CN113603790A

CN113603790A - RGD-anti-p 21Ras single-chain antibody fusion protein capable of entering tumor cells and preparation method thereof

Info

Publication number: CN113603790A
Application number: CN202110927679.9A
Authority: CN
Inventors: 杨举伦; 冯强; 黄晨晨; 刘芳枘; 潘鑫艳
Original assignee: 920th Hospital of the Joint Logistics Support Force of PLA
Current assignee: 920th Hospital of the Joint Logistics Support Force of PLA
Priority date: 2021-08-14
Filing date: 2021-08-14
Publication date: 2021-11-05

Abstract

The invention discloses a sequence, expression and purification preparation method and application of RGD-anti-p 21Ras single-chain antibody fusion protein capable of entering tumor cells. The invention establishes an optimal fusion protein prokaryotic recombinant expression system by screening different prokaryotic expression vectors and combinations of bacterial strains, optimizes expression conditions, and establishes a pilot-scale expression and purification process by using a fermentation tank and an AKTA chromatography system. Compared with the prior art, the invention improves the expression quantity and purity of the fusion protein, lays a foundation for large-scale production, and simultaneously, the RGD-anti-p 21Ras single-chain antibody fusion protein can enter tumor cells and is combined with p21Ras protein, thereby blocking Ras signal path and achieving the purposes of inhibiting the growth of the tumor cells and inducing the apoptosis of the tumor cells. The RGD-anti-p 21Ras single-chain antibody fusion protein has wide application prospect in the aspect of preparing preparations for treating Ras-related tumors.

Description

RGD-anti-p 21Ras single-chain antibody fusion protein capable of entering tumor cells and preparation method thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to RGD-anti-p 21Ras single-chain antibody fusion protein capable of entering tumor cells and a preparation method thereof.

Background

The ras gene is an important cellular proto-oncogene, named rat asrcoma from rat sarcoma. The ras gene family includes three major members: H-Ras, K-Ras and N-Ras, which are located on chromosomes 12, 11 and 1, respectively, encode proteins consisting of 188-189 amino acids with a molecular weight of about 21KD, and the three Ras proteins have 85% amino acid sequence homology.

Ras proteins regulate normal differentiation and proliferation of cells as extremely important signaling transport proteins. After the Ras protein is synthesized, the carboxyl terminal of the Ras protein must be subjected to complex post-translational modification so that the Ras protein is accurately positioned on the inner side surface of a cell membrane to play the biological function of the Ras protein. The Ras protein is combined with GTP under the stimulation of an extracellular growth signal to form an active Ras-GTP form, so that a plurality of signal channels at the downstream of the Ras protein are activated, and cell division, proliferation and malignant transformation are promoted.

When ras gene is mutated or overexpressed, binding to GTP becomes active, leading to malignant transformation of the cell and promoting infiltration and metastasis of malignant tumor cells. Studies have shown that ras gene mutations or overexpression of ras genes occur in about 30% of human tumors.

The single-chain antibody constructed by utilizing the genetic engineering technology is a linear fragment constructed by connecting a flexible oligonucleotide fragment with a variable region of a complete antibody, has the molecular weight of only 1/6 of the complete antibody, has strong penetrability, has short retention time in non-target tissues and is easy to remove from the body. And because there is no Fc fragment of the whole antibody, the immunogenicity is low, and the antibody can hardly generate anti-mouse antibody reaction when used in human body. Such antibodies inactivate a particular protein by binding to it within the cell, block its interaction with other proteins, or interfere with the normal intracellular localization of the protein, thereby preventing it from performing its normal biological function.

The invention utilizes the mixed primers of light and heavy chains of mouse antibodies to amplify from Balb/c mouse spleen B lymphocytes after Ras protein immunization to obtain the gene segments of light and heavy chain variable regions thereof. Through the overlap extension PCR technology, flexible oligonucleotide and the two fragments are connected to construct the single-chain antibody gene fragment of the anti-p 21Ras protein. In order to prepare the anti-p 21Ras single-chain antibody which can specifically enter tumor cells, the RGD transmembrane peptide and the anti-p 21Ras single-chain antibody gene are constructed into a fusion expression gene on the basis of the former period, the codon of the RGD-anti-p 21Ras single-chain antibody fusion gene is optimized, the optimized RGD-anti-p 21Ras single-chain antibody fusion protein gene is cloned to a pET-32a prokaryotic expression vector, and the recombinant expression vector is transferred into Origami (DE3) escherichia coli expression bacteria to construct an RGD-anti-p 21Ras single-chain antibody fusion protein recombinant expression system. Through a series of expression and purification conditions, RGD-anti-p 21Ras single-chain antibody fusion protein capable of penetrating tumor cell membranes and inhibiting tumor growth is prepared.

Disclosure of Invention

The invention aims to overcome the defects and provide an RGD-anti-p 21Ras single-chain antibody fusion protein capable of entering tumor cells, wherein the amino acid sequence of the fusion protein is shown as SEQ ID NO: 2, respectively.

The RGD-anti-p 21Ras single-chain antibody fusion protein is a fusion polypeptide consisting of RGD transmembrane peptide, a heavy chain variable region, a connecting peptide and a light chain variable region; the linker peptide is located between the heavy chain variable region and the light chain variable region as set forth in SEQ ID NO: 2 at position 136-150. The RGD transmembrane peptide sequence is located in SEQ ID NO: 2, 1-11 amino acid sequence.

The invention also aims to provide an RGD-anti-p 21Ras single-chain antibody fusion protein recombinant expression plasmid which is formed by cloning the RGD-anti-p 21Ras single-chain antibody fusion protein gene to a position between Kpn I and Hind III enzyme cutting sites of a prokaryotic expression plasmid pET-32 a. The RGD-anti-p 21Ras single-chain antibody fusion protein gene is shown as SEQ ID NO: 1 is shown.

The invention also aims to provide a prokaryotic expression system of RGD-anti-p 21Ras single-chain antibody fusion protein, which is formed by transforming the recombinant expression plasmid into an Escherichia coli expression bacterium Origami (DE 3).

The invention also aims to provide a fermentation tank induced expression condition of the RGD-anti-p 21Ras single-chain antibody fusion protein, which can be used for large-scale fermentation production of the expressed fusion protein.

The invention also aims to provide a purification method of the RGD-anti-p 21Ras single-chain antibody fusion protein, which comprises the steps of firstly carrying out ultrasonic disruption and recombination on the expression bacteria, centrifugally collecting the RGD-anti-p 21Ras single-chain antibody fusion protein existing in an inclusion body form, washing the fusion protein by using an inclusion body washing solution, then using a denaturant containing 8M urea to denature the fusion protein, purifying the denatured inclusion body fusion protein by using a nickel ion affinity chromatography column and an AKTA system, and finally adopting a urea gradient dialysis renaturation method to refold the denatured fusion protein to recover the bioactivity of the fusion protein so as to achieve the purpose of purifying the protein.

