CN111088204A - Recombinant escherichia coli expressing Caspase-3 recombinant scFv78 and functional verification method thereof - Google Patents
Recombinant escherichia coli expressing Caspase-3 recombinant scFv78 and functional verification method thereof Download PDFInfo
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Abstract
The invention provides a recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and a functional verification method thereof. According to the technical scheme, firstly, a recombinant single-chain antibody scFv78 encoding gene integrated with a Caspase-3 encoding gene is connected with a pET302 vector by a method of enzyme digestion and enzyme ligation, and then the obtained recombinant plasmid is converted into escherichia coli Nissle1917, so that a strain capable of expressing Caspase-3 recombinant scFv78 is obtained. According to the invention, the characteristics of intracellular invasion and aggregation of escherichia coli Nissle1917 and the fixed-point targeting characteristic of scFv78 on TEM1 positive cells are adopted, so that the site specificity of the human toxin Caspase-3 protein is exerted, the TEM1 positive tumor cells are killed more accurately, and the damage to normal cells of an organism is reduced. On the basis, the recombinant bacteria are used for expressing human toxin Caspase-3 recombinant single-chain antibody scFv78 protein, and the protein and MS1-TEM1 cells are incubated together in an in vitro environment, so that the affinity and the lethality of the protein to tumor cells are verified.
Description
Technical Field
The invention relates to the technical field of molecular biology, further relates to the biological pharmacy and tumor medicine technology, and particularly relates to recombinant escherichia coli expressing Caspase-3 recombinant scFv78 and a function verification method thereof.
Background
At present, the treatment aiming at the tumor comprises chemical, physical and biological methods, but the above-mentioned treatments can not achieve the ideal effect of treating the tumor all the time, so that the search for a safer and more effective novel tumor treatment method is urgent. Since William Coley firstly utilizes bacteria to treat tumors, the bacteria targeted treatment of tumors has become a hot spot of international research in recent years, a plurality of bacteria are found to specifically invade tumor tissues of organisms and multiply, and the idea of utilizing viable bacteria to treat tumors is generated.
The targeted therapy means that the antitumor drug can be specifically combined with a specific site of a tumor, so that the tumor cell killing effect is achieved, the healthy tissue is basically not affected, the current ideal treatment mode is achieved, and the future trend of tumor treatment is represented. They are mainly classified into monoclonal Antibody drugs and Antibody Drug Conjugates (ADCs). ADC drugs are a novel class of therapeutic drugs that are gaining increasing attention from pharmaceutical companies worldwide. The ADC drug is formed by coupling a monoclonal antibody and a potent toxic drug (toxin drug) through a bioactive connector (linker), and is a potent anticancer drug targeting cancer cells at a fixed point. Due to the accurate identification of the target and no influence on non-cancer cells, the medicine effect is greatly improved and the toxic and side effects are reduced.
Endosialin (also known as tumor endothelial cell marker molecule 1, TEM1/CD248) is a type I transmembrane protein, which is composed of a glycosylation-modified 80.9kDa protein core region, and the mature glycoprotein after modification is about 175 kDa. TEM1 is one of the recently discovered human tumor markers that play an important role in tumor cell growth, angiogenic infiltration, and metastasis. Tumor endothelial cell marker molecule 1 (TEM1/CD248) is highly expressed on various solid tumors (endothelial cells and stromal cells), such as sarcoma, brain tumor, breast cancer, skin cancer, colon cancer, and experiments show that in ovarian cancer, the expression of TEM1 is closely related to tumor infiltration and prognosis. Therefore, TEM1 is not expressed or is low expressed in normal tissues, but is high expressed in tumor vascular tissues, so TEM1 can be used as a biomarker for tumor blood vessels in cancer therapy. Single Chain antibody Single Chain Variable region fragments (scFv78) specific to TEM1 were obtained from previous studies. scFv78 is a single chain antibody with high affinity and has a higher affinity for both human and murine, and thus TEM1 can be considered as a target molecule in preclinical models.
