CN111944056A - Apoptosis protein fusion type anti-HER-2 single-chain antibody and preparation method and application thereof - Google Patents

Apoptosis protein fusion type anti-HER-2 single-chain antibody and preparation method and application thereof Download PDF

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CN111944056A
CN111944056A CN202010678663.4A CN202010678663A CN111944056A CN 111944056 A CN111944056 A CN 111944056A CN 202010678663 A CN202010678663 A CN 202010678663A CN 111944056 A CN111944056 A CN 111944056A
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chain antibody
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apoptosis
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CN111944056B (en
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郭军
陆丹丹
郭艺辰
郑子慧
陈佳慧
王婷婷
王敏
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Nanjing University of Chinese Medicine
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Abstract

The invention discloses an apoptosis protein fusion type anti-HER-2 single-chain antibody and a preparation method and application thereof, the apoptosis protein fusion type anti-HER-2 single-chain antibody is formed by coupling anti-HER-2 single-chain antibodies with apoptosis proteins in different serial connection modes, the apoptosis proteins are cytochrome C or DNA fragmentation factors 40, and the apoptosis protein fusion type anti-HER-2 single-chain antibody can be specifically coupled with anti-HER-2 single-chain antibodies into DFF40-scFv fusion type single-chain antibodies and nCytc (serial) -scFv fusion type single-chain antibodies. The apoptosis protein fusion type anti-HER-2 single-chain antibody is inserted into scFv upstream through a cDNA sequence of an apoptosis protein and cloned into an expression vector to construct a recombinant plasmid, the recombinant plasmid is transferred into cells to induce and express the apoptosis protein fusion type anti-HER-2 single-chain antibody, the preparation method is easy to construct and express, and the constructed apoptosis protein fusion type anti-HER-2 single-chain antibody can specifically target malignant tumors with high expression of HER-2 and mediate cancer cell apoptosis and can be used for preparing a medicament for targeted treatment of HER-2 high expression cancers.

Description

Apoptosis protein fusion type anti-HER-2 single-chain antibody and preparation method and application thereof
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to an apoptosis protein fusion type anti-HER-2 single-chain antibody, and a preparation method and application thereof.
Background
Breast cancer is the most common malignant tumor of women all over the world, and the incidence rate of breast cancer all over the world is on the rising trend from the end of the 70 th 20 th century. In 2018, 3, 23 days, the latest data released by the national cancer center shows that new cases of breast cancer of women all over the country account for 16.51 percent of the incidence of malignant tumors of women, and are located at the 1 st of the incidence of the malignant tumors of women. HER-2(Human Epidermal Growth Factor receptor 2, also known as neu, ErBb-2, HER-2) is a member of Epidermal Growth Factor receptor superfamily, is one of the most frequently changed oncogenes in Human tumors, and the gene expression level and the gene copy number of the HER-2 are obviously increased in several Human tumor cells, particularly in breast cancer, ovarian cancer and gastric cancer. Research shows that over 30% of human tumors have HER-2 gene amplification/overexpression (such as breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer and the like), and HER-2 overexpressed breast cancer patients have rapid disease progression, short chemotherapy remission period, poor endocrine treatment effect, disease-free survival and low overall survival rate. The overexpression of HER-2 gene is not only related to the generation and development of tumor, but also is an important target for the selection of tumor-targeted therapeutic drugs.
The vast majority of HER-2 targeted drugs currently in clinical approval have focused on breast cancer, such as lapatinib (tulisamide), trastuzumab (herceptin), T-DM1, pertuzumab. The FDA approved Trastuzumab (Trastuzumab) in 1998 for the treatment of breast cancer with overexpression of HER-2. The trastuzumab Chinese trade name 'herceptin' is an anti-HER-2 monoclonal antibody, prevents the attachment of human epidermal growth factor on HER-2 by attaching the herceptin to HER-2, thereby blocking the growth of cancer cells, and the herceptin can also stimulate the immune cells of the body to destroy the cancer cells. The genetically engineered antibody is a third generation antibody following a polyclonal antibody and a monoclonal antibody, and mainly comprises a chimeric antibody, a humanized antibody, a fully humanized antibody, a single chain antibody (scFv), a bispecific antibody and the like, wherein the single chain antibody (scFv) is a small molecule antibody fragment formed by connecting antibody heavy chain and light chain variable region fragments by using an elastic short peptide. Compared with the intact antibody molecule, the single-chain antibody has the following characteristics: (1) does not contain constant region fragments of antibody molecules, so the immunogenicity is weak, and the anti-mouse antibody can hardly be generated when the antibody is used for a human body; (2) has higher affinity and specificity to corresponding antigen; (3) the relative molecular mass is small, the penetrating power is strong, the stay time in vivo is short, and the method is suitable for immune imaging diagnosis and guided therapy of diseases; (4) because of the relatively small molecular mass, functional antibody molecules can be formed without glycosylation modification, and thus can be expressed in prokaryotic expression systems and are readily available. The anti-HER-2 single-chain antibody has been successfully prepared for targeting tumor, however, the single-chain antibody has poor stability, easy aggregation and short half-life period, and the effect is not obvious.
The single-chain antibody scFv is also used as a drug delivery platform to deliver effector molecules to kill target cells, but the killing functional domain of many existing immunotoxins is mainly derived from plant or bacterial toxins, has too large molecular weight and low tissue permeability, has high immunogenicity and strong side effects, and limits the clinical application of the immunotoxins. In order to solve the problems, a series of endogenous apoptosis-related molecules are reported to replace exogenous toxins, and a large number of literature researches show that cytochrome C (Cytc) and DNA fragmentation factor 40(DFF40) play important roles in apoptosis pathways, cytochrome C released to cytoplasm by Cytc can be combined with apoptosis-related factor 1(Apaf-1) in the presence of dATP to form multimer, caspase-9 is promoted to be combined with the cytochrome C to form apoptotic bodies, caspase-9 is activated, and activated caspase-9 can activate other caspases such as caspase-3 and the like, so as to induce apoptosis; DFF40, upon caspase-3 activation in apoptotic cells, cleaves the nucleosome-internuclear DNA, resulting in a characteristic DNA ladder, and since DFF40 is a downstream component in the apoptotic cascade, its expression and activation will lead to irreversible DNA damage, leading to definitive cell death.
Based on the research, the invention couples the anti-HER-2 single-chain antibody scFv with apoptosis protein DFF40 or nCytc (n is more than or equal to 1), delivers DFF40 or nCytc to target cells through scFv, inhibits the activity of tumor cells by activating an endogenous apoptosis pathway after the cells are internalized, and greatly reduces immunogenicity and toxic and side effects. By comparing the activity difference of different protein constructs, the preparation of the single-chain antibody immune apoptosis with the best anti-tumor activity and the minimum side effect is expected to play an important role in the diagnosis and treatment of the breast cancer.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an apoptosis protein fusion type anti-HER-2 single-chain antibody, a preparation method and application thereof aiming at the defects of the prior art.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows: an apoptosis protein fusion type anti-HER-2 single chain antibody is prepared by coupling anti-HER-2 single chain antibody with apoptosis protein in different series connection modes, wherein the anti-HER-2 single chain antibody comprises a heavy chain variable region VH shown in a sequence SEQ ID NO.1 and a light chain variable region VL shown in a sequence SEQ ID NO.2, and the heavy chain variable region and the light chain variable region are respectively connected with a flexible peptide (G) shown in a sequence SEQ ID NO.34S)3The upstream of VH of the heavy chain variable region contains a 6 × his label, the amino acid sequence of the anti-HER-2 single-chain antibody is shown as SEQ ID NO.4, the cDNA sequence of the anti-HER-2 single-chain antibody is shown as SEQ ID NO.5, and the apoptosis protein is cytochrome C tandem aggregate (nCytc, n is more than or equal to 1) or DNA fragmentation factor 40(DFF 40).
Furthermore, the amino acid sequence of the cytochrome C is shown as SEQ ID NO.6, and the cDNA sequence for coding the cytochrome C is shown as SEQ ID NO. 7.
Furthermore, the amino acid sequence of the DNA fragmentation factor 40 is shown as SEQ ID NO.8, and the cDNA sequence coding the DNA fragmentation factor 40 is shown as SEQ ID NO. 9.
Further, the apoptosis protein fused anti-HER-2 single-chain antibody is a Cytc-scFv fused single-chain antibody, the anti-HER-2 single-chain antibody is coupled with a cytochrome C according to claim 3, the cytochrome C is connected between a 6 x his tag and the anti-HER-2 single-chain antibody, and the amino acid sequence of the Cytc-scFv fused single-chain antibody is shown as SEQ ID NO. 10.
Furthermore, the apoptin-fused anti-HER-2 single-chain antibody is an nCytc-scFv-fused single-chain antibody, the anti-HER-2 single-chain antibody is coupled with a cytochrome C tandem aggregate, the cytochrome C tandem aggregate is connected between a 6 x his tag and the anti-HER-2 single-chain antibody in series, the cytochrome C tandem aggregate is connected by a plurality of cytochrome C through connecting peptides, the connecting peptides are Caspase-3 enzyme cutting sites DEVD, and the cDNA sequence of the DEVD is shown as SEQ ID No. 11.
Further, the apoptin fused anti-HER-2 single-chain antibody is a DFF40-scFv fused single-chain antibody, the anti-HER-2 single-chain antibody is connected with the DNA fragmentation factor 40 of claim 4, the DNA fragmentation factor 40 is connected between a 6 x his tag and the anti-HER-2 single-chain antibody, and the amino acid sequence of the DFF40-scFv fused single-chain antibody is shown as SEQ ID NO. 12.
The invention also provides a preparation method of the apoptosis protein fusion type anti-HER-2 single-chain antibody, which comprises the following steps:
(1) synthesizing a cDNA sequence of the anti-HER-2 single-chain antibody and a cDNA sequence of the apoptosis protein shown in SEQ ID NO. 5;
(2) constructing a recombinant plasmid: respectively connecting the cDNA sequence of the anti-HER-2 single-chain antibody coded in the step (1) and the cDNA sequence of the apoptosis protein coded in the step (1) into a pET-32a (+) expression vector to construct a recombinant plasmid;
(3) transforming the recombinant plasmid obtained in the step (2) into DH5 alpha competent cells, screening recombinant plasmid with correct sequencing into BL21(DE3) competent cells, and inducing and expressing the apoptosis protein fusion type anti-HER-2 single-chain antibody.