The RGD-anti-p 21Ras single-chain antibody fusion protein can widely antagonize three p21Ras proteins of H-Ras, K-Ras and N-Ras; the fusion protein can penetrate through the cell membrane of a tumor cell with high integrin expression to enter the tumor cell and cannot penetrate through the cell membrane of a normal cell to enter the normal cell, and can be used for diagnostic research, pathogenesis research or therapeutic research of ras gene-related tumors.

The above object of the present invention is achieved by the following scheme:

the invention adds RGD peptide sequence at the N-terminal of the anti-p 21Ras single-chain antibody, so that the antibody has the capability of specifically penetrating tumor cell membranes, thereby being combined with p21Ras protein in tumor cells and blocking Ras signal path.

The invention optimizes the codon of the RGD-anti-p 21Ras single-chain antibody fusion protein gene, changes the DNA sequence of the original fusion protein, ensures that the codon sequence is more suitable for being expressed in escherichia coli, and improves the expression quantity of the RGD-anti-p 21Ras single-chain antibody fusion protein in the escherichia coli.

The invention determines the optimal combination of the prokaryotic expression plasmid of the RGD-anti-p 21Ras single-chain antibody fusion protein and the Escherichia coli expression strain through experiments.

The invention determines the optimal induction expression condition of the RGD-anti-p 21Ras single-chain antibody fusion protein through experiments, and determines the optimal condition for purifying the RGD-anti-p 21Ras single-chain antibody fusion protein through nickel ion affinity chromatography by an AKTA chromatography system, thereby further improving the expression quantity and the protein purity of the protein.

The invention relates to an RGD-anti-p 21Ras single-chain antibody fusion protein capable of specifically entering tumor cells and a preparation method thereof, wherein the preparation method of the fusion protein comprises the following steps:

1. construction of anti-p 21Ras Single-chain antibody Gene

(1) The light and heavy chain variable region gene segments of Balb/c mice immunized by p21Ras protein are obtained by multiple amplification of B lymphocytes of the spleen by using mouse antibody light and heavy chain mixed primers. And connecting a flexible oligonucleotide chain (linker) with the two fragments by overlap extension PCR to construct a single-chain antibody gene fragment.

(2) Introducing different enzyme cutting sites at two ends of the gene fragment of the single-chain antibody respectively and connecting the two enzyme cutting sites with a phagemid expression vector pCANTAB-5E subjected to synchronous double enzyme cutting to obtain the recombinant phagemid. And transforming the recombinant phagemid with correct identification connection into escherichia coli TG1, rescuing by using an auxiliary phage M13K07, carrying out fusion expression on a target single-chain antibody gene fragment and a gIII gene in an expression vector by using a phage display technology, displaying on the tail surface of the phage to obtain a fusion-expressed single-chain antibody, detecting the fusion-expressed single-chain antibody by using an indirect ELISA (enzyme-linked immunosorbent assay) experiment, and screening out the positive recombinant phagemid expressing the anti-p 21Ras single-chain antibody.

(3) And (3) converting the positive recombinant phagemid obtained by screening into escherichia coli BL21(DE3) for soluble expression, thereby obtaining a soluble anti-p 21Ras single-chain antibody which is expressed in a fusion manner with the E-tag label, detecting the specificity and the affinity of the target single-chain antibody by using the single-chain antibody as a primary antibody and the E-tag antibody as a secondary antibody by adopting an indirect ELISA (enzyme-linked immunosorbent assay) experiment and an immunocytochemistry method, and confirming that the single-chain antibody can specifically recognize three Ras proteins.

Construction of RGD-anti-Ras single-chain antibody fusion protein recombinant expression vector

Because the anti-p 21Ras single-chain antibody constructed before can not directly penetrate cell membranes, the DNA sequence of the anti-p 21Ras single-chain antibody is improved, the DNA sequence of RGD membrane-penetrating peptide is added at the 5' end of the DNA sequence of the anti-p 21Ras single-chain antibody, and the RGD membrane-penetrating peptide and the anti-p 21Ras single-chain antibody are subjected to fusion expression in a prokaryotic expression system, so that the RGD-anti-p 21Ras single-chain antibody fusion protein can penetrate the cell membranes of tumor cells with high integrin expression, is combined with p21Ras protein in the tumor cells, but does not enter normal cells. Meanwhile, the codon of the RGD-anti-p 21Ras single-chain antibody fusion protein DNA sequence is optimized to improve the expression amount of the protein in escherichia coli. Finally, the optimized DNA fragment of the RGD-anti-p 21Ras single-chain antibody fusion protein is cloned into pET-32a prokaryotic expression plasmid, and the RGD-anti-p 21Ras single-chain antibody fusion protein recombinant expression plasmid is constructed.

3, establishment of RGD-anti-p 21Ras single-chain antibody fusion protein expression and purification process

(1) Constructing an RGD-anti-p 21Ras single-chain antibody fusion protein prokaryotic expression system: the RGD-anti-p 21Ras single-chain antibody fusion protein recombinant expression plasmid is transformed into Escherichia coli Origami (DE3), a positive clone is screened out through an LB plate containing ampicillin, and PCR identification is carried out to determine that the positive clone contains RGD-anti-p 21Ras single-chain antibody fusion protein gene.

(2) Establishment of RGD-anti-p 21Ras single-chain antibody fusion protein prokaryotic expression process: in a 1L triangular shake flask, the shake flask expression conditions are optimized by setting the concentration gradient, the induction time gradient, the induction mode and the like of an inducer IPTG through a single-factor variable, and the optimal induction culture medium and the optimal induction expression conditions of the RGD-anti-p 21Ras single-chain antibody fusion protein are determined. And finally, fermenting and expressing the RGD-anti-p 21Ras single-chain antibody fusion protein by using a fermentation tank under the optimal culture condition, and establishing an expression process of the RGD-anti-p 21Ras single-chain antibody fusion protein.

(3) Establishment of RGD-anti-p 21Ras single-chain antibody fusion protein purification process: collecting the bacteria liquid after induction expression, carrying out ultrasonic crushing on thalli after centrifugation, and centrifugally collecting the precipitated inclusion body protein after ultrasonic crushing; washing the inclusion body protein, and then using a denaturant containing urea to denature the inclusion body protein; purifying the denatured inclusion body protein by using an AKTA chromatography system through a nickel ion affinity chromatography column; urea gradient is adopted, and dialysis renaturation is carried out step by step to ensure that the denatured inclusion body protein is refolded and the biological activity of the inclusion body protein is recovered.