Caspase-3 (Caspase-3) is a key enzyme in the process of apoptosis execution and effect in mammalian cells, and the cascade activation reaction of the Caspase-3 is an important information transmission path of apoptosis, the equilibrium state of cell proliferation and apoptosis is an indispensable stable state for keeping normal survival of organisms and maintaining dynamic balance of cells in organisms, the disorder of an apoptosis regulation system is closely related to the formation and development of tumors, and a large amount of researches prove that Caspase-3 normally exists in plasma in a form of zymogen (32KDa), when activated, two subunits of large (17KDa) and small (12KDa) are released after two times of decomposition (sites are both in peptide bonds at the ends of aspartic acid), the released large and small subunits are recombined and combined to form a new dimer structure (a β sheet layer is taken as a core and two sides are α helices), the two dimers are assembled into a tetramerized active enzyme with 2 substrate binding sites and catalytic sites, thus a tetrapassage protein β is caused by a tetrapassamily-derived from Caspase 26, the principle that the Caspase-3 and a targeting apoptosis signal transduction pathway of a Caspase-3, thus the apoptosis-3 and the final targeting protein receptor of a tumor-mediated protein receptor, the apoptosis pathway of a tumor-mediated protein, and a targeting protein receptor of a tumor-mediated protein receptor, wherein the apoptosis-activating peptide is obtained by the three pathways of a targeting peptide-activating protein targeting peptide and a targeting motif of a targeting apoptosis-activating protein.
However, although the targeting of Caspase-3 is realized through the carrier effect of the scFv78, the scFv78 cannot be specifically aggregated in the tumor in the conventional administration mode, so that the targeting of the scFv78 is not improved. In this case, if the above recombinant antibody can be structurally modified to achieve its specificity for a solid tumor or tumor cells, it is expected to further enhance the antitumor effect. However, to achieve this goal, many problems remain to be solved, including whether the chemical coupling method is a microbial recombination method, what kind of carrier is selected, and how the two are connected at the molecular level.
As shown by research, Escherichia coli is sensitive to serum, has preference for solid tumors and can proliferate in large quantities in the solid tumors, and in view of the characteristic, the engineering bacteria capable of expressing exogenous gene drugs are constructed by taking Escherichia coli as host bacteria to specifically treat cancers. The nonpathogenic Escherichia coli Nissle1917(O6: K5: H1) belongs to probiotics and is parasitic in human and animal bodies, and a large amount of experimental and clinical data show that the bacterial medicament is effective and safe. In addition, E.coli Nissle1917 is an extracellular bacterium, which is more susceptible to antibiotic clearance than intracellular bacteria; the genome and genetic background of the gene are relatively clear and are easier to modify; it is a facultative anaerobe, which can accumulate both in necrotic areas of tumors and in oxygen-rich sites.
Disclosure of Invention
The invention aims to provide a recombinant escherichia coli expressing Caspase-3 recombinant scFv78 and a function verification method thereof aiming at the technical defects of the prior art, so as to solve the technical problem that the human toxin Caspase-3 recombinant single-chain antibody (scFv78) in the prior art can not be specifically aggregated in tumors.
Another technical problem to be solved by the invention is that the Escherichia coli Nissle1917 with preference for solid tumors can not express the human toxin Caspase-3 recombinant single-chain antibody (scFv78) in tumors.
The invention also aims to solve the technical problem of how to verify the anti-tumor efficacy of the recombinant microorganism carrying the gene medicine.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
recombinant escherichia coli for expressing Caspase-3 recombinant scFv78 is constructed by the following method:
1) connecting the encoding gene fragment 78-Caspase-3 of the human toxin Caspase-3 recombinant single-chain antibody scFv78 to pET302 plasmid by a method of enzyme digestion and enzyme ligation to obtain pET302-78-Caspase-3 recombinant plasmid;
2) and (3) converting the pET302-78-Caspase-3 recombinant plasmid into Escherichia coli Nissle1917 to obtain recombinant Escherichia coli E.coli Nissle1917-pET302-78-Caspase-3, namely the recombinant Escherichia coli for expressing Caspase-3 recombinant scFv 78.
Preferably, the coding gene fragment 78-Caspase-3 of the recombinant single-chain antibody scFv78 of the human toxin Caspase-3 in the step 1) is a DNA fragment with the nucleotide sequence shown as SEQ ID No. 1.
Preferably, the gene segment 78-Caspase-3 encoding the recombinant single-chain antibody scFv78 of the human toxin Caspase-3 in step 1) is obtained by artificial synthesis.