Further, in the step (2), the cDNA fragment of the anti-HER-2 single-chain antibody shown in SEQ ID NO.5 and the cDNA fragment of the pET-32a (+) expression vector are digested with restriction enzymes Nco I and BamH I, and then the cDNA fragment of the anti-HER-2 single-chain antibody is ligated between the Nco I and BamH I cleavage sites of the pET-32a (+) expression vector; the apoptosis protein is cytochrome C or DNA fragmentation factor 40, a cDNA fragment of a pET-32a (+) expression vector is cut by restriction enzymes Bgl II and Nco I, a cDNA sequence of Cytc shown in SEQ ID NO.7 or a cDNA sequence of DFF40 shown in SEQ ID NO.9, then the cDNA sequence of Cytc or the cDNA sequence of DFF40 is connected between the Bgl II and Nco I cutting sites of the pET-32a (+) expression vector, wherein the Nco I cutting site used on the pET-32a (+) expression vector when the cDNA fragment coding the anti-HER-2 single-chain antibody is inserted and the cDNA fragment coding the apoptosis protein is inserted is the same cutting site.
Further, when the apoptotic protein is cytochrome C and the number of cytochrome C is more than one, each cytochrome C is connected in series via Caspase-3 cleavage site DEVD as shown in SEQ ID NO. 11.
Further, the method also comprises a step (4) of purifying the apoptosis protein fusion type anti-HER-2 single-chain antibody obtained in the step (4) by using a Ni-NTA column.
The invention also provides application of the anti-HER-2 single-chain antibody or the apoptosis protein fusion type anti-HER-2 single-chain antibody in preparing a medicament for targeted therapy of HER-2 high-expression cancer.
Compared with the prior art, the invention has the beneficial effects that: in preclinical studies, many recombinant immunotoxins bind to different cell surface antigens associated with tumor cells, leading to cell death through toxin-mediated translational inhibition, and are potent anticancer agents. However, the reasons that limit the effectiveness of these molecules in clinically treating solid cancers include mainly low specificity and high toxicity. Toxin molecules in immunotoxins are mainly plant or bacterial toxins, such as pseudomonas exotoxin, staphylococcus aureus enterotoxin, diphtheria toxin, actinomycin, gelonin and the like, and have side effects of possibly causing gastrointestinal toxicity, hepatotoxicity and the like, and in addition, the toxin molecules have large molecular weight, low permeation efficiency, high immunogenicity, short half-life and the like. The invention takes HER-2 high expression type breast cancer surface HER-2 antigen as a molecular target, utilizes the advantages of high specificity, low immunogenicity and the like of a single chain antibody, takes anti-HER-2 single chain antibody scFv as a drug delivery platform, uses endogenous apoptosis related molecules to replace exogenous toxins, and avoids the generation of toxic and side effects by activating an endogenous apoptosis pathway; combining the targeting property of the single-chain antibody with the apoptosis-promoting activity of the apoptosis protein, designing and constructing three apoptosis protein fusion type single-chain antibodies: DFF40-scFv, Cytc-scFv and nCytc-scFv (n >1, taking the example n as an example 3), in vitro and in vivo experiments all prove that the fusion type single-chain antibody of the apoptosis protein can specifically target HER-2 over-expression breast cancer and mediate apoptosis, but has no toxicity to HER-2 negative breast cancer cells, and the activity difference of the three fusion type single-chain antibodies is DFF40-scFv >3Cytc-scFv > Cytc-scFv. To our knowledge, this was the first study to combine the apoptotic protein DFF40 with single chain antibodies for the treatment of HER-2 positive breast cancer, and more importantly, the apoptotic protein fusion-type single chain antibody protein construct (nCytc-scFv) that introduced the specific cleavage site DEVD of the apoptosis-enforcing protein Caspase3, released more effector molecules by enzymatic cleavage, increased antitumor activity, and was an important complement to the strategy of treating tumors via the endogenous apoptotic pathway. The nCytc-scFv is an ideal immune apoptosis molecule discovered for the first time in the research, comprises a skillful virtuous cycle, limits tumor killing to apoptosis while expanding anti-tumor activity, accords with a high-efficiency low-toxicity treatment concept, and is a novel and effective treatment way. In a word, the invention can realize good combination of targeting and anti-tumor activity when the fusion expression of the apoptosis-promoting protein and the single-chain antibody is used for treating the cancer, and provides a new idea for the targeted immunotherapy of the cancer and the research and development of antibody medicaments.
Drawings
FIG. 1 is a schematic diagram of a two-dimensional structure of a single-chain antibody protein;
FIG. 2 is a plasmid map of the recombinant plasmids pET-32a (+) -scFv, pET-32a (+) -DFF40-scFv, pET-32a (+) -Cytc-scFv and pET-32a (+) -3Cytc-scFv constructed in examples 1 and 2;
FIG. 3 shows the results of the protein expression purification identification of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv of example 3, wherein: a is the SDS-PAGE gel electrophoresis result of purified scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv proteins, M: a protein Marker; b is a Western Blot identification result graph of purified scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv proteins, anti-6His tag is used as primary antibody, and Marker and lane settings of B are the same as those of A;
FIG. 4 is an immunofluorescence image of the four single-chain antibody proteins of example 4 (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) incubated with SK-BR-3, MDA-MB-231 and MCF-7, respectively, wherein green is FITC fluorescent secondary antibody labeled single-chain antibody protein (outside the bright color in the figure), blue is DAPI staining core, and the scale bar is 25 μm;
FIG. 5 shows the results of measuring the activity of HER-2 overexpressing breast cancer cells by the four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv, and DFF40-scFv) of example 5, wherein: a is the cytotoxicity data of the single-chain antibody protein with different concentrations on SK-BR-3, B is the cytotoxicity data of the single-chain antibody protein with different concentrations on MDA-MB-231, C is the cytotoxicity data of the single-chain antibody protein with different concentrations on MCF-7, n is 3, compared with a control group, P is less than 0.05, P is less than 0.01, P is less than 0.001, ns: is not significant; d is the result of crystal violet staining after incubation of four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) with MDA-MB-231 and DFF40-scFv with MDA-MB-231 and MCF-7 respectively for 48 h;
FIG. 6 shows the results of detecting Caspase3 protein activity of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion-type single-chain antibodies of example 6 after induction of HER-2 overexpressed breast cancer cells, wherein: a is the detection result of Caspase3 activity after SK-BR-3 respectively interacts with four antibody proteins, B and C are the detection results of Caspase3 activity after MDA-MB-231 and MCF-7 respectively interact with four antibody proteins, n is 5, P is 0.05, P is 0.01, P is 0.001, ns: is not significant;
FIG. 7 is a graph showing the results of analyzing the change in the expression level of apoptosis-related protein by immunoblotting in example 7, in which: a is that after four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) act on SK-BR-3 cells, the content change detection results of total protein, Bcl-2 and Bax are extracted, alpha-tubulin is used as an internal reference, B is the ImageLab quantitative result of a protein band in A, the lower section in each column diagram represents Bcl-2, the upper section represents Bax, and the total amount of the two is 1;
FIG. 8 is a graph showing the results of Hoechst nuclear staining and cell morphology change in example 8, in which: a is a result graph of the cell nucleus fixation condition observed by an inverted fluorescence microscope after four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) are respectively incubated with SK-BR-3 cells for 48 hours, and the color is blue: hoechst; white: a pycnotic nucleus (bright white portion in the figure); b is a cell image obtained by respectively incubating four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) observed by a living cell long-time dynamic image imaging analyzer with SK-BR-3 cells for 0h, 24h and 48 h;
fig. 9 is a statistical analysis of the apoptosis rate of the four single-chain antibody proteins of example 8 (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) after incubation with three breast cancer cells (SK-BR-3, MDA-MB-231, MCF-7) for 48h, wherein n is 3, P <0.05, > P <0.01, > P <0.001, ns: is not significant;
FIG. 10 is a graph of the fusion protein-induced apoptosis assay of example 9 by flow cytometry, wherein: a is the apoptosis result detected by a flow cytometer immediately after the four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) are respectively incubated with SK-BR-3 cells for 48h, Annexin V-FITC/PI double staining and then incubated for 10min in dark, B-D: for quantitative analysis of apoptosis data in a, n-3, with scFv action as control, P <0.05, P <0.01, P <0.001, ns: is not significant;
FIG. 11 is the result of in vivo activity assay of the apoptotic protein fusion-type anti-HER-2 single chain antibody of example 10, wherein: a is a tumor volume change diagram in 30 days after four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) carry out protein drug therapy on a nude mouse xenograft model, the tumor volume is measured once every 3 days and calculated, and B is a tumor tissue shooting image separated from the nude mouse xenograft model of a scFv therapy group, a Cytc-scFv therapy group, a 3Cytc-scFv therapy group and a DFF40-scFv therapy group after the antibody protein drug therapy is finished; c is the tumor tissue mass results isolated from the nude mouse xenograft models of the scFv treated group, Cytc-scFv treated group, 3Cytc-scFv treated group, and DFF40-scFv treated group after the antibody protein drug treatment was completed, with scFv treated group as control, P <0.05, P <0.001, ns: not significant.
FIG. 12 shows Tunel staining of paraffin sections of tumor tissues of example 10, in which apoptotic cells were shown to be dark brown (dark spots in the figure).
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described below by using specific examples.
The experimental procedures used in the examples below are, unless otherwise specified, conventional procedures and the reagents, methods and equipment used are, unless otherwise specified, conventional in the art.
