower floor is a red corpuscle.Buffy coat at upper strata and middle interlayer is a peripheral blood mononuclear cell.Draw peripheral blood mononuclear cell to the 50ml centrifuge tube, add an amount of RPMI1640 dilution, then 2000rpm is centrifugal 5 minutes.Abandon or adopt supernatant, add an amount of RPMI1640 re-suspended cell, centrifugal 5 minutes of 1800rpm.Abandon or adopt supernatant, cell precipitation is resuspended with an amount of RPMI1640, centrifugal 5 minutes of 1500rpm.Supernatant discarded, cell is resuspended with RPMI1640 (containing 10% FBS (foetal calf serum) (river, Heilungkiang marine life engineering company)), adjusts cell concn to 1 * 10 ⁶, 37 ℃, 5%CO ₂Incubator is cultivated standby.

(2) chemotactic test

Cell migration test in 48 porocyte chemotactic cells, carry out (Neuroprobe, USA).Peripheral blood mononuclear cell (PBMC) suspension culture stimulated 3 days through PHA (20mg/L) respectively in RPMI1640 (containing 10%), and IL-2 (1000kU/L) stimulated 2 days, and fully use the activation back.The lower floor hole adds the various laboratory samples that 30 μ l contain different concns, and the hole, upper strata adds 4 * 10 of 50 μ l ⁶The cell suspension of cell/ml.Hole, upper strata and lower floor hole are that (Neuroprobe USA) separates for the polycarbonate membrane of no Povidone in 5 μ m apertures.Face towards the lower floor hole wraps by 2 hours in room temperature in advance with 5 μ g/ml, four collagen types in advance, uses the aqua sterilisa thorough washing then.Test (contains 5% CO in 37 ℃ of moistening environment ₂) carried out 4 hours.Then the polycarbonate membrane after the test is washed, and is fixing, with Liu solution test kit dyeing (Baso Diagnostic Inc.Taiwan).The cell quantity of 5 visuals field of (160 *) picked at random record chemotactic under high power lens.Calculate chemotactic index (CI), the cell count of chemotactic under chemotactic index=chemokine effect/the move to cell count of control wells.The results are shown in Figure 4.As seen from the figure: N-terminal ten octapeptides of SLC and the fusion rotein of ovarian cancer bispecific antibody have stronger chemotactic ability to peripheral blood lymphocyte.

The antigen-binding activity of N-terminal ten octapeptides of embodiment 6:ELISA method detection chemokine SLC and the fusion rotein of ovarian cancer bispecific antibody

The preparation of Jurkat membrane antigen: collect Jurkat cell (people's acute leukemia lymphoma cell) (available from American type culture collection (American Type Culture Collection, ATCC), sequence number: TIB-152) about 5 * 10 ⁶, 1,000g is suspended in cell precipitation among the 0.5ml PBS after centrifugal 10 minutes, carries out ultrasonication.With ultrasonication liquid 12, the 000rpm room temperature keeps ultrasonic supernatant after centrifugal 10 minutes, and it is quantitative that use Bradford method is carried out protein concentration.Add NaN then ₃To final concentration 0.05% (W/V), as sanitas; Add trehalose (available from Institute of Microorganism, Academia Sinica) to final concentration 0.15mol/L, as stablizer.After to be packed as 100 μ l/ parts frozen in-80 ℃, stand-by.SKOV3 (available from American type culture collection (American TypeCulture Collection, ATCC), sequence number :) and PBL (peripheral blood lymphocyte) with reference to the preparation of Jurkat membrane antigen

The ELISA operation steps:

(1) bag quilt: antigen concentration is respectively 10 μ g/ml SKOV3 membrane antigens; 10 μ g/ml Jurkat membrane antigens; 10ug/ml PBL membrane antigen.Bag is 100 μ l/ holes bag quilts by volume.Bag is cushioned liquid formula: 1.36g Na ₂CO ₃, 7.35g NaHCO ₃, 950ml water uses the NaOH of 1mol/L HCl or 1mol/L to transfer pH9.2, and moisturizing is to 1L.PBS37 ℃ of bag spent the night by 2 hours or 4 ℃ of bags.