Preferably, the coding gene fragment 78-Caspase-3 of the recombinant single-chain antibody scFv78 of the human toxin Caspase-3 in the step 1) is obtained by PCR amplification by using DNA fragments with nucleotide sequences shown as SEQ ID No.3 and SEQ ID No.4 as primers.
Preferably, the pET302-78-Caspase-3 recombinant plasmid used in the step 2) is obtained by transferring the pET302-78-Caspase-3 recombinant plasmid obtained in the step 1) into E.coli Top10, and then culturing and amplifying the E.coli Top 10.
Preferably, the enzyme in step 1) is double-digested by Xho I and Avr II, and the digested product is purified and then ligated into pET302 plasmid by using T4 ligase.
Preferably, step 2) comprises: melting 100 μ L of bacterial liquid of Escherichia coli Nissle1917 competent cells on ice, adding 2 μ L of the pET302-78-Caspase-3 recombinant plasmid, mixing, incubating on ice for 30min, immersing the bacterial liquid in 42 ℃ water bath for heat shock for 60-90s, then immediately placing on ice and standing for 2min, adding 900 μ L SOC culture solution, shaking and incubating at 37 deg.C on a constant temperature shaker at 22rpm for 1h, then centrifuging the bacterial liquid at the rotating speed of 4000rpm for 3min, discarding redundant liquid, suspending the bacterial cells by using the residual 100 mu L of culture medium, adding all the bacterial liquid to an LB solid culture plate containing ampicillin resistance, carrying out inverted culture at 37 ℃ overnight, screening positive clones, thus obtaining the recombinant Escherichia coli E.coli Nissle1917-pET302-78-Caspase-3, namely the recombinant Escherichia coli for expressing the Caspase-3 recombinant scFv 78.
Preferably, the escherichia coli Nissle1917 competent cell is prepared by the following method: coli Nissle1917 was streaked on an LB plate without resistance and cultured overnight at 37 ℃; selecting a single colony, inoculating the single colony in 5mL LB culture medium, and carrying out shaking culture at 37 ℃ for 12 h; inoculating the strain into 100mL LB culture medium according to the proportion of 1:100, and performing shaking culture until the OD value of bacteria is 0.4; ice-cooling for 20min, and centrifuging at 4 deg.C and 3000rpm for 10 min; washing thallus precipitate twice with 1/10 volume pre-cooled sterile deionized water, centrifuging at 4 deg.C and 3000rpm for 10 min; washing thallus with 1/100 volume precooled 10% glycerol, centrifuging at 4 deg.C and 3000rpm for 10 min; and (3) suspending the thallus precipitate in 1/100 volume of precooled 10% glycerol to obtain the escherichia coli Nissle1917 competent cell.
Preferably, the pET302 plasmid is a DNA molecule having the nucleotide sequence shown in SEQ ID No. 2.
Preferably, the conversion to shock is as described in step 2).
On the basis of the technical scheme, the invention further provides an anti-tumor function verification method of the recombinant escherichia coli, which comprises the steps of expressing the human toxin Caspase-3 recombinant single-chain antibody scFv78 by using the recombinant escherichia coli E.coli Nissle1917-pET302-78-C aspase-3, co-incubating the human toxin Caspase-3 recombinant single-chain antibody scFv78 with TEM1 positive cells, and detecting the affinity of the human toxin Caspase-3 recombinant single-chain antibody scFv78 with the TEM1 positive cells through living cell ELISA.
On the basis of the technical scheme, the invention further provides an anti-tumor function verification method of the recombinant escherichia coli, which comprises the following steps: coating a 96-well plate with 2% gelatin for 30min at 37 ℃, removing the gelatin, and connecting the MS1-TEM1 cell and the MS1 cell for overnight growth at 37 ℃; incubating the human toxin Caspase-3 recombinant single-chain antibody scFv78 diluted by PBS with cells for 2h at 4 ℃, then incubating the secondary antibody for 1h at 4 ℃, performing color development to measure OD value, and detecting the affinity of the human toxin Caspase-3 recombinant single-chain antibody scFv78 with the MS1-TEM1 by using a living cell ELISA method.