Example 1: construction of pET-32a (+) -scFv recombinant plasmid
First, obtaining of anti-HER-2 single chain antibody scFv homologous recombination gene fragment
1.1 heavy chain variable region VH, light chain variable region VL and Flexible peptide (G)4S)3Acquisition of sequences
Comparing the trastuzumab single-chain antibody sequence and the linker sequence through NCBI and Uniprot database search and literature retrieval, determining the amino acid sequence of the heavy chain variable region VH shown as SEQ ID NO.1, the amino acid sequence of the light chain variable region VL shown as SEQ ID NO.2 and the flexible peptide (G) shown as SEQ ID NO.34S)3The amino acid sequence of (a);
heavy chain variable region VH sequence (SEQ ID No. 1): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, respectively;
light chain variable region VL sequence (SEQ ID No. 2): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR, respectively;
flexible peptide (G)4S)3Sequence (SEQ ID NO. 3): GGGGSGGGGSGGS;
the amino acid sequence (VH-linker-VL) of the anti-HER-2 single-chain antibody is shown in SEQ ID NO.4 (the two-dimensional structure of the anti-HER-2 single-chain antibody scFv is shown in FIG. 1, and a 6 × his tag is contained at the upstream of the VH of the heavy chain variable region for nickel column purification), and the cDNA sequence for coding the anti-HER-2 single-chain antibody scFv is shown in SEQ ID NO. 5;
SEQ ID NO.4 specifically is: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR underlined is a flexible peptide (G)4S)3A sequence;
SEQ ID NO.5 specifically is: 5'-GAAGTTCAGCTGGTTGAATCTGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCGCTGCTTCTGGTTTCAACATCAAAGACACCTACATCCACTGGGTTCGTCAGGCTCCGGGTAAAGGTCTGGAATGGGTTGCTCGTATCTACCCGACCAACGGTTACACCCGTTACGCTGACTCTGTTAAAGGTCGTTTCACCATCTCTGCTGACACCTCTAAAAACACCGCTTACCTGCAGATGAACTCTCTGCGTGCTGAAGACACCGCTGTTTACTACTGCTCTCGTTGGGGTGGTGACGGTTTCTACGCTATGGACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCTTCTGGGGGCGGGGGCTCTGGGGGCGGGGGCTCTGGGGGCGGGGGCTCTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAACACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGACCTTTGGCCAGGGCACCAAAGTGGAAATTAAACGC-3', respectively;
1.2 Polymerase Chain Reaction (PCR) acquisition of scFv genes for homologous recombination
Designing and synthesizing PCR primers scFv-F (shown as SEQ ID NO. 14) and scFv-R (shown as SEQ ID NO. 15) of scFv gene sequence homologous recombination with pET-32a (+) vector according to cDNA sequence of scFv and sequence on pET-32a (+) vector, synthesizing nucleotide fragment containing restriction enzyme sites Nco I and BamH I and complementary base by chemical synthesis method, and amplifying homologous recombination sequence of scFv by primers scFv-F and scFv-R
scFv-F(SEQ ID NO.14)：5’-CGACGACGACGACAAGGCCATGGCTGAAGTTCAGCTGGTTGAATCTGG-3', wherein the pET-32a (+) vector homology sequence is underlined and the restriction enzyme site Nco I is in italics;
scFv-R(SEQ ID NO.15)：5’-CGACGGAGCTCGAATTCGGATCCTTAGCGTTTAATTTCCACTTTGGTGC-3', wherein the homologous sequence of the pET-32a (+) vector is underlined and the restriction site BamH I is in italics;
Figure BDA0002585061130000081
the PCR product is a single-chain antibody scFv gene fragment with both ends containing vector homologous sequences and enzyme cutting sites, and is used for carrying out homologous recombination with the enzyme-cut pET-32a (+) vector.
Secondly, construction of pET-32a (+) -scFv recombinant plasmid
The scFv homologous recombination gene fragment recovered from the gel is connected with pET-32A (+) plasmid after double enzyme digestion (Nco I and BamH I) through homologous recombination, the connection product is transformed into E.coli DH5 alpha, recombinant plasmid pET-32A (+) -scFv is extracted after the amplification culture of bacterial liquid and sent to the marine engineering biology company Limited for sequencing, and the result shows that the sequence inserted into the positive recombinant plasmid pET-32A (+) -scFv is consistent with the experimental design, thereby successfully constructing and obtaining the recombinant plasmid pET-32A (+) -scFv (as shown in figure 2A).
Example 2: construction of recombinant plasmids of pET-32a (+) -Cytc-scFv, pET-32a (+) -3Cytc-scFv and pET-32a (+) -DFF40-scFv
First, experimental material
1. Primers for PCR
Searching and determining a cytochrome C (Cytc) amino acid sequence shown as SEQ ID NO.6 and a cDNA sequence which is shown as SEQ ID NO.7 and codes the cytochrome C through NCBI and Uniprot databases; determining the amino acid sequence of the DNA fragmentation factor 40(DFF40) shown as SEQ ID NO.8 and the cDNA sequence of the DNA fragmentation factor 40 shown as SEQ ID NO.9 by NCBI and Uniprot database search; PCR primers Cytc-F and Cytc-R for amplifying the gene sequence of Cytc for homologous recombination with pET-32a (+) vector are designed and synthesized based on the cDNA sequences of Cytc and DFF40 and the sequence on pET-32a (+) vector; and PCR primers DFF40-F and DFF40-R for amplifying gene sequences of DFF40 for homologous recombination with the vector, wherein the specific sequence information is as follows:
amino acid sequence of cytochrome C (SEQ ID NO. 6): MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE, respectively;
cDNA sequence of cytochrome C (SEQ ID NO. 7): 5'-AGATCTCATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCACGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACACAGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGACCATGG-3', respectively;
a forward primer Cytc-F sequence for amplifying a Cytc homologous recombination sequence (SEQ ID No. 16): 5' -CCAGCACATG GACAGCCCAGATCTCATGGGTGATGTTGAGAAAGGC-3', wherein the homologous sequence of the pET-32a (+) vector is underlined and the cleavage site BglII is in italics;
a reverse primer Cytc-R sequence for amplifying a Cytc homologous recombination sequence (SEQ ID No. 17): 5' -CAACCAGCTG AACTTCAGCCATGGTCTCATTAGTAGCTTTTTTGAGATAAGC-3', wherein the pET-32a (+) vector homology sequence is underlined and the restriction enzyme site Nco I is in italics;
amino acid sequence of DFF40 (SEQ ID NO. 8): MLQKPKSVKLRALRSPRKFGVAGRSCQEVLRKGCLRFQLPERGSRLCLYEDGTELTEDYFPSVPDNAELVLLTLGQAWQGYVSDIRRFLSAFHEPQVGLIQAAQQLLCDEQAPQRQRLLADLLHNVSQNIAAETRAEDPPWFEGLESRFQSKSGYLRYSCESRIRSYLREVSSYPSTVGAEAQEEFLRVLGSMCQRLRSMQYNGSYFDRGAKGGSRLCTPEGWFSCQGPFDMDSCLSRHSINPYSNRESRILFSTWNLDHIIEKKRTIIPTLVEAIKEQDGREVDWEYFYGLLFTSENLKLVHIVCHKKT THKLNCDPSRIYKPQTRLKRKQPVRKRQ, respectively;
cDNA sequence of DFF40 (SEQ ID NO. 9): 5'-ATGCTCCAGAAGCCCAAGAGCGTGAAGCTGCGGGCCCTGCGCAGCCCGAGGAAGTTCGGCGTGGCTGGCCGGAGCTGCCAGGAGGTGCTGCGCAAGGGCTGTCTCCGCTTCCAGCTCCCTGAGCGCGGTTCCCGGCTGTGCCTGTACGAGGATGGCACGGAGCTGACGGAAGATTACTTCCCCAGTGTTCCCGACAACGCCGAGCTGGTGCTGCTCACCTTGGGCCAGGCCTGGCAGGGCTATGTGAGCGACATCAGGCGCTTCCTCAGTGCATTTCACGAGCCACAGGTGGGGCTCATCCAGGCCGCCCAGCAGCTGCTGTGTGATGAGCAGGCCCCACAGAGGCAGAGGCTGCTGGCTGACCTCCTGCACAACGTCAGCCAGAACATCGCGGCCGAGACCCGGGCTGAGGACCCGCCGTGGTTTGAAGGCTTGGAGTCCCGATTTCAGAGCAAGTCTGGCTATCTGAGATACAGCTGTGAGAGCCGGATCCGGAGTTACCTGAGGGAGGTGAGCTCCTACCCCTCCACGGTGGGTGCGGAGGCTCAGGAGGAATTCCTGCGGGTCCTCGGCTCCATGTGCCAGAGGCTCCGGTCCATGCAGTACAATGGCAGCTACTTCGACAGAGGAGCCAAGGGCGGCAGCCGCCTCTGCACACCGGAAGGCTGGTTCTCCTGCCAGGGTCCCTTTGACATGGACAGCTGCTTATCAAGACACTCCATCAACCCCTACAGTAACAGGGAGAGCAGGATCCTCTTCAGCACCTGGAACCTGGATCACATAATAGAAAAGAAACGCACCATCATTCCTACACTGGTGGAAGCAATTAAGGAACAAGATGGAAGAGAAGTGGACTGGGAGTATTTTTATGGCCTGCTTTTTACCTCAGAGAACCTAAAACTAGTGCACATTGTCTGCCATAAGAAAACCACCCACAAGCTCAACTGTGACCCAAGCAGAATCTACAAACCCCAGACAAGGTTGAAGCGGAAGCAGCCTGTGCGGAAACGCCAG-3', respectively;
forward primer for amplification of DFF40 DFF40-F sequence (SEQ ID NO. 18): 5' -CCAGCACATGGACAGCCCAGATCTCATGCTCCAGAAGCCCAAGAGC-3', wherein the homologous sequence of the pET-32a (+) vector is underlined and the cleavage site BglII is in italics;
reverse primer DFF40-R sequence for amplification of DFF40 (SEQ ID NO. 19): 5' -CAACCAGCTGAACTTCAGCCATGGT CTGGCGTTTCCGCACAGGCTG-3', wherein the pET-32a (+) vector homology sequence is underlined and the restriction enzyme site Nco I is in italics;
2. plasmid: pET-32a (+) E.coli expression vectors were purchased from Addgene plasmid libraries;
second, Experimental methods
1. Obtaining a cytochrome C homologous recombination gene segment: synthesizing a nucleotide fragment with enzyme cutting sites Bgl II and Nco I and supplementary bases added at two ends of a cDNA sequence of Cytc shown in SEQ ID NO.7 by a chemical synthesis method, and then amplifying a Cytc homologous recombination sequence by primers Cytc-F and Cytc-R;
2. obtaining of fragment of homologous recombination gene of DFF 40: synthesizing a nucleotide fragment with enzyme cutting sites Bgl II and Nco I and supplementary bases added at two ends of a cDNA sequence of DFF40 shown in SEQ ID NO.9 by a chemical synthesis method, and then amplifying a DFF40 homologous recombination sequence by primers DFF40-F and DFF 40-R;
3. construction of pET-32a (+) -Cytc-scFv recombinant plasmid
Connecting the Cytc homologous recombination gene with pET-32a (+) -scFv recombinant plasmid subjected to double enzyme digestion (Bgl II and Nco I) through homologous recombination, and transforming a connection product to E.coli DH5 alpha; after the recombinant bacteria are expanded and cultured, the recombinant bacteria are sent to Shanghai biological engineering company Limited for sequencing, the result shows that the positive recombinant plasmids are completely consistent with the expected cDNA sequence, the amino acid sequence of the inserted Cytc-scFv fusion type single-chain antibody is shown as SEQ ID NO.10, and the recombinant plasmids pET-32a (+) -Cytc-scFv (shown in figure 2) are constructed.