After sealing: PBS washes plate 1-2 time, add confining liquid PBSA (PBS-1%BSA (W/V)), 200 μ l/ holes, 37 ℃ were sealed 1-2 hour.

(3) after application of sample: PBS washes plate three times, add purification of samples, hatched 1-2 hour for 37 ℃ in 100 μ l/ holes.Sample using method: as initial concentration, carry out 7 gradients of doubling dilution, each gradient three multiple hole with the fusion rotein of N-terminal ten octapeptides of the purifying chemokine SLC of 20 μ g/ml and bispecific antibody.

(4) add after first antibody: PBST (PBS-0.05%Twen-20 (V/V)) washes plate three times,, hatched 1-2 hour for 37 ℃ with confining liquid 1/1000 dilution self-control rabbit anti-antibody.

(5) add after second antibody: PBST washes plate three times, with the goat anti-rabbit igg (available from magnificent company) of confining liquid 1/1000 dilution mark, hatched 1-2 hour for 37 ℃ in 100 μ l/ holes.

(6) after colour developing: PBST washes plate 5 times, add colour developing liquid (48.6ml 0.1M citric acid, 51.4ml0.2M Na ₂HPO ₄Add water to 1L, pH5.0 is made into substrate buffer solution; Adding 4mg OPD in the 10ml substrate buffer solution is O-Phenylene Diamine ((Sigma company), 15 μ l 30%H ₂O ₂Be made into colour developing liquid), 100 μ l/ holes, room temperature lucifuge colour developing 15-30 minute.

(7) termination reaction: add stop buffer (1mol/L HCl), 50 μ l/ holes.

(8) measurement result: read light absorption value at 490nm.

ELISA the results are shown in Figure 5-7.As seen from the figure: for the SKOV3 membrane antigen, N-terminal ten octapeptides of SLC have the binding ability similar to the ovarian cancer bispecific antibody to the fusion rotein of ovarian cancer bispecific antibody, for Jurkat and PBL membranin, the binding ability of N-terminal ten octapeptides of SLC and the fusion rotein of ovarian cancer bispecific antibody is better than the ovarian cancer bispecific antibody.

Embodiment 7:FACS (flow cytometry) method detects fusion rotein and at first adopts indirect FACS method to detect the combination of fusion rotein to tumour cell SKOV3 with combining of tumour cell is active.

Concrete operations are as follows:

(1) cultivate, collect tumour cell SKOV3: the culture condition of cell is: liquid nutrient medium RPMI1640 (GIBCO company), 10%FBS (river, Heilungkiang marine life engineering company), 5%CO ₂, 37 ℃ of incubators are cultivated.After cell grows to logarithmic phase, collecting cell 5 * 10 ⁵Individual.

(2) with above-mentioned cell 1,000g is after centrifugal 10 minutes, uses the PBS suspension cell, and once more 1,000g is after centrifugal 10 minutes, and cell precipitation is suspended among the PBS that 100 μ l contain 10 μ g/ml fusion roteins and bispecific antibody BHL, places 30 minutes for 4 ℃.Every kind of cell is all set up the homotype contrast that does not add antibody, and following each step of this contrast all operates equally with testing sample.

Behind centrifugal 10 minutes of (3) 1, the 000g, cell precipitation is suspended in 100 μ l contains among the 1/10000 dilution rabbit anti-antibody PBS, continue 4 ℃ and placed 30 minutes.

Behind centrifugal 10 minutes of (4) 1, the 000g, with cell precipitation be suspended in 100 μ l contain 1/1000 the dilution FITC coupling sheep anti-mouse igg (BD company) PBS in, continues 4 ℃ placements 30 minutes.

(5) flow cytometer (BD company, FACS Calibur) detects, and exciting light is 488nm, receives 10,000 cells at every turn.

Above-mentioned experimental result is seen Fig. 8.As seen from the figure: N-terminal ten octapeptides of SLC have similar binding ability embodiment 8:FACS (flow cytometry) method detection fusion rotein active with combining of peripheral blood lymphocyte (PBL) and Jurkat cell with the fusion rotein of ovarian cancer bispecific antibody and ovarian cancer bispecific antibody to the SKOV3 cell

It is active to the combination of peripheral blood lymphocyte (PBL) and Jurkat cell that we also adopt the method for direct FACS to detect fusion rotein.Concrete experimental implementation is with reference to embodiment 7.