The invention provides a recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and a functional verification method thereof. According to the technical scheme, firstly, a recombinant single-chain antibody scFv78 encoding gene integrated with a Caspase-3 encoding gene is connected with a pET302 vector by a method of enzyme digestion and enzyme ligation, and then the obtained recombinant plasmid is converted into escherichia coli Nissle1917, so that a strain capable of expressing Caspase-3 recombinant scFv78 is obtained. According to the invention, the characteristics of intracellular invasion and aggregation of escherichia coli Nissle1917 and the fixed-point targeting characteristic of scFv78 on TEM1 positive cells are adopted, so that the site specificity of the human toxin Caspase-3 protein is exerted, the TEM1 positive tumor cells are killed more accurately, and the damage to normal cells of an organism is reduced. On the basis, the recombinant bacteria are used for expressing human toxin Caspase-3 recombinant single-chain antibody scFv78 protein, and the protein and MS1-TEM1 cells are incubated together in an in vitro environment, so that the affinity and the lethality of the protein to tumor cells are verified.
The technical scheme constructs pET302-78-Caspase-3 recombinant plasmid, utilizes pET302 vector to carry recombinant antibody, and uses Escherichia coli Nissle1917 as a therapeutic vector, comprehensively uses the characteristics of Escherichia coli Nissle1917 intracellular invasion and the property of specificity aggregation in solid tumor, and further expands the specificity of tumor killing in the invention. The invention combines the early experimental basis and provides a certain theoretical basis and data support for the development of ADC medicament for treating tumors.
According to the invention, the single-chain antibody (scFv78) which is discovered in the early stage of a laboratory and specifically recognizes the TEM1 protein is combined with the characteristics of tumor specific aggregation and intracellular invasion of escherichia coli Nissle1917, and carries the human-derived toxin Caspase-3 to achieve the effect of specifically killing tumors. The technical advantages are focused on the following aspects: the invention utilizes the invasion and aggregation characteristics of escherichia coli Nissle1917 in solid tumor and the targeting characteristics of single-chain antibody (scFv78) to TEM1 positive cells, combines the cell killing effect of human toxin Caspase-3, belongs to a novel therapeutic drug of antibody coupling drug, can better realize the effect of specifically killing tumor cells and can reduce the damage to normal cells of an organism; the invention adopts the Escherichia coli Nissle1917 as a drug expression vector to mediate prokaryotic plasmid expression, thereby greatly reducing the drug cost; and the escherichia coli Nissle1917 is probiotics and is safe and nontoxic; escherichia coli Nissle1917 belongs to extracellular bacteria and is easier to be cleared by antibiotics than intracellular bacteria; the genome and genetic background of the Escherichia coli Nissle1917 are relatively clear and are easier to modify; escherichia coli Nissle1917 is facultative anaerobe, is less limited by factors such as tumor size and microenvironment in tumors in the aspect of tumor selection, and has a greater application value.
Drawings
FIG. 1 is a diagram showing the PCR verification result after extraction of plasmids from positive clones in example 1 of the present invention; m: Marker-DL 2000; 1-3: plasmid pET302-78-Caspase-3 in coli Nissle1917-pET 302-78-Caspase-3; 1 shows that the construction was successful.
FIG. 2 is a microscopic image of the cell killing ability of Caspase-3 protein in example 1 of the present invention.
FIG. 3 is a graph showing the results of the affinity ELISA detection of the recombinant bacteria on TEM1 positive cells in example 1 of the present invention.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Approximating language, as used herein in the following examples, may be applied to identify quantitative representations that could permissibly vary in number without resulting in a change in the basic function. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The original plasmids pET302, E.coli Top10, E.coli Nissle1917, restriction enzyme, T4DNA ligase, LB solid medium and liquid medium used in the following experiments were all conventional biochemical experimental materials and were purchased from the market.
Example 1
1. Using pET302-78-Caspase-3 as a template and SEQ ID No.3 and SEQ ID No.4 sequences as primers, and carrying out PCR amplification to obtain a 78-Caspase-3 fragment shown in SEQ ID No. 1.