SEQ ID NO.10 specifically is: MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNETMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKR
4. Construction of pET-32a (+) -DFF40-scFv recombinant plasmid
Connecting the homologous recombination gene of DFF40 with pET-32a (+) -scFv recombinant plasmid subjected to double enzyme digestion (Bgl II and Nco I) through homologous recombination, and transforming a connection product into E.coli DH5 alpha; sequencing, wherein the result shows that the positive recombinant plasmid is completely consistent with the expected cDNA sequence, the amino acid sequence of the inserted DFF40-scFv fusion type single-chain antibody is shown as SEQ ID NO.12, and the recombinant plasmid pET-32a (+) -DFF40-scFv (shown in figure 2) is constructed;
SEQ ID NO.12 specifically is: MLQKPKSVKLRALRSPRKFGVAGRSCQEVLRKGCLRFQLPERGSRLCLYEDGTELTEDYFPSVPDNAELVLLTLGQAWQGYVSDIRRFLSAFHEPQVGLIQAAQQLLCDEQAPQRQRLLADLLHNVSQNIAAETRAEDPPWFEGLESRFQSKSGYLRYSCESRIRSYLREVSSYPSTVGAEAQEEFLRVLGSMCQRLRSMQYNGSYFDRGAKGGSRLCTPEGWFSCQGPFDMDSCLSRHSINPYSNRESRILFSTWNLDHIIEKKRTIIPTLVEAIKEQDGREVDWEYFYGLLFTSENLKLVHIVCHKKTTHKLNCDPSRIYKPQTRLKRKQPVRKRQTMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR
5. Construction of pET-32a (+) -3Cytc-scFv recombinant plasmid
Connecting three sections of Cytc gene fragments by using a Caspase-3 enzyme cutting site DEVD sequence shown as SEQ ID NO.11 to form a 3Cytc gene fragment, obtaining a 3Cytc homologous recombinant gene according to a method similar to the Cytc, then connecting the 3Cytc homologous recombinant gene with pET-32a (+) -scFv recombinant plasmids subjected to double enzyme cutting (Bgl II and Nco I) through homologous recombination, and transforming a connection product to E.coli DH5 alpha; sequencing, wherein the result shows that the positive recombinant plasmid is completely consistent with the expected cDNA sequence, the amino acid sequence of the inserted 3Cytc-scFv fusion type single-chain antibody is shown as SEQ ID NO.13, and the recombinant plasmid pET-32a (+) -3Cytc-scFv (figure 2) is constructed;
cDNA sequence of DEVD (SEQ ID NO. 11): 5'-GATGAAGTTGAT-3', respectively;
SEQ ID NO.13 specifically is: MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNEDEVDMGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNEDEVDMGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNETMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR, wherein DEVD is underlined;
furthermore, the number of the apoptosis proteins Cytc is not limited to 1 or 3, but may be 2 or more than 3, each Cytc is connected with the DEVD sequence of the Caspase-3 cleavage site, and the cDNA sequence of the DEVD is shown in SEQ ID NO. 11.
Example 3: inducible expression and identification of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv proteins
First, experimental material
Competent cells of E.coli expressing strains BL21(DE3) were purchased from Beijing Tiangen Biochemical technology; isopropyl-L-thio- β -D-galactopyranoside (IPTG) was purchased from among Aladdin; NI-NTA Resin was purchased from Beijing Quanyujin Biotech;
second, Experimental methods
1. Inducible expression of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv proteins
The positive plasmid pET28a-scFv obtained in example 1 and the positive plasmids pET-32a (+) -Cytc-scFv, pET-32a (+) -3Cytc-scFv and pET-32a (+) -DFF40-scFv obtained in example 2 were transformed into E.coli expression strain competent cell BL21(DE3), respectively, expression of the proteins scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv was induced by IPTG induction and corresponding condition search, and the proteins were purified by NI-NTA Resin affinity chromatography, and the two-dimensional structures of the proteins scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv are shown in FIG. 1.
2. Identification of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv proteins
The purified scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv proteins were subjected to SDS-PAGE for detection: mixing the protein solution with SDS-PAGE sample buffer solution at a certain proportion, heating in metal bath for 10min, cooling to room temperature, loading, and performing 100V electrophoresis for 70 min; after finishing, cutting off the gel, immersing the gel into Coomassie brilliant blue staining solution for dyeing for 25min, then decoloring for 3h by using a decoloring solution in a shaking table at room temperature, and identifying the purification effect by using the molecular weight position indicated by a marker and whether more impurity bands exist in the background of a corridor;
western Blot analysis was performed on the purified scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv proteins: preparing a protein sample by boiling denaturation as described above, loading the cooled sample into a gel hole, performing electrophoresis at 100V for 70min, cutting off the gel at a corresponding position according to an indicator band of a marker after the electrophoresis is finished, transferring the protein onto a PVDF membrane by using a bio-rad semi-dry transfer instrument, sealing with 5% skimmed milk powder for 1h, and then incubating at 4 ℃ overnight by taking a murine 6His Tag antibody as a primary antibody; the following day, the membrane was washed with PBST at pH7.6, followed by incubation at room temperature for 2h with goat anti-mouse IgG-FITC as a secondary antibody, followed by exposure using an enhanced chemiluminescence kit.
Third, results and analysis
The correctly sequenced recombinant plasmids pET-32a (+) -scFv, pET-32a (+) -Cytc-scFv and pET-32a (+) -DFF40-scFv are respectively transformed into BL21(DE3) competent cells, recombinants integrated with plasmids are screened out on an LB solid culture plate by ampicillin, the expression of fusion genes is controlled by a phage T7 promoter on pET32a (+), induction is carried out by using a lactose operon, the bacteria are cultured by using a medicine bottle at 37 ℃, then IPTG is added, the optimal induction condition is determined to be 0.5mM IPTG by a controlled variable method, and the reaction is carried out for 12 hours at 20 ℃.
In order to obtain scFv proteins and Cytc-scFv, 3Cytc-scFv and DFF40-scFv recombinant proteins for protein identification, bacterial precipitates of pET-32a (+) -scFv/BL21(DE3), pET-32a (+) -Cytc-scFv/BL 21(DE3), pET-32a (+) -3 Cytc-scFv/BL 21(DE3) and pET-32a (+) -DFF 40-scFv/BL 21(DE3) under the condition of inducing reaction at 20 ℃ for 12h at 0.5mM IPTG were subjected to ultrasonic disruption, supernatant of the disrupted suspension was taken to purify proteins by NI-NTA Resin affinity chromatography, and then eluted by eluates with different imidazole concentrations, and the target proteins were mainly eluted in an eluent of 100mM imidazole.
The purification effect of the protein was verified by SDS-PAGE, and as shown in FIG. 3A, the molecular weights of the scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv proteins were scFv: 46kDa, DFF 40-scFv: 84kDa, Cytc-scFv: 58kDa, 3 Cytc-scFv: 83kDa, and after Coomassie brilliant blue staining, each protein has a band at a corresponding position according to different molecular weights, and the result is consistent with an expected result, and the background is clean and has no foreign protein, which indicates that the purification effect is better.
Further, protein expression verification of scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv was carried out by Western Blot assay, and as shown in FIG. 3B, bioluminescence imaging was carried out after antigen-antibody binding, and the results were consistent with the results of gel staining, and bands were detected at the corresponding positions.
The above results indicate that the recombinant proteins scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv have been successfully induced to express and purify.
Example 4: activity measurement of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion type single-chain antibody against breast cancer cells in vitro
First, experimental material
Human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were purchased from American type culture Collection (American type culture Collection) ATCC; the 6His Tag antibody was purchased from Shanghai assist in san Francisco Biotech; goat anti-mouse IgG-FITC was purchased from Kyoto Chimona Jinqiao Biotechnology, Inc. of Beijing.
Second, immunofluorescence experiment
1. Grouping experiments: HER-2 high expressing cells SK-BR-3 as experimental group, HER-2 negative cells MDA-MB-231 and MCF-7, used as control, four antibody constructs (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) were incubated with three cells for 30min at 37 ℃.
2. And (3) experimental operation:
(1) cell preparation: respectively inoculating the three breast cancer cells into a 12-hole plate culture dish which is pre-placed with a sterile slide, respectively adding 5ug/ml of different antibodies after the cells adhere to the wall, and adding the different antibodies into the culture dish in 5% CO2Culturing at 37 deg.C for 30min in incubator;
(2) fixing: absorbing the culture medium, washing the cells for three times by PBS buffer solution, fixing the cells by using fresh 4% paraformaldehyde, shaking the cells for 30min at room temperature, and moving the cells to a microscope to observe whether the cells are completely fixed or not after the completion of the shaking;
(3) permeability: the fixative was aspirated off and the fixed cells were washed 3 times with PBS for 5min each. The cells were permeabilized in PBST solution (PBS + 0.1% Triton X-100) at 4 ℃ for 10min in a refrigerator, and after completion, the cells were washed 3 times with PBS for 5min each time;
(4) and (3) sealing: adding 5% BSA blocking solution, shaking the mixture at room temperature for 30min, and blocking the non-specific binding sites;
(5) primary antibody binding: the primary antibody is diluted by using a murine 6His Tag antibody according to the dilution ratio of the if experiment specified by the antibody specification, and the primary antibody dilution is added and incubated for 2h at room temperature or overnight at 4 ℃. PBS wash 3 times, each time for 5 min;
(6) and (3) binding of a secondary antibody: using goat anti-mouse IgG-FITC as the secondary antibody, incubating for 2h at room temperature in a dark place, and washing for 5min with PBS 3 times;
(7) dyeing the core: adding DAPI staining solution, combining in dark for 10min, washing with PBS for 5min for 3 times;
(8) sealing and detecting: and (3) dripping a drop of the sealing tablet on the glass slide, taking out the climbing film, contacting the cell surface with the sealing tablet to seal the climbing film, and observing and taking an image by a confocal fluorescence microscope after the climbing film is dried and fixed.
Third, results and analysis
When observed by using 630-fold confocal microscopy, as shown in FIG. 4, independent scFv or apoptosis protein fusion type single-chain antibodies DFF40-scFv, Cytc-scFv and 3Cytc-scFv are combined to the surface of SK-BR-3 cells, in contrast, no green target protein is observed on the surfaces of HER-2 negative cells MDA-MB-231 and MCF-7, namely, scFv and apoptosis protein fusion type scFv are combined with HER-2 overexpression type breast cancer cells SK-BR-3 surface HER-2 antigen specifically; therefore, the anti-HER-2 single-chain antibody scFv prepared by a prokaryotic expression system specifically binds to a breast cancer cell surface antigen HER-2, has equivalent targeting activity with a monoclonal antibody, and has no damage on the binding activity after being coupled with an apoptosis protein DFF40 or Cytc.
Example 5: validation of Effect of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion type Single chain antibody on cell viability of HER-2 overexpression type breast cancer
First, experimental material
CCK-8 cell viability assay kit was purchased from Sigma, USA; the human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were derived from the same sources as in example 4; the crystal violet staining solution was purchased from beijing solibao; 4% paraformaldehyde from Sigma, usa;
second, Experimental methods
The CCK-8 cell viability detection kit contains WST-8, and in the presence of an electron carrier 1-Methoxy PMS, intracellular dehydrogenase reduces the WST-8 into water-soluble orange formazan dye, the water-soluble orange formazan dye can be dissolved in a culture medium and can detect absorbance at 450nm through an enzyme labeling instrument, and the generation amount of the water-soluble orange formazan dye is in direct proportion to the number of living cells. The crystal violet staining solution is an alkaline staining solution, can stain the cell nucleus into dark purple, and is commonly used for cell counting and cell proliferation analysis. The invention determines the in vitro cytotoxicity effect of the immune cell apoptosis protein by performing CCK-8 analysis and cell proliferation analysis on SK-BR-3, MDA-MB-231 and MCF7 cell lines.