(4) Identification of RGD-anti-p 21Ras single-chain antibody fusion protein: the purity of the RGD-anti-p 21Ras single-chain antibody fusion protein was identified by SDS-PAGE, the concentration of the RGD-anti-p 21Ras single-chain antibody fusion protein was detected by a spectrophotometer using the BCA method, and the immunological activity of the RGD-anti-p 21Ras single-chain antibody fusion protein was detected by WB and ELISA.

The RGD-anti-p 21Ras single-chain antibody fusion protein capable of entering tumor cells is realized by the following method for inhibiting the tumor cells:

(1) the membrane penetration capability of the fusion protein to tumor cells with high expression of integrin is detected through immunocytochemistry and immunofluorescence experiments.

(2) RGD-anti-p 21Ras single-chain antibody fusion protein is used for in vitro tumor inhibition experiments. The killing effect of the fusion protein on the tumor is detected by an MTT method, the influence of the fusion protein on the tumor migration capacity is detected by a cell scratch experiment, the influence of the fusion protein on the tumor proliferation capacity is detected by a plate clone, and the apoptosis inducing effect of the fusion protein on the tumor cell is detected by TUNEL.

The invention has the following beneficial results: the invention creatively fuses an anti-p 21Ras single-chain antibody constructed in the early stage with RGD cell-penetrating peptide, enables the antibody to be expressed in a large amount in an escherichia coli expression system through codon optimization, and obtains RGD-anti-p 21Ras single-chain antibody fusion protein through a series of purification, so that the anti-p 21Ras single-chain antibody has the capacity of penetrating the cell membrane of a tumor cell, thereby enabling the anti-p 21Ras single-chain antibody to be combined with the p21Ras protein in the tumor cell, further blocking a Ras signal path, and achieving the purposes of inhibiting the growth of the tumor cell and inducing the apoptosis of the tumor cell. The RGD-anti-p 21Ras single-chain antibody fusion gene is subjected to codon optimization and is respectively cloned into three prokaryotic expression plasmids of pET-28a, pET-32a and pET-22b, then the three recombinant expression plasmids are respectively transformed into prokaryotic expression bacteria of escherichia coli BL21(DE3), Origami (DE3) and Origami (DE3), 9 RGD-anti-p 21Ras single-chain antibody fusion protein prokaryotic expression systems are constructed, and recombinant expression plasmids and expression strain combinations with the highest expression quantity are screened out from the prokaryotic expression systems. And finally, optimizing the fermentation conditions of the shake flask and the fermentation tank, and further improving the yield of the RGD-anti-p 21Ras single-chain antibody fusion protein in the fermentation tank by using the self-induction culture medium, so that the finally expressed protein yield reaches 30 times of that of the shake flask IPTG/LB culture medium. Finally, an AKTA system is used for exploring and determining the purification parameters of nickel ion affinity chromatography through experiments, and the RGD-anti-p 21Ras single-chain antibody fusion protein with the purity of 85 percent is finally obtained. The expression and purification conditions of the RGD-anti-p 21Ras single-chain antibody fusion protein determined by the invention can be linearly amplified, and can be directly used for industrial production of the RGD-anti-p 21Ras single-chain antibody fusion protein. The RGD-anti-p 21Ras single-chain antibody fusion protein is proved to be capable of specifically penetrating cell membranes of tumor cells with high integrin expression through in vitro membrane penetration experiments and in vitro tumor cell inhibition experiments, remarkably inhibiting migration and proliferation of the tumor cells in vitro, killing and inducing apoptosis of the tumor cells, and providing a new targeted medicament for future tumor treatment.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention adds RGD cell-penetrating peptide gene sequence at the 5' end of the single-chain antibody gene obtained at the earlier stage, so that the anti-p 21Ras single-chain antibody can specifically penetrate the cell membrane of tumor cells, and can be combined with p21Ras protein in the tumor cells to play a role in blocking Ras signal path.

(2) The RGD-anti-p 21Ras single-chain antibody fusion protein is obtained by a prokaryotic expression system, and compared with the prior art (the adenovirus carries the anti-p 21Ras single-chain antibody gene), the RGD-anti-p 21Ras single-chain antibody fusion protein prepared by the invention is easier to prepare and easier to realize industrial production.

(3) The invention optimizes the codon of the RGD-anti-p 21Ras single-chain antibody fusion gene, simultaneously determines the optimal prokaryotic expression system by screening different prokaryotic expression plasmids and prokaryotic expression strains, and determines the optimal culture medium for induced expression and the optimal fermentation condition through experiments, so that the expression quantity of the RGD-anti-p 21Ras single-chain antibody fusion protein is improved by 60 times compared with the prior art.

Drawings

FIG. 1 is a schematic diagram of three recombinant prokaryotic expression plasmids of the present invention;

FIG. 2 is an electrophoretogram of RGD-anti-p 21Ras single-chain antibody fusion gene in the PCR-identified recombinant prokaryotic expression strain of the present invention; DL2000, lanes 1-2: pET28a-RGD-p21Ras scfv/BL21(DE3), lane 3: pET28a-RGD-p21Ras scfv/Origami (DE3), lane 4: pET28a-RGD-p21Ras scfv/Origami B (DE3), lane 5: pET32a-RGD-p21Ras scfv/BL21(DE3), lane 6: pET32a-RGD-p21Ras scfv/Origami (DE3), lane 7: pET32a-RGD-p21Ras scfv/Origami B (DE3), lane 8: pET22b-RGD-p21Ras scfv/BL 27 (DE3), lane 9: pET 22-b-p 21Ras scfv/Ras 4642, lane 10: pET 4642-RGD-p 4680/Ras 51 (DE 3);

FIG. 3 is an electrophoretogram of SDS-PAGE identifying the expression level of a target protein under different induction conditions; m is protein Marker, lanes 1-2, expression level of RGD-anti-p 21Ras single-chain antibody fusion protein in pET32a-RGD-p21Ras scfv/Origami (DE3) recombinant bacteria under self-induction conditions, lane 3, expression level of RGD-anti-p 21Ras single-chain antibody fusion protein in pET32a-RGD-p21Ras scfv/Origami (DE3) recombinant bacteria under IPTG induction conditions, lane 4, non-induced pET32a-RGD-p21Ras scfv/Origami (DE3) recombinant bacteria;

FIG. 4 is an electrophoretogram of the present invention monitoring the purification process of a target protein by SDS-PAGE; m is protein Marker, lane 1, unpurified inclusion body RGD-anti-p 21Ras single-chain antibody fusion protein, lane 2, purified soluble RGD-anti-p 21Ras single-chain antibody fusion protein, lane 3, purified inclusion body RGD-anti-p 21Ras single-chain antibody fusion protein, lane 4, inclusion body RGD-anti-p 21Ras single-chain antibody fusion protein after dialysis renaturation;