Wherein 78 is a single-chain antibody which is screened and obtained in the earlier stage of the laboratory and can specifically recognize TEM1, and is named as 78; caspase3 is an apoptosis inducing factor, and gene sequence of Caspase3 fragment is obtained through GeneBank, and then the gene sequence is handed over to a company (Nanjing Kingsry) for complete sequence chemical synthesis to obtain pUC 57-78-Csepase 3 recombinant plasmid or pET302-78-Caspase-3 recombinant plasmid. And then using pET302-78-Caspase-3 or pUC 57-78-Csepase 3 as a template and SEQ ID No.3 and SEQ ID No.4 sequences as primers, and carrying out PCR amplification to obtain a 78-Caspase-3 fragment shown in SEQ ID No. 1.
2. The obtained 78-Caspase-3 fragment and plasmid pET302 are subjected to double enzyme digestion by Xho I and Avr II, after enzyme digestion products are purified, T4 ligase is adopted to be connected into plasmid pET302(SEQ ID No.2), and the recombinant plasmid is named as pET 302-78-Caspase-3. The results are shown in FIG. 1.
3. Escherichia coli Nissle1917 stored at-80 ℃ is streaked and inoculated on an LB plate without resistance, and cultured overnight at 37 ℃; picking a single colony in 5mL LB, and carrying out shake culture at 37 ℃ for 12 h; inoculating the strain into 100mL LB according to the proportion of 1:100, and performing shaking culture until the OD of the bacteria is about 0.4; after ice-bath for 20min, centrifuging at 4 ℃ and 3000rpm for 10 min; washing thallus precipitate twice with 1/10 volume of precooled sterile deionized water, and centrifuging at 4 ℃ and 3000rpm for 10 min; washing thallus with 1/100 volume precooled 10% glycerol, centrifuging at 4 deg.C 3000rpm for 10 min; the pellet was resuspended in 1/100 volumes of pre-cooled 10% glycerol to make E.coli Nissle1917 competent, dispensed and retained at-80 ℃ for use.
4. Transforming the obtained recombinant plasmid pET302-78-Caspase-3 into escherichia coli Nissle1917, melting competent cells (100 mu L) of the escherichia coli Nissle1917 on ice, adding the recombinant plasmid (2 mu L) to pat the tube wall uniformly, and placing on the ice for incubation for 30 min; soaking the bacteria solution in 42 deg.C water bath, thermally shocking for 60-90s (no vibration), immediately placing on ice, and standing for 2 min; adding 900 μ L SOC culture solution, and incubating for 1h (220rpm) on a constant temperature shaker at 37 deg.C; and (3) centrifuging the bacterial liquid at 4000rpm for 3min, discarding redundant liquid, suspending the bacterial liquid by using the residual 100 mu L of culture medium, adding all the bacterial liquid to an LB solid culture plate containing ampicillin resistance, placing the bacterial liquid on a 37 ℃ inversion culture plate, and screening positive clones overnight to obtain recombinant escherichia coli Nissle1917, which is named as E.
5. The 96-well plates were coated with 2% gelatin (37 ℃, 30min), and after gelatin was spun off, MS1-TEM1 cells and MS1 cells were plated together (37 ℃, overnight growth). scFv78 diluted with PBS was incubated with cells (4 ℃ C., 2h), followed by incubation of secondary antibody at 4 ℃ for 1h, OD was measured by color development, and affinity of 78-Caspase-3 to TEM1 positive cells was examined by live cell ELISA. The experimental results are shown in fig. 2 and fig. 3, and the recombinant escherichia coli E.coli Nissle1917-pET302-78-Caspase-3 has good affinity and lethality to TEM1 positive cells, so that the recombinant bacterium has an exact anti-tumor effect.
Example 2
Recombinant escherichia coli Nissle1917 for expressing Caspase-3 recombinant scFv78, wherein the recombinant escherichia coli Nissle1917 is constructed by the following method:
1) connecting the encoding gene fragment 78-Caspase-3 of the human toxin Caspase-3 recombinant single-chain antibody scFv78 to pET302 plasmid by a method of enzyme digestion and enzyme ligation to obtain pET302-78-Caspase-3 recombinant plasmid;
2) and (3) converting the pET302-78-Caspase-3 recombinant plasmid into Escherichia coli Nissle1917 to obtain recombinant Escherichia coli E.coli Nissle1917-pET302-78-Caspase-3, namely the recombinant Escherichia coli Nissle1917 for expressing Caspase-3 recombinant scFv 78.