1. Cell viability assay
Experimental grouping: HER-2 high expression type cell SK-BR-3 is used as an experimental group, HER-2 negative cells MDA-MB-231 and MCF-7 are used as a control, and the in vitro cytotoxicity effect of the immune cell apoptosis protein is determined by performing CCK-8 analysis and cell proliferation analysis on SK-BR-3, MDA-MB-231 and MCF7 cell lines.
(II) cell viability detection:
(1) at 104～105Cell/well Density cells were seeded in 96-well plates at 37 ℃ with 5% CO2Culturing the cells in an incubator for 24 hours;
(2) washing the cells with PBS, arranging blank wells (culture medium + CCK8, without cells and proteins), control wells (cells + culture medium + CCK8, without proteins) and experimental wells (cells + culture medium + CCK8+ proteins), adding target proteins with different concentrations into the experimental wells in groups, and continuously incubating for 48h in an incubator; (3 auxiliary holes per set)
(3) A10 μ LCCK-8 solution was added to each well of the plate using a line gun.
(4) Incubating for 1-4 hours at 37 ℃ until the color changes obviously;
(5) taking out the plate, coating with tinfoil and keeping out of the sun, and gently mixing on an oscillator for 1min at room temperature to ensure uniform color distribution;
(6) immediately measuring the absorbance value of each hole at 450nm by using a microplate reader;
(7) cell viability calculation formula: cell viability ═ [ (As-Ab)/(Ac-Ab) ] × 100% (As for experimental wells, Ac for control wells, Ab for blank wells).
2. Crystal violet staining test
(1) At 104～105Cell/well Density cells were seeded in 6-well plates at 37 ℃ with 5% CO2Culturing the cells in an incubator for 24 hours;
(2) washing cells with PBS, adding apoptosis protein fusion type single-chain antibody proteins (Cytc-scFv, 3Cytc-scFv and DFF40-scFv) with different concentrations into each experimental group by taking the scFv acting group as a control group, and continuously incubating for 48h in an incubator;
(3) sucking out the culture medium, washing twice with PBS, fixing with 4% paraformaldehyde shaking table for 10min at room temperature, washing with distilled water for 2min, switching to fresh distilled water, and washing for 2 min;
(4) adding 1ml of 0.1% crystal violet staining solution into each well, incubating for 10min, washing with distilled water, and observing and photographing.
Third, results and analysis
1. Cell viability assay experiment 10-2
As shown in FIG. 5A, four antibody constructs (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) were incubated with SK-BR-3 cells at a concentration gradient of 0.01 to 1000nM, and it was found that compared with scFv, the apoptotic protein fusion type single-chain antibodies Cytc-scFv, 3Cytc-scFv and DFF40-scFv were effective in suppressing the activity of SK-BR-3 cells having the highest HER-2 expression level, and the suppression effect was concentration-dependent. Cell viability was significantly reduced under stimulation with the same concentration of protein at 100 nM: the toxicity of the DFF40-scFv is highest, the cell viability is reduced by about 80%, and the 3Cytc-scFv is secondly used, while the Cytc-scFv is low in toxicity and is only about half of that of the 3Cytc-scFv, and the 3Cytc-scFv is obviously increased in toxicity due to the release of a plurality of Cytc. Furthermore, it was observed that the cell viability of MDA-MB-231 and MCF7 cells expressing low levels of HER-2 was hardly affected compared to SK-BR-3 cells (FIG. 5B, FIG. 5C), which clearly indicates that the presence of higher levels of HER-2 on the cell surface is essential for the specific cytotoxicity of the apoptotic protein fusion type single chain antibody.
2. Crystal violet staining test
The HER-2 high-expression cell SK-BR-3 is stimulated by using four antibody constructs (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv), and similarly, the cell number of the SK-BR-3 cell after the scFv action is the largest and the cell proliferation activity is basically not influenced as can be seen from the cell staining result shown in FIG. 5D; compared with scFv, the cell colony numbers of the apoptosis protein fusion type single-chain antibodies Cytc-scFv, 3Cytc-scFv and DFF40-scFv are obviously reduced, which indicates that the fusion proteins Cytc-scFv, 3Cytc-scFv and DFF40-scFv can obviously inhibit cell growth, and the activity difference is consistent with the cell viability detection experiment result: the number of cells is the least after the DFF40-scFv acts, and the cytotoxicity is the strongest; the number of 3Cytc-scFv cells is less than that of Cytc-scFv cells, and the activity is better; in addition, both control cells MDA-MB-231 and MCF7 were stimulated with DFF40-scFv and found to be unaffected in cell proliferation activity.
In summary, we can obtain: due to the presence of scFv, DFF40 or Cytc fusion proteins (Cytc-scFv, 3Cytc-scFv and DFF40-scFv) are specifically toxic to cells that highly express HER-2.
Example 6: detection of Caspase3 protein activity of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion type single-chain antibody after HER-2 over-expression type breast cancer cell induction
First, experimental material
Caspase3 activity detection kit purchased from Nanjing institute of bioengineering; the human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were derived from the same sources as in example 4;
secondly, detecting the activity of Caspase3 protein
Caspase3 is an apoptosis executive protein, playing a key role in the apoptotic pathway. The detection kit for Caspase3 activity is characterized in that a polypeptide (Ac-DEVD-PNA) with Caspase3 sequence specificity is coupled to a yellow group pNA: when the substrate is cut by Caspase3, the yellow group pNA is released, and the yellow group pNA has strong absorption near 405nm and can be detected by a microplate reader, so that the activation degree of Caspase3 can be indirectly examined. Furthermore, the specific cleavage site DEVD of the apoptosis-performing protein Caspase3 introduced into the 3Cytc-scFv protein construct, and activation and cleavage of Caspase3 released more Cytc from the fusion protein once internalized by the cell.
The invention respectively stimulates SK-BR-3, MDA-MB-231 and MCF7 cell lines through scFv, Cytc-scFv, 3Cytc-scFv or DFF40-scFv fusion type single-chain antibodies, and detects the activity of the induced Caspase3 protein by adopting a Caspase3 activity detection kit, and the specific steps are as follows:
(1) after the cells are stimulated by antibody protein, the cells are digested by pancreatin and centrifuged at 800g for 5min to be collected;
(2) washing the cell precipitate once with PBS, centrifuging as above, removing supernatant, adding lysis solution at a ratio of 50 μ l lysis solution per 200 ten thousand cells, resuspending the precipitate, performing ice bath lysis for 3min, and performing vortex oscillation for 3-4 times (10 s each time) or freeze thawing for 2-3 times;
(3) centrifuge at 12000rpm for 15min at 4 ℃ and carefully pipette the supernatant into a fresh clean centrifuge tube. Setting a control group and an experimental group according to the requirements of a Caspase3 activity detection kit, and respectively adding corresponding amounts of reaction liquid, lysate, a sample to be detected, Caspase3 enzyme digestion substrate and the like, wherein the reaction system is shown in Table 1;
TABLE 1
Figure BDA0002585061130000181
(4) Uniformly mixing the reaction system, continuously incubating in an incubator for 4 hours, and measuring the absorbance value at 405nm when the color change is obvious;
(5) by ODExperimental group/ODControl groupTo reflect the extent of Caspase3 activation.
Third, results and analysis
As shown in FIG. 6A, after the apoptosis protein fusion type scFv (Cytc-scFv, 3Cytc-scFv and DFF40-scFv) and SK-BR-3 cells were incubated together, the Caspase3 protein activity was significantly enhanced, and compared with scFv (scFv) alone, the Caspase3 activation processes after the action of DFF40-scFv, Cytc-scFv and 3Cytc-scFv were respectively 6 times, 3 times and 4.5 times; however, for HER-2 negative cells MDA-MB-231 and MCF-7, no activation of Caspase3 protein was detected following the action of all antibody constructs (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) (FIG. 6B, FIG. 6C).
The results show that the single scFv has no apoptosis-promoting activity, and the fusion expression of the scFv and apoptosis-related proteins DFF40 and Cytc can effectively induce HER-2 overexpression type breast cancer cell apoptosis in vitro.
Example 7: determination of ratio of apoptosis inhibitory protein Bcl-2 to apoptosis promoting protein Bax after induction of HER-2 overexpression type breast cancer cells SK-BR-3 by scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion type single-chain antibody
First, experimental material
BCA concentration detection kits were purchased from american CST; the human breast cancer cell line SK-BR-3 was derived from example 4;
second, analysis of expression level of apoptosis protein
The Bcl-2 gene is called B-lymphocytoma-2, and the Bcl-2 family is one of the most important regulatory factors in regulating apoptosis-related genes. Bcl-2 and Bax are both Bcl-2 protein family members, the former being anti-apoptotic proteins and the latter pro-apoptotic proteins, both of which have been shown to play a major role in the mitochondrial apoptotic pathway. Bcl-2 regulates cell death by controlling mitochondrial membrane permeability, inhibits caspase activity by preventing cytochrome C release from mitochondria and/or binding to apoptosis-activating factor (Apaf-1), thereby inhibiting apoptosis; bax can induce cytochrome C release and promote activation of Caspase3, so that apoptosis is formed. Bcl-2 can form a dimer with Bax, and if the Bax expression level is higher than that of Bcl-2, apoptosis is promoted; and if the expression level of Bcl-2 is higher than that of Bax, the apoptosis is inhibited. Therefore, we can judge the occurrence of apoptosis by detecting the expression changes of Bcl-2 and Bax and comparing the ratio changes of the Bcl-2 and Bax.
1. Total protein extraction
(1) Preparing cell lysate (RIPA: Cocktail: PMSF ═ 100:1:1) and placing on ice for standby;
(2) culturing cells with a 12-hole plate, after the stimulation of antibody protein is finished, removing the culture medium by suction, washing the cells for 2 times by ice-cold PBS, immediately placing the plate on ice, adding about 100 mu l of prepared lysate into each hole, repeatedly and softly blowing and beating the lysate for several times by using a pipette gun to ensure that the lysate is fully contacted with the bottom surface, and standing and cracking the lysate for 30min on the ice;
(3) after the specified time, using a cell scraper to scrape the lysate containing cell debris, transferring the lysate into a clean EP tube, and centrifuging the lysate for 15min at 4 ℃ and 12000 rpm;
(4) the supernatant was carefully aspirated, i.e., total cytosolic protein. The subsequent experiments were immediately carried out, the remainder being stored in a refrigerator at-80 ℃.