FIG. 5 shows immunoreactivity of a WB-detected RGD-anti-p 21Ras single-chain antibody fusion protein; m is protein Marker, 1 is uninduced pET32a-RGD-p21Ras scfv/Origami (DE3), 2 is purified soluble RGD-anti-p 21Ras single-chain antibody fusion protein, 3 is purified inclusion body RGD-anti-p 21Ras single-chain antibody fusion protein, and 4 is dialysis renatured inclusion body RGD-anti-p 21Ras single-chain antibody fusion protein;

FIG. 6 shows the potency of the RGD-anti-p 21Ras single-chain antibody fusion protein detected by ELISA according to the present invention;

FIG. 7 shows the in vitro membrane penetration effect of the immunohistochemical detection RGD-anti-p 21Ras single-chain antibody fusion protein of the present invention; bar 1 shows that the RGD-anti-p 21Ras single-chain antibody fusion protein enters SW480 cells, and bar 2 shows that the anti-p 21Ras single-chain antibody alone cannot enter SW480 cells;

FIG. 8 shows the membrane penetration effect of the immunofluorescence assay RGD-anti-p 21Ras single-chain antibody fusion protein of the present invention; wherein, the red signal is RGD-anti-p 21Ras single-chain antibody fusion protein, the blue signal is cell nucleus, the horizontal row 1 shows that the RGD-anti-p 21Ras single-chain antibody fusion protein enters SW480 cells, and the horizontal row 2 shows that the single anti-p 21Ras single-chain antibody can not enter SW480 cells;

FIG. 9 shows the effect of RGD-anti-p 21Ras single-chain antibody fusion protein on the migration ability of tumor cells detected by the scratching experiment of the present invention;

FIG. 10 is a graph showing the effect of RGD-anti-p 21Ras single-chain antibody fusion protein on the proliferation ability of tumor cells in a plate clone colony assay of the present invention;

FIG. 11 shows the detection of the killing effect of RGD-anti-p 21Ras single-chain antibody fusion protein on tumor cells by MTT assay of the present invention;

FIG. 12 shows the effect of the RGD-anti-p 21Ras single-chain antibody fusion protein on the apoptosis induction of tumor cells detected by the TUNEL assay of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following drawings and examples, but the scope of the present invention is not limited thereto, and the method used in the present invention is a general method unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

Example 1: preparation of Single chain antibody Gene fragment

1.1 p21Ras protein immunization of Balb/c mice: 5 Balb/c mice (purchased from Beijing Wintolite Hua laboratory animal technology Co., Ltd.) aged 6-8 weeks were injected with 100. mu.g of purified p21Ras-K protein expressed in pronucleus in each laboratory (see the paper "expression, identification and purification of recombinant p21Ras protein and preparation of polyclonal antibody" for the preparation of p21Ras-K protein "), injected with equal amount of Freund's complete adjuvant for the first time, and injected at 5 o' clock under the skin. Two weeks later, the second injection is given at the same dose as the first injection, and the same amount of incomplete Freund's adjuvant is added, and the injection is performed at 5 points subcutaneously. The third injection is carried out two weeks later, the dosage is the same as that of the first injection, adjuvant is not added, and the injection is carried out in the abdominal cavity. The fourth injection is carried out two weeks later, the dosage is the same as that of the first injection, adjuvant is not added, and the injection is carried out in the abdominal cavity. After 3 days, the spleen was removed and the ground spleen was rinsed with 10ml of sterile D-Hank's solution. And (3) sucking the cell suspension in the culture dish by using a dropper, transferring the cell suspension into a 50ml centrifuge tube, centrifuging for 10 minutes at 1000g, discarding supernatant, and precipitating to obtain the required mouse spleen B lymphocyte.

1.2 extraction and reverse transcription of total RNA from mouse splenic B lymphocytes to synthesize cDNA: the isolated mouse spleen B lymphocytes were subjected to a conventional Trizol method to extract total RNA. The specific steps of extraction are referenced to the molecular cloning guidelines (third edition). The extracted RNA was subjected to electrophoresis on a 1% agarose gel at 90V for 30 minutes. The result shows that the 28S, 18S and 5S subunits of the extracted total RNA have correct sizes, and the band is clear without obvious bands, so that the extracted total RNA can be used for downstream reverse transcription experiments. The reverse transcription was performed using a reverse transcription kit from Fermentas, according to the protocol.

1.3 overlap extension PCR Synthesis of Single chain antibody Gene fragments: the mouse light and heavy chain variable region primers and Linker primers for single-chain Antibody construction by overlap extension PCR were amplified using Recombinant Phage Antibody System (RPAS) available from GE healthcare. Firstly, the following reagents are added into a PCR tube for carrying out light chain variable region amplification: mouse spleen B lymphocyte cDNA 4u l; 10 × PCR Buffer 5 μ l; dNTP (10mM) 5. mu.l; 1 mul of light chain primer mixed solution; rTaq enzyme 0.5. mu.l; sterilized deionized water 34.5. mu.l. The other PCR tube was added with the following reagents for heavy chain variable region amplification: mouse spleen B lymphocyte cDNA 4u l; 10 × PCR Buffer 5 μ l; dNTP (10mM) 5. mu.l; heavy chain primer mixture 1. mu.l; rTaq enzyme 0.5. mu.l; sterilized deionized water 34.5. mu.l. The system is prepared and then put into a PCR instrument to be processed for 5 minutes at 95 ℃; (94 ℃ 30 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minute, 30 cycles); the reaction was terminated at 72 ℃ for 10 minutes. The amplification product was subjected to electrophoresis on a 1.5% agarose gel at 90V for 30 minutes. The results showed that the size of the amplified heavy chain variable region was about 350bp and the size of the light chain variable region was about 330bp, consistent with the expectations.

Performing gel cutting and purification on the bands with correct sizes, and performing gel recovery according to the instructions of the Tiangen centrifugal column type DNA purification kit; the purified band was again subjected to 1.5% agarose gel electrophoresis using 2. mu.l of the gel under 90V for 30 minutes to determine the mass and approximate concentration of DNA after gel recovery.