Wherein, the coding gene fragment 78-Caspase-3 of the recombinant single-chain antibody scFv78 of the human toxin Caspase-3 in the step 1) is a DNA fragment with the nucleotide sequence shown as SEQ ID No. 1.
The coding gene fragment 78-Caspase-3 of the recombinant single-chain antibody scFv78 of the human toxin Caspase-3 in the step 1) is obtained by PCR amplification by taking DNA fragments with nucleotide sequences shown as SEQ ID No.3 and SEQ ID No.4 as primers.
The pET302-78-Caspase-3 recombinant plasmid used in the step 2) is obtained by transferring the pET302-78-Caspase-3 recombinant plasmid obtained in the step 1) into E.coli Top10, and then culturing and amplifying the E.coli Top 10.
The enzyme digestion enzyme in the step 1) is subjected to double enzyme digestion by Xho I and Avr II, and after the enzyme digestion product is purified, T4 ligase is adopted to be connected into pET302 plasmid.
The step 2) comprises the following steps: melting 100 μ L of bacterial liquid of Escherichia coli Nissle1917 competent cells on ice, adding 2 μ L of the pET302-78-Caspase-3 recombinant plasmid, mixing, incubating 30min on ice, immersing the bacterial liquid into 42 ℃ water bath for heat shock for 60-90s, then immediately placing on ice and standing for 2min, adding 900 μ L SOC culture solution, shaking and incubating at 37 deg.C on a constant temperature shaker at 22rpm for 1h, then centrifuging the bacterial liquid at the rotating speed of 4000rpm for 3min, discarding redundant liquid, suspending the bacterial cells by using the residual 100 mu L of culture medium, adding all the bacterial liquid to an LB solid culture plate containing ampicillin resistance, carrying out inverted culture at 37 ℃ overnight, screening positive clones, thus obtaining the recombinant Escherichia coli E.coli Nissle1917-pET302-78-Caspase-3, namely the recombinant Escherichia coli Nissle1917 for expressing the Caspase-3 recombinant scFv 78.
The escherichia coli Nissle1917 competent cell is prepared by the following method: coli Nissle1917 was streaked on an LB plate without resistance and cultured overnight at 37 ℃; selecting a single colony, inoculating the single colony in 5mL LB culture medium, and carrying out shaking culture at 37 ℃ for 12 h; inoculating the strain into 100mL LB culture medium according to the proportion of 1:100, and performing shaking culture until the OD value of bacteria is 0.4; ice-cooling for 20min, and centrifuging at 4 deg.C and 3000rpm for 10 min; washing thallus precipitate twice with 1/10 volume pre-cooled sterile deionized water, centrifuging at 4 deg.C and 3000rpm for 10 min; washing thallus with 1/100 volume precooled 10% glycerol, centrifuging at 4 deg.C and 3000rpm for 10 min; and (3) suspending the thallus precipitate in 1/100 volume of precooled 10% glycerol to obtain the escherichia coli Nissle1917 competent cell.
The pET302 plasmid is a DNA molecule with a nucleotide sequence shown as SEQ ID No. 2.
Next, the tumor cell killing function of recombinant E.coli Nissle1917 can be verified by the following method A or B:
the method A comprises the following steps: expressing the recombinant human toxin Caspase-3 single-chain antibody scFv78 by using the recombinant Escherichia coli E.coli Nissle1917-pET302-78-Caspase-3, co-incubating the recombinant human toxin Caspase-3 single-chain antibody scFv78 with TEM1 positive cells, and detecting the affinity of the recombinant human toxin Caspase-3 single-chain antibody scFv78 with the TEM1 positive cells by live cell ELISA.
The method B comprises the following steps: coating a 96-well plate with 2% gelatin for 30min at 37 ℃, removing the gelatin, and connecting the MS1-TEM1 cell and the MS1 cell for overnight growth at 37 ℃; incubating the human toxin Caspase-3 recombinant single-chain antibody scFv78 diluted by PBS with cells for 2h at 4 ℃, then incubating the secondary antibody for 1h at 4 ℃, performing color development to measure OD value, and detecting the affinity of the human toxin Caspase-3 recombinant single-chain antibody scFv78 with the MS1-TEM1 by using a living cell ELISA method.