2. BCA protein concentration assay
(1) Preparing a BCA working solution according to the BCA concentration detection kit specification for later use: solution A: the ratio of the solution B to the solution B is 50:1,
(2) diluting the standard substance to a final concentration of 0.5mg/ml for later use,
(3) setting a reaction system comprising a standard curve hole and an experimental sample hole;
TABLE 2
Figure BDA0002585061130000191
(4) Sequentially adding each group of reagents into a 96-well plate in sequence, and gently mixing the reagents in each well through a 200-microliter pipette gun, wherein each group of the sample group is repeatedly provided with three auxiliary wells;
(5) putting the pore plate into a preheated constant-temperature incubator at 37 ℃ for incubation for 30min, taking out after the incubation is finished, and immediately detecting the absorbance value at 570nm by using an enzyme-labeling instrument;
(6) and establishing a standard curve according to the absorbance value, and calculating the protein concentration of each hole according to the standard curve.
3. Immunoblotting
Heating and denaturing each protein sample, calculating sample loading volume according to different concentrations, ensuring consistent sample loading amount of each hole, completing Western blot related experimental steps according to the Western blot method, and performing gray scale analysis.
Third, results and analysis
As shown in FIG. 7, four fusion proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) were allowed to act on SK-BR-3 cells, and total proteins were extracted to detect changes in the contents of the apoptosis inhibitory protein Bcl-2 and the apoptosis-promoting protein Bax, and the results are shown in FIG. 7A, using α -tubulin as an internal reference; as shown in FIG. 7B, four fusion proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) acted on SK-BR-3 cells, respectively, and were quantified by ImageLab, Bcl-2/Bax ratio: scFv > Cytc-scFv >3Cytc-scFv > DFF 40-scFv.
It can be seen that the scFv fused to the apoptotic protein DFF40 (DFF40-scFv) had the best apoptosis-inducing activity, while the three Cytc tandem fusion scFv (3Cytc-scFv) were superior in effect to the single Cytc fusion scFv (Cytc-scFv).
Example 8: morphological observation of HER-2 high expression type breast cancer cell SK-BR-3 apoptosis characteristics after co-incubation with scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion type single chain antibody
First, experimental material
Hoechst staining fluid was purchased from beijing solibao; the human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were derived from the same sources as in example 4;
second, analysis of expression level of apoptosis protein
Hoechst is a blue fluorescent dye and can mark DNA, and as the cell undergoes apoptosis, chromatin is condensed, so that the nucleus of the apoptotic cell is obviously different from that of the normal cell.
1. Hoechst nuclear staining
(1) Placing the clean slide in a six-well plate, inoculating cells and growing overnight to fill it with about 50% -80%;
(2) after the fusion protein (scFv, Cytc-scFv, 3Cytc-scFv or DFF40-scFv) stimulates apoptosis, the culture medium is aspirated, 0.5ml of fixing solution is added, and fixation is performed for 10 min;
(3) removing the fixative and washing twice with PBS, then adding 0.5ml Hoechst 33258 staining solution and staining for 5min in a shaking table at room temperature;
(4) the staining solution was removed, washed with PBS, and then a drop of anti-fluorescence quencher was applied to the slide, and the slide was covered to allow the cell surface to contact the slide and avoid air bubbles as much as possible. Then, the fluorescence is observed under a confocal fluorescence microscope, and the excitation wavelength is about 350 nm.
2. Long-time imaging observation of cells
SK-BR-3 cells interacted with different protein constructs (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) in a live cell workstation environment for 48 hours, during which time all morphological changes during cell growth were recorded continuously using a 20 Xmagnification microscope. The difference in cell morphology between 0h, 24h and 48h was compared to assess the extent of apoptosis and to quantify.
Third, results and analysis
As shown in FIG. 8A, which is a fluorescence image of SK-BR-3 cells after Hoechst nucleus staining, it can be seen that nuclei still appear normal blue under the action of scFv, while nuclei appear broken block-shaped dense staining and are whitish (bright color in the figure) under the action of DFF40-scFv, and in contrast, 3Cytc-scFv also appears whitish but the degree is not as much as that of DFF40-scFv, and Cytc-scFv is slightly whitish; the results show that after the apoptosis protein fusion type single-chain antibody acts, the cells have different degrees of nuclear compaction due to different apoptosis titer induced by each protein.
FIG. 8B is a time-lapse image of a live cell long-time dynamic imaging analyzer, which records the growth change of the antibody protein incubated with SK-BR-3 cells within 48h, and shows that after the scFv acted, the cells within 24h and 48h were intact, no solid shrinkage occurred, no surface bleb occurred, and no apoptotic bodies were formed; in contrast, under the action of DFF40-scFv, the cell volume is obviously reduced in 24h, the cells are solidified and shrunk, and the bubbling on the cell surface can be seen in 48h, so that an apoptotic body is formed and is in a fragment shape; under the action of 3Cytc-scFv, the cytoplasm is compact for 24h, and the cytoplasm also bleeds and apoptotic bodies appear for 48 h; finally, the cells after Cytc-scFv acted were well-shaped for 24h, and cell shrinkage was observed for 48 h.
FIGS. 9A-D show the statistics of the apoptosis rate of different proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) incubated with three breast cancer cells (SK-BR-3, MDA-MB-231, MCF-7) for 48h, and the effect of SK-BR-3 cells is consistent with that described in FIG. 8B; for the HER-2 negative cells MDA-MB-231 and MCF-7 used as controls, no significant apoptosis occurred after induction by any of the four antibody proteins.
In conclusion, the fusion type scFv of the apoptosis protein can effectively induce the apoptosis of target cells with high HER-2 expression, the activity of the DFF40-scFv is strongest, the onset time is fastest, the activity of the Cytc-scFv is lower after 3Cytc-scFv takes place, a small amount of apoptosis can be detected after 48 hours, and the fusion type scFv of the apoptosis protein has no effect on cells without specific antigens on the surface.
Example 9: flow cytometry detects that HER-2 high-expression breast cancer cell SK-BR-3 is obviously apoptotic after fusion protein action
First, experimental material
An Annexin V-FITC/PI double-staining apoptosis detection kit is purchased from Nanjing to build a bioengineering institute; the human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were derived from the same sources as in example 4;
two, flow cytometry
(1) The pro-apoptotic activity of the group on which the fusion-type single-chain antibody of the apoptotic protein had been administered (experimental group, Cytc-scFv, 3Cytc-scFv or DFF40-scFv) was compared with that of the group on which the scFv had been administered as a control group. Culturing cells in a six-well plate, stimulating apoptosis of fusion protein (scFv, Cytc-scFv, 3Cytc-scFv or DFF40-scFv) for 48h, aspirating the cell culture medium into a suitable centrifuge tube, washing the cells 3 times with PBS, and digesting the cells with trypsin without EDTA;
(2) after the cells are digested, adding the cell culture solution collected in the step (1), slightly mixing uniformly, transferring the mixture into a new clean centrifugal tube, centrifuging for 5min at 1000g, removing supernatant, gently resuspending the cells by PBS and counting;
(3) take 1.5X 105Resuspended cells, centrifuged at 1000g for 5min, the supernatant discarded, 500. mu.l binding solution (from Annexin V-FITC/PI double-stain apoptosis assay kit) added to gently resuspend cells,
(4) adding 5 μ l Annexin V-FITC, mixing gently, adding 5 μ l Propidium Iodide (PI), mixing gently,
(5) incubate at room temperature (20-25 ℃) in the dark (coated with tinfoil) for 10 min. Then, the detection is carried out by a flow cytometer, Annexin V-FITC is green fluorescence, and PI is red fluorescence. The two regions on the right side of the flow quadrant are shown as apoptotic/necrotic cells.
Third, results and analysis
As shown in FIG. 10A, a flow chart was generated using FITC as the abscissa and PI as the ordinate, the first column was a flow quadrant graph of scFv acting on three breast cancer cell lines, respectively, and the cell types were substantially distributed in the lower left quadrant, i.e., Annexin V-/PI-Normal growth of cells and little freshnessApoptosis of the cell; the second column shows the result of action of DFF40-scFv, and shows that the HER-2 high expression cell SK-BR-3 has Annexin V in the lower right quadrant under the action of DFF40-scFv+/PI-And the upper right quadrant Annexin V+/PI+The total cell amount of (A) reaches 50%, namely 50% of cells show apoptosis to different degrees; HER-2 negative cells MDA-MB-231, MCF-7 used for control did not show apoptosis; the third column shows the result of Cytc-scFv action, under the action of Cytc-scFv, HER-2 negative cells MDA-MB-231 and MCF-7 used for control do not undergo apoptosis except that SK-BR-3 cells have apoptosis rate of about 15%; the rightmost column shows the effect of 3Cytc-scFv, the apoptosis rate of SK-BR-3 cells is about twice that of Cytc-scFv, and MDA-MB-231 and MCF-7 cells do not undergo apoptosis.
FIG. 10B is a graph of the quantification of apoptosis data in FIG. 10A, consistent with the results shown in the quadrant graph.
In conclusion, from the above flow cytometry experiment results, we can conclude that the apoptotic protein fusion-type scFv can efficiently induce apoptosis of HER-2 highly expressed target cells, has no effect on cells without surface specific antigen, and is active DFF40-scFv >3Cytc-scFv > Cytc-scFv compared to scFv alone, in agreement with the aforementioned in vitro experiments.
Example 10: in vivo activity validation of apoptotic protein fusion type anti-HER-2 single chain antibody
The above-mentioned various in vitro experiments fully verify the targeting anti-tumor activity of the fusion type single-chain antibody, and then we discuss whether the fusion protein can exert the consistent activity in vivo and in vitro by establishing animal models.
Materials and reagents
The Tunel cell apoptosis in-situ detection kit is purchased from Nanjing to build a bioengineering institute; human breast cancer cell line SK-BR-3 was purchased from American Type Culture Collection (ATCC); animals: 20 BALB/C female nude mice, 6 weeks old, weighing 16-18g, provided by the university of Nanjing model animal research center; and the animal is fed and dissected according to the national animal experiment operating specification strictly, and is approved by animal ethics committee of Nanjing university of traditional Chinese medicine.