Connecting the amplified light chain variable region and the heavy chain variable region by using a Linker primer, and introducing Sfi I and Not I enzyme cutting sites at two ends of a connecting product: the purified light and heavy chain variable region fragments with the same molar weight are connected by a Linker mixture through an overlap extension PCR method, and the connection product is added with Sfi I restriction sites at the 5 'end of a heavy chain and added with Not I restriction sites at the 3' end of a light chain under the action of RS Primers (restriction site Primers), so that the connection product can be used for subsequent connection with an expression vector pCANTAB-5E (purchased from Pharmacia) with the same restriction sites. The specific steps refer to the specification of the RPAS system of GE healthcare. The PCR product was subjected to 1.5% agarose gel electrophoresis, and the visible single-chain antibody band size was about 780bp, consistent with expectations, concentrated, clear and free of bands. And (5) performing gel cutting purification on the target band and measuring the concentration of the purified target band. The constructed single-chain antibody was named anti-p 21 Ras-ScFv.

1.4 cloning of Single chain antibody Gene fragments: the constructed single-chain antibody fragment anti-p 21Ras-ScFv was ligated to pMD-18T vector (purchased from TAKARA) to construct a pMD-ScFv recombinant plasmid. The following ligation system was prepared in 200. mu.l PCR tubes: 1. mu.l of pMD-18T vector; 0.1pmol to 0.3pmol of the target fragment DNA; solution I make up to 10. mu.l. The metal bath was reacted at 16 ℃ for 4 hours. The ligation was added to 100. mu.l DH 5. alpha. competence and ice-cooled for 30 min. The mixture was heat-shocked at 42 ℃ for 90 seconds and immediately ice-cooled for 90 seconds. 900. mu.l of LB liquid medium was added thereto, and the mixture was cultured at 37 ℃ and 80rpm with shaking for 1 hour. 200 μ l of the culture was plated on LB/Amp plates containing X-Gal and cultured in an inverted state at 37 ℃ for 10 hours.

1.5 PCR identification of positive recombinants by bacterial liquid: single colonies on LB/Amp plates were picked and dissolved in 50. mu.l of ddH₂And in O, thermally cracking at 98 ℃ for 10 minutes, and then centrifuging at 13000rpm for 1 minute to obtain a supernatant, namely the PCR template. Adding 2.5 mul of 10 XPCR Buffer into a 200 microliter PCR reaction tube; dNTP Mix (2.5mM each) 2. mu.l; M13F (10. mu.M) 0.5. mu.l, M13R (10. mu.M) 0.5. mu.l; 5 mul of bacterial liquid; rTaq enzyme 0.5. mu.l; ddH₂O14. mu.l. The PCR reaction was programmed to pre-denaturation 94 ℃ for 4 min. (94 ℃ for 1min, 57 ℃ for 1min, 72 ℃ for 1min, 30 cycles), extension at 72 ℃ for 10min, storage at 4 ℃. The PCR product was subjected to 1% agarose gel electrophoresis, and a band of the expected 930bp was observed, thereby determining that the clone was a positive recombinant clone.

Example 2: establishment of single-chain antibody library and screening identification

2.1 construction of recombinant phagemids

2.1.1 double digestion of recombinant pMD-ScFv vector and expression vector: the plasmid is extracted from the positive pMD-ScFv clone identified by PCR, and the extraction steps are operated according to the instructions of the Tiangen plasmid miniprep kit. The recombinant pMD-ScFv plasmid and the expression vector plasmid pCANTAB-5E (purchased from Pharmacia) are subjected to Sfi I enzyme digestion, and 30 mul of each expression vector plasmid/pMD-ScFv vector is added into a 200 mul PCR reaction tube; sfi I enzyme (10U/. mu.l) 4. mu.l; 10 × Buffer M5 μ l, ddH₂O11. mu.l, and reacting at 50 ℃ for 4 hours after the system is prepared. After the enzyme digestion product is recovered and purified by glue, 30 mul of the purified product and 2 mul of Not I enzyme (10U/mul) are added into a new 200 mul PCR reaction tube, 10 in the production lineBuffer H 5μl，BSA 2μl，Trion X-1002μl，ddH₂O9. mu.l, and reacting at 37 ℃ for 4 hours after the system is prepared. And (3) carrying out 1% agarose gel electrophoresis on all the enzyme digestion products, carrying out double digestion on the recombinant pMD-ScFv plasmid to obtain a target band at 780bp, and carrying out double digestion on the expression vector plasmid to obtain a target band at 3.5 kb. And respectively cutting and purifying target bands by gel, and performing the steps according to the instructions of a Tiangen centrifugal column type DNA purification kit.

2.1.2 ligation of recombinant expression vectors: the expression vector pCANTAB-5E subjected to synchronous double enzyme digestion by Sfi I and Not I is connected with the target fragment of ScFv according to the molar ratio of 1:5, so that the recombinant expression vector pCANTAB-ScFv containing the single-chain antibody gene is constructed. The ligation was performed in a total volume of 10. mu.l at 16 ℃ for 4 hours. The ligation products were all added to 100. mu.l of TG1 competent and ice-cooled for 30 min. The mixture was heat-shocked at 42 ℃ for 90 seconds and then ice-cooled for 90 seconds. 900. mu.l of LB liquid medium was added thereto, and the mixture was cultured at 37 ℃ and 80rpm with shaking for 1 hour. 200. mu.l of the culture was plated on LB/Amp plates and cultured in an inverted state at 37 ℃ for 10 hours.

2.1.3 identification of recombinant expression vectors: single colonies on LB/Amp plates were picked and dissolved in 50. mu.l of ddH₂And in O, thermally cracking at 98 ℃ for 10 minutes, and then centrifuging at 13000rpm for 1 minute to obtain supernatant, namely the template of the PCR reaction. PCR identification of the inserts the universal primers for the expression vector pCANTAB-5E, S1F: CAACGTGAAAAAATTATTATTCGC, S6R: GTAAATGAATTTTCTGTATGA-GG, were used. Adding 2.5 mul of 10 XPCR Buffer into a 200 microliter PCR reaction tube; dNTPs (2.5mM each) 2. mu.l; 0.5. mu.l of S1F (10. mu.M), 0.5. mu.l of S6R (10. mu.M); 5 mul of bacterial liquid; rTaq enzyme 0.5. mu.l; ddH₂O14. mu.l. The PCR reaction was programmed to pre-denaturation 94 ℃ for 4 min. (94 ℃ for 1min, 58 ℃ for 1min, 72 ℃ for 1min, 30 cycles), extension at 72 ℃ for 10min, storage at 4 ℃. The PCR product was electrophoresed on a 1% agarose gel, and a band of 950bp was observed. The recombinant expression vector pCANTAB-ScFv is subjected to Sfi I and Not I step-by-step double enzyme digestion, and enzyme digestion bands are formed at 3.5kb and 780bp positions, so that the recombinant expression vector is determined to be a positive recombinant clone.