The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.
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gcaccaagct gaccgtccta ggcggtggtt cctctagatc ttcctcctct ggtggcggtg 300
gctcgggcgg tggtgggcag gtgcagctgc aggagtcggg gggaaccttg gtacagcctg 360
gtaggtccct gagactctct tggaaagcct ctggattcac ctttagcaac tatgccatgg 420
gctgggtccg ccagactcca ggaaaggggc tggagtggct gtcggctatt cgtaaaagtg 480
gtcctaccac atactacgcg gactccgtga agggccggtt catcatctcc agagacaatt 540
ccaagaacac cctgtatctg caaatgaata ggctgagagt cggcgacacg gccacttatt 600
actgtgcgac tcaccccatc gcgggctact ggggccaggg aaccc 645
<210>2
<211>5712
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>2
gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat 60
tcccctctag aaataatttt gtttaaactt taagaaggag atatacatat gcatcatcat 120
catcatcacg tgaattcgct cgagatcgat gatattcgag cctaggtata atcggatccg 180
gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta 240
gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact 300
atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag 360
catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg 420
cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctag cttatcgatg 480
ataagctgtc aaacatgaga attaattctt gaagacgaaa gggcctcgtg atacgcctat 540
ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 600
gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 660
tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 720
ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 780
ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 840
gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 900
gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 960
acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 1020
actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 1080
ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 1140
cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 1200
gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag 1260
caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 1320
aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 1380
ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 1440
tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 1500
ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 1560
ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 1620
ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1680
tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1740
cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1800
taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 1860
gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 1920
acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 1980
ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2040
ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2100
cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2160
aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2220
gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2280
gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2340
gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 2400
ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 2460
ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 2520
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct 2580
cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 2640
gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 2700
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 2760
cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg 2820
tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc 2880
tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc 2940
tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg 3000
atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg 3060
gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact 3120
cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag 3180
catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga 3240
ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg 3300
cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg 3360
caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc 3420
caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg 3480
gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct 3540
ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg 3600
tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc 3660
ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga 3720
tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc 3780
cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc 3840
cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca 3900
gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga 3960
aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 4020
ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 4080
cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 4140
cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 4200
agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc 4260
gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4320
aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt 4380
gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 4440
tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata 4500
taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc 4560
agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 4620
atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 4680
gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 4740
tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg 4800
atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg 4860
gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca 4920
ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc 4980
agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 5040
cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc 5100
gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 5160
aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc 5220
atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 5280
cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt 5340
ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag 5400
gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat 5460
taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc 5520
atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc 5580
gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc 5640
gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc 5700
gtagaggatc ga 5712
<210>3
<211>22
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>3
cttcctcgag atgttggttc ct 22
<210>4
<211>22
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>4
tattcctagg gggttccctg gc 22
Claims (10)
1. Recombinant escherichia coli expressing Caspase-3 recombinant scFv78, which is characterized in that the recombinant escherichia coli is constructed by the following method:
1) connecting the encoding gene fragment 78-Caspase-3 of the human toxin Caspase-3 recombinant single-chain antibody scFv78 to pET302 plasmid by a method of enzyme digestion and enzyme ligation to obtain pET302-78-Caspase-3 recombinant plasmid;
2) and (3) converting the pET302-78-Caspase-3 recombinant plasmid into Escherichia coli Nissle1917 to obtain recombinant Escherichia coli E.coli Nissle1917-pET302-78-Caspase-3, namely the recombinant Escherichia coli for expressing Caspase-3 recombinant scFv 78.
2. The recombinant Escherichia coli expressing Caspase-3 recombinant scFv78 according to claim 1, wherein the gene fragment 78-Caspase-3 encoding the recombinant single chain antibody scFv78 of human toxin in step 1) is a DNA fragment having a nucleotide sequence shown in SEQ ID No. 1.
3. The recombinant Escherichia coli expressing Caspase-3 recombinant scFv78 of claim 2, wherein the encoding gene segment 78-Caspase-3 of the recombinant single chain antibody scFv78 of human toxin Caspase-3 in step 1) is obtained by PCR amplification using DNA segments with nucleotide sequences shown in SEQ ID No.3 and SEQ ID No.4 as primers.