Second, Experimental methods
1. Establishing nude mouse xenotransplantation model
(1)75mm2SK-BR-3 breast cancer cells are cultured in a culture bottle in a large quantity, are re-suspended in DMEM after being digested, are mixed with matrigel (4 ℃) at a ratio of 4:1 and are used for planting tumor cells,
(2) the right upper axilla of a six-week-old female nude mouse was subcutaneously inoculated with 1X 107An SK-BR-3 cell;
(3) when the tumor reached an average volume of about 50mm3When the method is used, the mice are randomly divided into four groups, namely an scFv action group, a DFF40-scFv action group, a 3Cytc-scFv action group and a Cytc-scFv action group, wherein each group comprises 5 mice;
(4) the mice of the above groups were treated by intraperitoneal injection (10mg/kg) of the fusion protein every three days, and the tumor size was measured every three days using calipers, according to the standard formula: v (mm)3)＝width2(mm2) X length (mm)/2 tumor volume;
(5) the experiment was terminated on day 30, mice were euthanized, and tumors were excised, photographed, and weighed.
2. Tunel staining
Making the separated tumor into a paraffin section, carrying out Tunel staining by using a Tunel cell apoptosis in-situ detection kit, observing and photographing under an optical microscope (apoptotic cells are dark brown), and analyzing the apoptosis condition of tumor tissues.
Third, results and analysis
As shown in fig. 11A, tumor volume changes were monitored every 3 days, mice in the scFv-affected group increased in tumor volume day by day, and tumor growth was not inhibited; the tumor volume growth amplitude of the mice under the action of the DFF40-scFv action group, the 3Cytc-scFv action group and the Cytc-scFv action group is reduced to different degrees, wherein the tumor growth of the DFF40-scFv action group is greatly inhibited, and the change of the tumor volume during protein injection treatment is very little; the tumor of the 3Cytc-scFv action group is also inhibited, the volume is slightly larger than that of the DFF40-scFv action group, and the growth of the tumor of the Cytc-scFv action group is obvious and slightly inhibited compared with that of the scFv of the control group. After the experiment, tumor bodies were isolated, and the tumors in each group were arranged in order and kept in photographs for comparison, and it was found that the difference between the tumor volume and the final mass was consistent with that recorded during the experiment (fig. 11B, 11C).
As shown in FIG. 12, the Tunel staining of paraffin section of tumor tissue showed that no apoptosis was detected in tumor cells in mice of the scFv-affected group, compared with significant apoptosis in mice of the DFF 40-scFv-affected group, and the apoptosis rate in 3 Cytc-scFv-affected group was second, while only a small amount of apoptosis occurred in Cytc-scFv.
In conclusion, the nude mouse xenograft model successfully verifies that the apoptosis protein fusion type anti-HER-2 single-chain antibody can effectively target HER-2 over-expression type breast cancer tumor in vivo and induce tumor cell apoptosis on the animal level, and the activity of the three apoptosis protein fusion type anti-HER-2 single-chain antibodies (DFF40-scFv, 3Cytc-scFv and Cytc-scFv) is consistent with that of in vitro tests; from this we can derive: the fusion expression of the apoptosis-promoting protein and the single-chain antibody for treating the cancer can realize good combination of targeting property and anti-tumor activity, and provides a new idea for the targeted immunotherapy of the cancer.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.
Sequence listing
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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser
130 135 140
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
145 150 155 160
Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly
165 170 175
Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu
195 200 205
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
210 215 220
Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
225 230 235 240
Ile Lys Arg
<210> 5
<211> 729
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaagttcagc tggttgaatc tggtggtggt ctggttcagc cgggtggttc tctgcgtctg 60
tcttgcgctg cttctggttt caacatcaaa gacacctaca tccactgggt tcgtcaggct 120
ccgggtaaag gtctggaatg ggttgctcgt atctacccga ccaacggtta cacccgttac 180
gctgactctg ttaaaggtcg tttcaccatc tctgctgaca cctctaaaaa caccgcttac 240
ctgcagatga actctctgcg tgctgaagac accgctgttt actactgctc tcgttggggt 300
ggtgacggtt tctacgctat ggactactgg ggtcagggta ccctggttac cgtttcttct 360
gggggcgggg gctctggggg cgggggctct gggggcgggg gctctgatat tcagatgacc 420
cagagcccga gcagcctgag cgcgagcgtg ggcgatcgcg tgaccattac ctgccgcgcg 480
agccaggatg tgaacaccgc ggtggcgtgg tatcagcaga aaccgggcaa agcgccgaaa 540
ctgctgattt atagcgcgag ctttctgtat agcggcgtgc cgagccgctt tagcggcagc 600
cgcagcggca ccgattttac cctgaccatt agcagcctgc agccggaaga ttttgcgacc 660
tattattgcc agcagcatta taccaccccg ccgacctttg gccagggcac caaagtggaa 720
attaaacgc 729
<210> 6
<211> 105
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser
1 5 10 15
Gln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn
20 25 30
Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser
35 40 45
Tyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr
50 55 60
Leu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys
65 70 75 80
Met Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile
85 90 95
Ala Tyr Leu Lys Lys Ala Thr Asn Glu
100 105
<210> 7
<211> 315
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgggtgatg ttgagaaagg caagaagatt tttattatga agtgttccca gtgccacacc 60
gttgaaaagg gaggcaagca caagactggg ccaaatctcc acggtctctt tgggcggaag 120
acaggtcagg cccctggata ctcttacaca gccgccaata agaacaaagg catcatctgg 180
ggagaggata cactgatgga gtatttggag aatcccaaga agtacatccc tggaacaaaa 240
atgatctttg tcggcattaa gaagaaggaa gaaagggcag acttaatagc ttatctcaaa 300
aaagctacta atgag 315
<210> 8
<211> 338
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Leu Gln Lys Pro Lys Ser Val Lys Leu Arg Ala Leu Arg Ser Pro
1 5 10 15
Arg Lys Phe Gly Val Ala Gly Arg Ser Cys Gln Glu Val Leu Arg Lys
20 25 30
Gly Cys Leu Arg Phe Gln Leu Pro Glu Arg Gly Ser Arg Leu Cys Leu
35 40 45
Tyr Glu Asp Gly Thr Glu Leu Thr Glu Asp Tyr Phe Pro Ser Val Pro
50 55 60
Asp Asn Ala Glu Leu Val Leu Leu Thr Leu Gly Gln Ala Trp Gln Gly
65 70 75 80
Tyr Val Ser Asp Ile Arg Arg Phe Leu Ser Ala Phe His Glu Pro Gln
85 90 95
Val Gly Leu Ile Gln Ala Ala Gln Gln Leu Leu Cys Asp Glu Gln Ala
100 105 110
Pro Gln Arg Gln Arg Leu Leu Ala Asp Leu Leu His Asn Val Ser Gln
115 120 125
Asn Ile Ala Ala Glu Thr Arg Ala Glu Asp Pro Pro Trp Phe Glu Gly
130 135 140
Leu Glu Ser Arg Phe Gln Ser Lys Ser Gly Tyr Leu Arg Tyr Ser Cys
145 150 155 160
Glu Ser Arg Ile Arg Ser Tyr Leu Arg Glu Val Ser Ser Tyr Pro Ser
165 170 175
Thr Val Gly Ala Glu Ala Gln Glu Glu Phe Leu Arg Val Leu Gly Ser
180 185 190
Met Cys Gln Arg Leu Arg Ser Met Gln Tyr Asn Gly Ser Tyr Phe Asp
195 200 205
Arg Gly Ala Lys Gly Gly Ser Arg Leu Cys Thr Pro Glu Gly Trp Phe
210 215 220
Ser Cys Gln Gly Pro Phe Asp Met Asp Ser Cys Leu Ser Arg His Ser
225 230 235 240
Ile Asn Pro Tyr Ser Asn Arg Glu Ser Arg Ile Leu Phe Ser Thr Trp
245 250 255
Asn Leu Asp His Ile Ile Glu Lys Lys Arg Thr Ile Ile Pro Thr Leu
260 265 270
Val Glu Ala Ile Lys Glu Gln Asp Gly Arg Glu Val Asp Trp Glu Tyr
275 280 285
Phe Tyr Gly Leu Leu Phe Thr Ser Glu Asn Leu Lys Leu Val His Ile
290 295 300
Val Cys His Lys Lys Thr Thr His Lys Leu Asn Cys Asp Pro Ser Arg
305 310 315 320
Ile Tyr Lys Pro Gln Thr Arg Leu Lys Arg Lys Gln Pro Val Arg Lys
325 330 335
Arg Gln
<210> 9
<211> 1014
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atgctccaga agcccaagag cgtgaagctg cgggccctgc gcagcccgag gaagttcggc 60
gtggctggcc ggagctgcca ggaggtgctg cgcaagggct gtctccgctt ccagctccct 120
gagcgcggtt cccggctgtg cctgtacgag gatggcacgg agctgacgga agattacttc 180
cccagtgttc ccgacaacgc cgagctggtg ctgctcacct tgggccaggc ctggcagggc 240
tatgtgagcg acatcaggcg cttcctcagt gcatttcacg agccacaggt ggggctcatc 300
caggccgccc agcagctgct gtgtgatgag caggccccac agaggcagag gctgctggct 360
gacctcctgc acaacgtcag ccagaacatc gcggccgaga cccgggctga ggacccgccg 420
tggtttgaag gcttggagtc ccgatttcag agcaagtctg gctatctgag atacagctgt 480
gagagccgga tccggagtta cctgagggag gtgagctcct acccctccac ggtgggtgcg 540
gaggctcagg aggaattcct gcgggtcctc ggctccatgt gccagaggct ccggtccatg 600
cagtacaatg gcagctactt cgacagagga gccaagggcg gcagccgcct ctgcacaccg 660
gaaggctggt tctcctgcca gggtcccttt gacatggaca gctgcttatc aagacactcc 720
atcaacccct acagtaacag ggagagcagg atcctcttca gcacctggaa cctggatcac 780
ataatagaaa agaaacgcac catcattcct acactggtgg aagcaattaa ggaacaagat 840
ggaagagaag tggactggga gtatttttat ggcctgcttt ttacctcaga gaacctaaaa 900
ctagtgcaca ttgtctgcca taagaaaacc acccacaagc tcaactgtga cccaagcaga 960
atctacaaac cccagacaag gttgaagcgg aagcagcctg tgcggaaacg ccag 1014
<210> 10
<211> 351
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser
1 5 10 15
Gln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn
20 25 30
Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser
35 40 45
Tyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr
50 55 60
Leu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys
65 70 75 80
Met Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile
85 90 95
Ala Tyr Leu Lys Lys Ala Thr Asn Glu Thr Met Ala Glu Val Gln Leu
100 105 110
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
115 120 125
Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp
130 135 140
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr
145 150 155 160
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe
165 170 175
Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn
180 185 190
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly
195 200 205
Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
210 215 220
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
225 230 235 240
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
245 250 255
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val
260 265 270
Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
275 280 285
Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
290 295 300
Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
305 310 315 320
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr
325 330 335
Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
340 345 350
<210> 11
<211> 12
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gatgaagttg at 12
<210> 12
<211> 584
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Met Leu Gln Lys Pro Lys Ser Val Lys Leu Arg Ala Leu Arg Ser Pro
1 5 10 15
Arg Lys Phe Gly Val Ala Gly Arg Ser Cys Gln Glu Val Leu Arg Lys
20 25 30
Gly Cys Leu Arg Phe Gln Leu Pro Glu Arg Gly Ser Arg Leu Cys Leu
35 40 45
Tyr Glu Asp Gly Thr Glu Leu Thr Glu Asp Tyr Phe Pro Ser Val Pro
50 55 60
Asp Asn Ala Glu Leu Val Leu Leu Thr Leu Gly Gln Ala Trp Gln Gly
65 70 75 80
Tyr Val Ser Asp Ile Arg Arg Phe Leu Ser Ala Phe His Glu Pro Gln
85 90 95
Val Gly Leu Ile Gln Ala Ala Gln Gln Leu Leu Cys Asp Glu Gln Ala
100 105 110