2.2 enrichment and screening of phage antibody libraries

2.2.1 enrichment of phage antibody library: the helper phage M13K07 (purchased from GE healthcare) was added to the recombinant bacterial solution containing pCANTAB-ScFv, which was identified as positive, in a ratio of the number of bacteria to the number of helper phage 1:20, and cultured for 2 hours at 37 ℃ and 150rpm in a constant temperature shaker. When the liquid became cloudy, the mixture was centrifuged at 1500g for 25 minutes at room temperature in a centrifuge, and the supernatant was discarded. The pellet was resuspended in 2 XYTAK solution, incubated at 37 ℃ and 200rpm overnight with shaking. The obtained liquid is the phage culture solution after enrichment.

2.2.2 Indirect ELISA method for screening the specificity of single-chain antibody: the resulting culture solution was centrifuged at low speed at room temperature for 25 minutes, and the supernatant was aspirated, added with 1/5 vol of 10% nonfat dry milk blocking solution, and left at room temperature for 10 minutes. The p21ras-H, N, K protein was diluted to 5. mu.g/ml with 0.05M carbonate buffer pH9.6, 100. mu.l of the diluted protein solution was added to each well of the microplate, and the wells were coated overnight in a refrigerator at 4 ℃. Discard the liquid in the wells the next day, add 0.15M PBS-Twenz (phosphate-Tween) washing buffer 300. mu.l per well, shake for 3 minutes on a shaking table, discard the liquid in the wells, repeat washing 3 times. 100. mu.l of 1% BSA-PBS blocking solution was added to each well, incubated in a 37 ℃ incubator for 1 hour, and the plates were washed three times. Adding 100 mul of appropriately diluted single-chain recombinant phage clone supernatant of fusion expression into each hole as a primary antibody, placing the primary antibody in a wet box, incubating for 1 hour at constant temperature at 37 ℃, washing the plate for three times, and setting blank, negative and positive controls at the same time. Adding 100 μ l of freshly prepared TMB (tetramethylbenzidine) substrate solution into each well, wherein the substrate solution is an enzyme-labeled secondary antibody (HRP-labeled anti-M13 g8p protein) and diluted at a ratio of 1:2000, keeping out of the sun for 5-10 min, and adding 2M H into each well when a positive control is obviously blue and a blank and a negative control is colorless₂SO₄The reaction was stopped with 50. mu.l. OD reading using microplate reader₄₅₀The value is positive when the value of the to-be-detected hole/the value of the negative control hole is more than or equal to 2.

2.3 soluble expression and identification of Single chain antibodies

2.3.1 soluble expression of Single chain antibodies: the bacterial liquid which is screened by ELISA and contains the positive recombinant phage is expanded and cultured again to OD₆₀₀0.8. Extracting plasmids from the cultured bacterial liquid, and carrying out the steps according to the instruction of a QIAGEN plasmid miniprep kit. 3. mu.l of each plasmid were transformed into 100. mu.l of BL21(DE3) respectivelyIn the state. Selecting positive monoclonal antibody, inoculating to 5ml LB/Amp liquid culture medium, culturing, adding the previous culture broth into 1L new LB/Amp liquid culture medium at 1/100 ratio, and culturing to OD₆₀₀0.8. The cultured cells were collected, and the cells were resuspended in sterile PBS buffer, 100U/. mu.l lysozyme was added to give a final concentration of 1U/. mu.l lysozyme, and the cells were left at room temperature for 15 minutes at 4 ℃ and 12000rpm, centrifuged for 30 minutes, and the supernatant was collected.

2.3.2 identification of soluble expressed Single chain antibodies

2.3.2.1 SDS-PAGE to determine the relative molecular weights of the single chain antibodies: adding a certain amount of 2 xSDS loading buffer into the supernatant obtained in the last step to ensure that the final concentration of the protein is 3-4mg/ml, heating the mixed solution in a boiling water bath for 10 minutes, and cooling to obtain the loaded solution for electrophoresis. And after electrophoresis, taking out the separation gel, putting the separation gel into a container filled with deionized water, and taking out the separation gel after heating and boiling. Adding a quick staining solution to immerse the separation gel, shaking on a decoloring shaking table for 10 minutes, and discarding the staining solution when a protein band is visible. About 50ml of water was added again, boiled for 2 minutes, and the heating was stopped and continued on a decolorizing shaker for 30 minutes before observing the results. The results show that the destination stripe appears at 30KDa, consistent with expectations.

2.3.2.2 detection of the binding specificity and sensitivity of the soluble expressed single-chain antibody and tumor cell line by immunocytochemistry: 10 tumor cell strains including a human liver cancer cell strain HepG2, a human liver cancer cell strain QGY-7703, a human gastric cancer cell strain BGC-853, a human gastric cancer cell strain MKN-28, a human colorectal cancer cell strain HCT116, a human ovarian cancer cell strain SKOV3, a human cervical cancer cell strain Hela, a human breast cancer cell strain MDA-MB-231, a human breast cancer cell strain MDA-MB-435 and a human breast cancer cell strain MCF-7 are adopted; collecting 10 tumor cell strains in a logarithmic growth phase in a centrifugal tube, centrifuging to remove supernatant, re-suspending cell precipitates by using physiological saline, centrifuging to remove supernatant, re-suspending cell precipitates by using 95% ethanol, fixing the cell precipitates in 95% ethanol for 3 hours after centrifugation, carefully taking out tumor cell precipitate blocks, dehydrating, clearing, waxing, embedding, slicing, dewaxing, hydrating and high-pressure antigen repairing according to conventional tissues, adding a prepared soluble single-chain antibody as a primary antibody, using an anti-E-tag antibody as a secondary antibody (purchased from Abcam company), and detecting the condition that the tumor cells express p21ras protein by using an SP method. The results show that the prepared soluble single-chain antibody can show positive reactions with all the tumor cell strains in different degrees, and shows good and wide anti-tumor cell strain pedigrees.