Above-mentioned experimental result as shown in Figures 9 and 10.As seen from the figure: to Jurkat and PBL cell, N-terminal ten octapeptides of SLC are stronger than ovarian cancer bispecific antibody with the binding ability of the fusion rotein of ovarian cancer bispecific antibody.

The activity that the fusion protein mediated T lymphocyte of embodiment 9:MTT kills and wounds ovarian cancer cell SKOV3

We are target cell (Target cells) with the ovarian cancer cell SKOV3 with bispecific antibody BHL generation specific combination, with PBL is effector cell (Effect cells), external according to certain effect target than (target cell/effector cell, E/T) mix, add certain density fusion rotein and ovarian cancer bispecific antibody BHL simultaneously, cultivated 48 hours at 37 ℃ then, adopt the MTT method to measure the survival condition of tumour cell again.Detect the activity that the two mediation T lymphocyte kills and wounds ovarian cancer cell SKOV3 with this.Concrete operations are as follows:

(1) extract collection PBMC, concrete operations are identical with embodiment 5 (1).

(2) cultivate collection SKOV3 cell, concrete operations are identical with embodiment 6.

(3) concentration of use RPMI1640 substratum (available from GIBCO company) equalizing effect cell (PBMC) and target cell (SKOV3): fixedly the density of SKOV3 cell is 1 * 10 ⁵Individual/ml, 100 μ l/ holes join in the 96 porocyte culture plates (Nunc company).Add PBL simultaneously, 100 μ l/ holes join in the above-mentioned Tissue Culture Plate, and making and imitating the target ratio is 10.Use the RPMI1640 substratum to adjust the concentration of 18TBHL and BHL simultaneously.Add with 50 μ l/ holes and to be added with in the 96 porocyte culture plates of effector cell and target cell, then at 37 ℃ CO ₂(5%) in the incubator, cultivated 48 hours.Every kind of sample is 4 multiple holes.Imitate target than all setting up the negative control that does not add antibody, the negative control of individual effect cell or target cell and the substratum negative control that does not add any cell for every kind simultaneously.

(4) cell culture medium is discarded after, use PBS (300 μ l/ hole) to wash plate once, add MTT (Sigma company) solution (concentration: 200 μ l/ holes) again, 37 ℃ hatch 4 hours after, use PBS (300 μ l/ hole) to wash plate once, after 30 minutes are hatched in adding DMSO (dimethyl sulfoxide (DMSO), 200 μ l/ holes) (Sigma company) 37 ℃, measure A ₆₀₀

(5) calculation formula of specific killing rate (specific cytolysis) is:

Specific killing rate (%)=[A ₆₀₀(E/T)-A ₆₀₀(E/T/A)]/[A ₆₀₀(E/T)-A ₆₀₀(M)] * 100%

A ₆₀₀(E/T): every kind of A of imitating target than the negative control hole that does not add antibody ₆₀₀Measured value;

A ₆₀₀(E/T/A): the A of every kind of sample ₆₀₀Measured value;

A ₆₀₀(M): the A that does not add the substratum negative control of any cell ₆₀₀Measured value.

The MTT Mortaility results is seen Figure 11.As seen from the figure: the function that N-terminal ten octapeptides of SLC and the fusion protein mediated peripheral blood lymphocyte of ovarian cancer bispecific antibody kill and wound the SKOV3 cell is better than the ovarian cancer bispecific antibody slightly.

Embodiment 9: streaming detects the activity that fusion protein mediated T lymphocyte kills and wounds ovarian cancer cell SKOV3

1. target cell mark PKH26

(1) uses fresh serum free medium (RPMI1640) washing target cell, centrifugal 5 minutes of room temperature 400g.The target cell precipitation is suspended in the resuspended damping fluid of 250 μ l, and piping and druming evenly becomes single cell suspension as far as possible.