4. The recombinant Escherichia coli expressing Caspase-3 recombinant scFv78 of claim 1, wherein the recombinant plasmid pET302-78-Caspase-3 used in step 2) is obtained by transferring the recombinant plasmid pET302-78-Caspase-3 obtained in step 1) into E.coli Top10, culturing and amplifying.
5. The recombinant Escherichia coli expressing Caspase-3 recombinant scFv78 of claim 1, wherein the enzyme in step 1) is Xho I and Avr II, and the enzyme is purified and ligated to pET302 plasmid using T4 ligase.
6. The recombinant E.coli expressing Caspase-3 recombinant scFv78 according to claim 1, wherein step 2) comprises: melting 100 μ L of bacterial liquid of Escherichia coli Nissle1917 competent cells on ice, adding 2 μ L of the pET302-78-Caspase-3 recombinant plasmid, mixing, incubating on ice for 30min, immersing the bacterial liquid in 42 ℃ water bath for heat shock for 60-90s, then immediately placing on ice and standing for 2min, adding 900 μ L SOC culture solution, shaking and incubating at 37 deg.C on a constant temperature shaker at 22rpm for 1h, then centrifuging the bacterial liquid at the rotating speed of 4000rpm for 3min, discarding redundant liquid, suspending the bacterial cells by using the residual 100 mu L of culture medium, adding all the bacterial liquid to an LB solid culture plate containing ampicillin resistance, carrying out inverted culture at 37 ℃ overnight, screening positive clones, thus obtaining the recombinant Escherichia coli E.coli Nissle1917-pET302-78-Caspase-3, namely the recombinant Escherichia coli for expressing the Caspase-3 recombinant scFv 78.
7. The recombinant E.coli expressing Caspase-3 recombinant scFv78 according to claim 6, wherein the E.coli Nissle1917 competent cell is prepared by the following method: coli Nissle1917 was streaked on an LB plate without resistance and cultured overnight at 37 ℃; selecting a single colony, inoculating the single colony in 5mL LB culture medium, and carrying out shaking culture at 37 ℃ for 12 h; inoculating the strain into 100mL LB culture medium according to the proportion of 1:100, and performing shaking culture until the OD value of bacteria is 0.4; ice-cooling for 20min, and centrifuging at 4 deg.C and 3000rpm for 10 min; washing thallus precipitate twice with 1/10 volume pre-cooled sterile deionized water, centrifuging at 4 deg.C and 3000rpm for 10 min; washing thallus with 1/100 volume precooled 10% glycerol, centrifuging at 4 deg.C and 3000rpm for 10 min; and (3) suspending the thallus precipitate in 1/100 volume of precooled 10% glycerol to obtain the escherichia coli Nissle1917 competent cell.
8. The recombinant Escherichia coli expressing Caspase-3 recombinant scFv78 according to claim 1, wherein the pET302 plasmid is a DNA molecule having the nucleotide sequence shown in SEQ ID No. 2.
9. The method for verifying the anti-tumor function of the recombinant escherichia coli of any one of claims 1 to 8, wherein the recombinant escherichia coli e.coli Nissle1917-pET302-78-Caspase-3 is used to express the recombinant human toxin Caspase-3 single-chain antibody scFv78, the recombinant human toxin Caspase-3 single-chain antibody scFv78 is incubated with TEM1 positive cells, and then the affinity of the recombinant human toxin Caspase-3 single-chain antibody scFv78 with the TEM1 positive cells is detected by live cell ELISA.
10. The method for verifying the antitumor function of recombinant Escherichia coli according to any one of claims 1 to 8, comprising the steps of: coating a 96-well plate with 2% gelatin for 30min at 37 ℃, removing the gelatin, and connecting the MS1-TEM1 cell and the MS1 cell for overnight growth at 37 ℃; incubating the human toxin Caspase-3 recombinant single-chain antibody scFv78 diluted by PBS with cells for 2h at 4 ℃, then incubating the secondary antibody for 1h at 4 ℃, performing color development to measure OD value, and detecting the affinity of the human toxin Caspase-3 recombinant single-chain antibody scFv78 with the MS1-TEM1 by using a living cell ELISA method.
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