2.4 sequencing the single-chain antibody with correct identification result: the bacterial liquid with the soluble expression result correctly containing the single-chain antibody gene recombination pMD-ScFv vector is sent to a sequencing company for sequencing, and the DNA sequencing result shows that the gene sequence arrangement mode of the single-chain antibody is V_H-Linker-V_LAnd after Kabat comparison with a mouse immunoglobulin variable region sequence database, finding that the sequence accords with the gene structure of a mouse light and heavy chain variable region, wherein the specific sequence is shown in SEQ ID NO: 5.

example 3 preparation of RGD-anti-p 21Ras Single-chain antibody fusion protein

3.1 design and construction of recombinant prokaryotic expression plasmid of RGD-anti-p 21Ras single-chain antibody

Directly adding RGD gene sequence (the sequence is shown in SEQ ID NO: 6) to the 5' end of the gene sequence of the anti-p 21Ras single-chain antibody obtained in the last step, and finally obtaining the gene sequence of the RGD-anti-p 21Ras single-chain antibody fusion protein, wherein the specific sequence is shown in SEQ ID NO: 7. the RGD-anti-p 21Ras single-chain antibody fusion gene sequence is subjected to codon optimization through an online website (http:// www.jcat.de /) according to the codon preference of escherichia coli, the optimized sequence (the specific sequence is shown as SEQ ID NO: 1) is sent to Kunming Pongku biological technology company Limited for chemical synthesis, Nde I is added at the 5 'end of one sequence, Hind III enzyme cutting sites are added at the 3' end, Kpn I is added at the 5 'end of the other sequence, Hind III enzyme cutting sites are added at the 3' end, and two RGD-anti-p 21Ras single-chain antibody fusion genes with different enzyme cutting sites at the tail ends are synthesized. And the RGD-anti-p 21Ras single-chain antibody fusion gene containing Nde I and Hind III enzyme cutting sites at two ends is respectively cloned into pET-28a (+) and pET-22b prokaryotic expression plasmids, the RGD-anti-p 21Ras single-chain antibody fusion gene containing Kpn I and Hind III enzyme cutting sites at two ends is cloned into pET-32a prokaryotic expression plasmids, 3 kinds of recombinant prokaryotic expression plasmids containing the RGD-anti-p 21Ras single-chain antibody fusion gene are constructed, and the part is completed by Kunming prokaryote science and technology limited company (the mode diagram of the constructed recombinant prokaryotic expression plasmid is shown in the attached figure 1 of the description). After the recombinant prokaryotic expression plasmid is constructed, subsequent experiments are correctly carried out through sequencing identification.

3.2 construction of recombinant prokaryotic expression bacteria

3.2.1 construction of BL21(DE3) recombinant expression bacteria

The three recombinant plasmids were transformed into BL21(DE3) competent plasmid, and the procedure was the same as that of E.coli DH5 alpha transformation. Separately, 500. mu.l of non-resistant LB liquid medium was added to BL21(DE3) recombinant expression bacteria transformed with the recombinant plasmid, and the mixture was subjected to shaking recovery at 37 ℃ and 180rpm for 60 min. Sucking 200ul of pET-28a (+) recombinant plasmid/BL 21(DE3) resuscitation solution and uniformly coating the resuscitation solution on an LB solid culture plate containing 50 mu g/ml kanamycin resistance; 200ul of recovery liquid of pET-32a recombinant plasmid/BL 21(DE3) and pET-22b recombinant plasmid/BL 21(DE3) were pipetted and applied evenly to LB solid culture plates containing 100. mu.g/ml ampicillin resistance. The cells were cultured overnight in a 37 ℃ incubator. When the monoclonal bacteria grow out, BL21 recombinant monoclonal is picked and amplified in LB liquid culture medium, and then PCR is carried out to identify positive clones. The PCR reaction system is as follows: 5 mul of recombinant bacterial liquid, 0.25ul of rTaq DNA polymerase, 2.5ul of 10 XPCR buffer, 2ul of dNTP mix, forward primer F11 ul, reverse primer R11 ul and ddH₂O13.25 ul, total volume 25 ul. The reaction conditions were as follows: 94 ℃ for 5 min; (94 ℃, 50 s; 55 ℃,1 min; 72 ℃, 45 s; for a total of 30 cycles); 72 ℃ for 10 min. Sequencing to identify the sequence without mutation and subsequent experiment. The PCR identification result is shown in the attached figure 2 in the specification.

3.2.2 construction of Origami (DE3) recombinant expression bacteria

The three recombinant plasmids were transformed into Origami (DE3) competent plasmid, and the procedure was the same as that of E.coli DH 5. alpha. transformation. Separately, 500. mu.l of a non-resistant LB liquid medium was added to Origami (DE3) recombinant expression bacteria transformed with the recombinant plasmid, and the mixture was subjected to shaking recovery at 37 ℃ and 180rpm for 60 min. Sucking 200ul of pET-28a (+) recombinant plasmid/Origami (DE3) recovery solution and uniformly coating the recovery solution on an LB solid culture plate containing 50 ug/ml kanamycin resistance; 200ul of the recovery solution of pET-32a recombinant plasmid/Origami (DE3) and pET-22b recombinant plasmid/Origami (DE3) were pipetted and applied evenly to LB solid plates containing 100. mu.g/ml ampicillin resistance, respectively. The cells were cultured overnight in a 37 ℃ incubator. When the monoclonal bacteria grow out, Origimi (DE3) recombinant monoclonal is picked out, and PCR and sequencing identification are carried out, wherein the steps are the same as the steps. The PCR identification result is shown in the attached figure 2 in the specification.

3.2.3 construction of OrigamiB (DE3) recombinant expression bacteria

The three recombinant plasmids were transformed into OrigamiB (DE3) competent cells, and the procedure was the same as that for e.coli DH5 α. Separately, 500. mu.l of a non-resistant LB liquid medium was added to OrigamiB (DE3) recombinant expression bacteria that had been transformed with the recombinant plasmid, and the mixture was subjected to shake recovery at 37 ℃ and 180rpm for 60 min. Sucking 200ul of pET-28a (+) recombinant plasmid/OrigamiB (DE3) resuscitating solution and uniformly spreading the resuscitating solution on an LB solid culture plate containing 50 ug/ml kanamycin resistance; 200ul of the recovery solution of pET-32a recombinant plasmid/OrigamiB (DE3) and pET-22b recombinant plasmid/OrigamiB (DE3) were pipetted and applied evenly to LB solid plates containing 100. mu.g/ml ampicillin resistance, respectively. The cells were cultured overnight in a 37 ℃ incubator. When the monoclonal bacteria grow out, OrigamiB (DE3) recombinant monoclonal is picked, and PCR and sequencing identification are carried out, wherein the steps are as above. The PCR identification result is shown in the attached figure 2 in the specification.

3.3 inducible expression of RGD-anti-p 21Ras Single-chain antibody fusion protein

3.3.1 Shake flask determination of optimal combination and conditions for inducible expression of RGD-anti-p 21Ras single-chain antibody fusion protein

3.3.1.1 optimal combinations for screening RGD-anti-p 21Ras Single chain antibody fusion proteins

And respectively selecting the successfully transformed recombinant expression bacteria, and culturing the monoclonal bacteria in an LB liquid culture medium. Inoculating the bacterial liquid into a 1L shake flask according to the proportion of 1:100, carrying out shake culture at 37 ℃ and 200rpm until the OD600 is about 0.6, adding a filter sterilized inducer IPTG to the final concentration of 1mM, inducing for 5 hours to express a target protein under the conditions of 37 ℃ and 160rpm, and finally identifying the expression condition of the target protein by SDS-PAGE. The results show that: the expression level of RGD-anti-p 21Ras single-chain antibody fusion protein was much higher in the combination of pET-32a/Origami (DE3) than in the other combinations. Therefore, the recombinant expression bacteria are selected to induce and express the target protein. In addition, the expression conditions of the target protein in the soluble supernatant and the inclusion body sediment of different strains are respectively compared, and the RGD-anti-p 21Ras single-chain antibody fusion protein mostly exists in the form of the inclusion body and the soluble supernatant is little in any combined expression bacteria, so that the target protein in the form of the inclusion body is collected in subsequent experiments.