(2) with PKH26 250 * the be diluted in resuspended damping fluid (10 of 250 μ l ^-6M)

(3) with after both mixing, piping and druming immediately is even, and room temperature is reversed repeatedly, mixes 2-5 minute.

(4) 1%BSA or the FBS of adding 500ul, abundant mixing, and placed 1 minute.

(5) add the 1ml perfect medium, contain 10%FBS.

(6) room temperature 400g is centrifugal 10 minutes, and it is inferior to use the 1ml perfect medium to give a baby a bath on the third day after its birth.At last with cell suspension in the 1ml perfect medium.

2. fragmentation test and apoptosis detect

(1) use fresh perfect medium (RPMI1640) with diluted sample to finite concentration, join 96 porocyte culture plates at the bottom of the U-shaped with certain volume.

(2) use fresh perfect medium (RPMI1640) that PKH26 mark and unlabelled target cell all are diluted to 10 ⁶Ml, and 10 μ l/ holes join in the U-shaped 96 porocyte culture plates.

(3) use fresh perfect medium (RPMI1640) that the effector cell is diluted to 2 * 10 ⁶/ ml, and join in the 96 porocyte culture plates with 50 μ l/ holes, make and imitate target than being 10:1.

(4) room temperature 250g is centrifugal 5 minutes, hatches 3-4 hour at 37 ℃.

(5) the parallel hole cell is incorporated in the centrifuge tube of a 1.5ml centrifugal 10 minutes of 400g.Cell precipitation uses PBS to wash once, and cell precipitation is suspended in the 100 μ l binding buffer liquid.

(6) (fluorescein isothiocyanate, Fluoresceinisothiocynate), the room temperature cell precipitation is suspended in the 400 μ l binding buffer liquid every pipe adding 3-5ul AnnexinV-FITC.

(7) FACS detects.FITC：FL1，PKH26：FL2。

The result is shown in Figure 12-13.As seen from the figure: the ability that fusion protein mediated peripheral blood lymphocyte kills and wounds ovarian cancer cell SKOV3 is better than the ovarian cancer bispecific antibody slightly.

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Sequence table

＜110〉Beijing Anbote Gene Engineering Co., Ltd.

＜120〉fusion rotein of a kind of chemoattracting small peptide and bispecific antibody

<130>IB055218

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1677

<212>DNA

<213>Artificial

<400>1

<210>2

<211>559

<212>PRT

<213>Artifical

<400>2

Claims (12)

1. the fusion rotein of chemoattracting small peptide and bispecific antibody, wherein said chemoattracting small peptide is N-terminal ten octapeptides of the chemokine SLC of CC family (secondary lymphoid-tissue chemokines).

2. the described fusion rotein of claim 1 is characterized in that bispecific antibody is antitumor * anti-CD3 bispecific antibody.

3. the described fusion rotein of claim 2, it is characterized in that described antitumor * anti-CD3 bispecific antibody is ovarian cancer resistance * anti-CD3 bispecific antibody.

4. the described fusion rotein of claim 1 is characterized in that this fusion rotein is N-terminal ten octapeptides, (GGGGS) by the chemokine SLC of CC family (secondary lymphoid-tissue chemokines) ₂Connection peptides, ovarian cancer resistance * anti-CD3 bispecific antibody are connected in sequence.

5. the described fusion rotein of claim 1, the aminoacid sequence that it is characterized in that described fusion rotein is shown in SEQ ID NO:2.

6. the described fusion rotein of claim 5, wherein said fusion rotein is coded by the nucleotide sequence shown in the SEQ ID NO:1.

7. the described fusion rotein of claim 5, its C-terminal can have C-myc and His ₆Label.

8. expression vector is characterized in that comprising the encoding gene of any one described fusion rotein among the claim 1-7.

9. the described carrier of claim 8, it is p18TBHL.

10. the host cell that contains claim 8 or 9 described carriers.

11. the host cell of claim 10, it is intestinal bacteria.

12. the method for any one described fusion rotein among the purifying claim 1-7 is characterized in that uniting and adopts DEAE anionite-exchange resin and Ni affinity column to carry out purifying.

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