Pro Gln Arg Gln Arg Leu Leu Ala Asp Leu Leu His Asn Val Ser Gln
115 120 125
Asn Ile Ala Ala Glu Thr Arg Ala Glu Asp Pro Pro Trp Phe Glu Gly
130 135 140
Leu Glu Ser Arg Phe Gln Ser Lys Ser Gly Tyr Leu Arg Tyr Ser Cys
145 150 155 160
Glu Ser Arg Ile Arg Ser Tyr Leu Arg Glu Val Ser Ser Tyr Pro Ser
165 170 175
Thr Val Gly Ala Glu Ala Gln Glu Glu Phe Leu Arg Val Leu Gly Ser
180 185 190
Met Cys Gln Arg Leu Arg Ser Met Gln Tyr Asn Gly Ser Tyr Phe Asp
195 200 205
Arg Gly Ala Lys Gly Gly Ser Arg Leu Cys Thr Pro Glu Gly Trp Phe
210 215 220
Ser Cys Gln Gly Pro Phe Asp Met Asp Ser Cys Leu Ser Arg His Ser
225 230 235 240
Ile Asn Pro Tyr Ser Asn Arg Glu Ser Arg Ile Leu Phe Ser Thr Trp
245 250 255
Asn Leu Asp His Ile Ile Glu Lys Lys Arg Thr Ile Ile Pro Thr Leu
260 265 270
Val Glu Ala Ile Lys Glu Gln Asp Gly Arg Glu Val Asp Trp Glu Tyr
275 280 285
Phe Tyr Gly Leu Leu Phe Thr Ser Glu Asn Leu Lys Leu Val His Ile
290 295 300
Val Cys His Lys Lys Thr Thr His Lys Leu Asn Cys Asp Pro Ser Arg
305 310 315 320
Ile Tyr Lys Pro Gln Thr Arg Leu Lys Arg Lys Gln Pro Val Arg Lys
325 330 335
Arg Gln Thr Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
340 345 350
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
355 360 365
Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
370 375 380
Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg
385 390 395 400
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
405 410 415
Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
420 425 430
Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
435 440 445
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
450 455 460
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
465 470 475 480
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
485 490 495
Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr
500 505 510
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
515 520 525
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly
530 535 540
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
545 550 555 560
Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln
565 570 575
Gly Thr Lys Val Glu Ile Lys Arg
580
<210> 13
<211> 569
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Met Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser
1 5 10 15
Gln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn
20 25 30
Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser
35 40 45
Tyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr
50 55 60
Leu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys
65 70 75 80
Met Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile
85 90 95
Ala Tyr Leu Lys Lys Ala Thr Asn Glu Asp Glu Val Asp Met Gly Asp
100 105 110
Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser Gln Cys His
115 120 125
Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn Leu His Gly
130 135 140
Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser Tyr Thr Ala
145 150 155 160
Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu Met Glu
165 170 175
Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys Met Ile Phe
180 185 190
Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala Tyr Leu
195 200 205
Lys Lys Ala Thr Asn Glu Asp Glu Val Asp Met Gly Asp Val Glu Lys
210 215 220
Gly Lys Lys Ile Phe Ile Met Lys Cys Ser Gln Cys His Thr Val Glu
225 230 235 240
Lys Gly Gly Lys His Lys Thr Gly Pro Asn Leu His Gly Leu Phe Gly
245 250 255
Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser Tyr Thr Ala Ala Asn Lys
260 265 270
Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu Met Glu Tyr Leu Glu
275 280 285
Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys Met Ile Phe Val Gly Ile
290 295 300
Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala Tyr Leu Lys Lys Ala
305 310 315 320
Thr Asn Glu Thr Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly
325 330 335
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
340 345 350
Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly
355 360 365
Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
370 375 380
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
385 390 395 400
Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
405 410 415
Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
420 425 430
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
435 440 445
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln
450 455 460
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
465 470 475 480
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp
485 490 495
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala
500 505 510
Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser
515 520 525
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
530 535 540
Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly
545 550 555 560
Gln Gly Thr Lys Val Glu Ile Lys Arg
565
<210> 14
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cgacgacgac gacaaggcca tggctgaagt tcagctggtt gaatctgg 48
<210> 15
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgacggagct cgaattcgga tccttagcgt ttaatttcca ctttggtgc 49
<210> 16
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ccagcacatg gacagcccag atctcatggg tgatgttgag aaaggc 46
<210> 17
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
caaccagctg aacttcagcc atggtctcat tagtagcttt tttgagataa gc 52
<210> 18
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ccagcacatg gacagcccag atctcatgct ccagaagccc aagagc 46
<210> 19
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
caaccagctg aacttcagcc atggtctggc gtttccgcac aggctg 46

Claims (9)

1. The apoptosis protein fusion type anti-HER-2 single-chain antibody is characterized in that the anti-HER-2 single-chain antibody is coupled with apoptosis proteins in different tandem modes, the anti-HER-2 single-chain antibody comprises a heavy chain variable region VH shown in a sequence SEQ ID NO.1 and a light chain variable region VL shown in a sequence SEQ ID NO.2, and the heavy chain variable region and the light chain variable region are respectively connected with a flexible peptide (G) shown in a sequence SEQ ID NO.34S)3And the VH upstream of the heavy chain variable region contains a 6 x his tag, and the amino acid sequence of the anti-HER-2 single-chain antibody is shown as SEQ ID NO.4, the cDNA sequence of the anti-HER-2 single-chain antibody is shown in SEQ ID NO.5, and the apoptosis protein is cytochrome C or DNA fragmentation factor 40.
2. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 1, wherein: the amino acid sequence of the cytochrome C is shown as SEQ ID NO.6, and the cDNA sequence of the cytochrome C is shown as SEQ ID NO. 7.
3. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 1, wherein: the amino acid sequence of the DNA fragmentation factor 40 is shown as SEQ ID NO.8, and the cDNA sequence for coding the DNA fragmentation factor 40 is shown as SEQ ID NO. 9.
4. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 2, wherein: the apoptosis protein fusion type anti-HER-2 single-chain antibody is a Cytc-scFv fusion type single-chain antibody, and is formed by coupling the anti-HER-2 single-chain antibody of claim 1 and cytochrome C of claim 3, wherein the cytochrome C is connected between a 6 x his tag and the anti-HER-2 single-chain antibody, and the amino acid sequence of the Cytc-scFv fusion type single-chain antibody is shown as SEQ ID NO. 10.
5. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 2, wherein: the apoptosis protein fusion type anti-HER-2 single-chain antibody is an nCytc-scFv fusion type single-chain antibody, and is formed by coupling the anti-HER-2 single-chain antibody of claim 1 and a cytochrome C tandem aggregate, wherein the cytochrome C tandem aggregate is connected between a 6 x his tag and the anti-HER-2 single-chain antibody in series, the cytochrome C tandem aggregate is formed by connecting a plurality of cytochrome C of claim 3 through a connecting peptide, the connecting peptide is Caspase-3 enzyme cutting site DEVD, and the cDNA sequence of the DEVD is shown in SEQ ID NO. 11.
6. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 3, wherein: the apoptin-fused anti-HER-2 single-chain antibody is a DFF 40-scFv-fused single-chain antibody, and is formed by connecting the anti-HER-2 single-chain antibody of claim 1 with the DNA fragmentation factor 40 of claim 4, wherein the DNA fragmentation factor 40 is connected between a 6 x his tag and the anti-HER-2 single-chain antibody, and the amino acid sequence of the DFF 40-scFv-fused single-chain antibody is shown as SEQ ID NO. 12.
7. The method for producing an apoptotic protein fusion-type anti-HER-2 single chain antibody according to any one of claims 1 to 6, comprising the steps of:
(1) synthesizing a cDNA sequence of the anti-HER-2 single-chain antibody and a cDNA sequence of the apoptosis protein shown in SEQ ID NO. 5;
(2) constructing a recombinant plasmid: respectively connecting the cDNA sequence of the anti-HER-2 single-chain antibody coded in the step (1) and the cDNA sequence of the apoptosis protein coded in the step (1) into a pET-32a (+) expression vector to construct a recombinant plasmid;
(3) transforming the recombinant plasmid obtained in the step (2) into DH5 alpha competent cells, screening recombinant plasmid with correct sequencing into BL21(DE3) competent cells, and inducing and expressing the apoptosis protein fusion type anti-HER-2 single-chain antibody.
8. The method of claim 7, wherein: in the step (2), a cDNA fragment encoding the anti-HER-2 single-chain antibody is inserted between the Nco I and BamH I cleavage sites of the pET-32a (+) expression vector, the apoptosis protein is cytochrome C as claimed in claim 3 or DNA fragmentation factor 40 as claimed in claim 4, a cDNA fragment encoding the apoptosis protein is inserted between Bgl II and Nco I cleavage sites of pET-32a (+) expression vector, and the restriction enzyme Nco I enzyme cutting site adopted on the pET-32a (+) expression vector when inserting the cDNA segment of the anti-HER-2 single-chain antibody and the cDNA segment of the coding apoptosis protein is the same enzyme cutting site, when the apoptosis protein is cytochrome C and the number of cytochrome C is more than one, the individual cytochrome C are connected in series by Caspase-3 cleavage site DEVD as shown in SEQ ID NO. 11.
9. Use of the anti-HER-2 single-chain antibody of claim 1 or the apoptotic protein fusion-type anti-HER-2 single-chain antibody of any one of claims 1 to 6 in the preparation of an anti-cancer medicament for the targeted treatment of HER-2 high-expression cancer.
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Publication number Priority date Publication date Assignee Title
CN116514998A (en) * 2023-05-12 2023-08-01 再少年(北京)生物科技有限公司 Chimeric antigen receptor, chimeric antigen receptor-natural killer cell and application thereof in preparation of antitumor drugs
CN116514998B (en) * 2023-05-12 2023-09-15 再少年(北京)生物科技有限公司 Chimeric antigen receptor, chimeric antigen receptor-natural killer cell and application thereof in preparation of antitumor drugs

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