3.3.1.2 determination of optimal expression conditions of RGD-anti-p 21Ras single-chain antibody fusion protein in IPTG/LB medium

pET32a-RGD-p21Ras scfv/Origami (DE3) monoclonal bacteria were picked up and cultured in LB liquid medium. Inoculating the bacterial liquid into a 1L shake flask according to the proportion of 1:100, carrying out shake culture at 37 ℃ and 200rpm until the OD600 is about 0.6, adding filter sterilized inducer IPTG to the final concentration of 0.2mM,0.4mM,0.6mM,0.8mM,1mM,1.2mM and 1.4mM respectively, setting the induction time gradients to be 4h, 6h, 8h, 10h, 12h and 20h respectively under the conditions of 37 ℃ and 160rpm, and carrying out induction expression on the target protein. Finally, SDS-PAGE identifies the expression of the target protein. The result shows that the target protein band is widest when the IPTG concentration is 0.6mM and the induction time is 10h, and the target protein expression level is highest.

3.3.1.3 RGD-anti-p 21Ras single-chain antibody fusion protein induced and expressed by self-induced culture medium

The self-induced medium was prepared according to the formulation in Table 1, and pET32a-RGD-p21Ras scfv/Origami (DE3) recombinant expression bacteria were inoculated at an inoculation rate of 1: 100. Performing shake culture at 37 ℃, culturing the bacterial liquid until OD is approximately equal to 0.6, then cooling to 20 ℃, performing shake culture at 200rpm for 20 hours, and using the culture solution to induce and express the target protein, wherein SDS-PAGE detects the expression condition of the target protein. The result shows that the thallus weight of the recombinant expression bacteria expressed by self-induction in the shake flask reaches 10.6 g/L. Compared with the optimal conditions for IPTG/LB culture medium induction, the target protein band induced and expressed by the self-induction culture medium is much wider than that induced by the IPTG/LB culture medium, and the expression level of the target protein induced and expressed by the self-induction culture medium is about 5 times that of the IPTG/LB culture medium. The comparison result of the target protein induced and expressed by the self-induced culture medium and the IPTG/LB culture medium is shown in the attached figure 3 of the specification.

TABLE 1 self-induction Medium formulation

3.3.2 fermentation-induced expression of RGD-anti-p 21Ras single-chain antibody fusion protein

And (3) determining the optimal conditions for the fermentation tank to amplify, induce and express the RGD-anti-p 21Ras single-chain antibody fusion protein by using the self-induction culture medium. pET32a-RGD-p21Ras scfv/Origami (DE3) secondary seed liquid is inoculated into 60L self-induction culture medium according to the proportion of 1:20, and fermentation is carried out according to the culture and expression conditions found in the previous period. Culturing at 37 deg.C and 200rpm for 8h to complete thallus amplification, inducing expression at 20 deg.C and 200rpm for 20h, feeding with lactose, peptone and yeast powder, and controlling pH at about 7.4 by feeding sodium hydroxide and 30% phosphoric acid. And (4) after fermentation, centrifugally collecting thalli, and detecting the expression condition of the target protein by SDS-PAGE. The result shows that the yield of the recombinant expression bacteria expressed by fermentation induction can reach 31.064g/L, which is 3 times of the yield of the shake flask self-induction expression bacteria. The successful establishment of pilot-scale fermentation induction conditions is shown, and a stable and efficient fermentation process of pET32a-RGD-p21Ras scfv/Origami (DE3) recombinant expression bacteria is established.

3.4 purification of RGD-anti-p 21Ras Single-chain antibody fusion protein

3.4.1 Collection of Inclusion body proteins

10ml of a cell washing solution (20mM Tris-HCl, pH8.0) was added to each gram of the cells collected by centrifugation, and the cells were resuspended at 4 ℃ for 10min and then centrifuged at 12000rpm for 15 min. Discarding the supernatant, collecting the thallus, adding 30ml of ultrasonic buffer solution (50mmol/L Tris-HCl, 0.1mmol/L EDTA, 5% glycerol, 0.1mmol/L DTT, 0.1mol/L NaCl) into each gram of thallus for resuspension, ultrasonically crushing on ice with the power of 60%, ultrasonically treating for 5s at intervals of 5s, and ultrasonically treating on ice for 30 min. Pure TritonX-100 to 1% (V/V) was added to the above ultrasonic lysate to break down cell membranes and dissolve membrane proteins. And (4) incubating on ice for 10min, then centrifuging at 12000rpm for 15min, and collecting the precipitate to obtain the inclusion body. Inclusion pellets were resuspended in 30ml of sonication buffer per gram at 12000rpm and centrifuged to remove TrintonX-100. The resulting precipitate was washed inclusion bodies. The pellet was resuspended in approximately 2 volumes of 20mM, pH8, Tris-HCl and the supernatant centrifuged at 12000g to remove EDTA.

3.4.2 RGD-anti-p 21Ras single-chain antibody fusion protein affinity chromatography

Adding 10ml of balance buffer (10mM imidazole/1 XPBS) into each 1g of inclusion body precipitate, suspending and mixing uniformly, shaking at room temperature for 30min-60min until the inclusion body precipitate is completely dissolved, centrifuging for 20min at 4 ℃, removing the precipitate, and collecting supernatant.

Purification of RGD-anti-p 21Ras single-chain antibody fusion protein was accomplished using the AKTA explorer 100 protein purification system. An Xk30/20 column was used, which had an internal diameter of 2cm and a height of 30 cm. 25ml of Ni Sepharose 6FF/HP chromatography packing were loaded onto the column. After the nickel column was equilibrated with 3 column volumes of equilibration buffer, the equilibration buffer containing the inclusion body proteins was loaded onto the nickel column, maintaining the pressure below 0.4MPa, assuming a flow rate of 10 ml/min. After loading, the hybrid proteins were washed with 20 column volumes of wash buffer (25mM imidazole/1 XPBS). When the effluent OD value was less than 0.01 by washing, the objective protein was eluted with 5 column volumes of elution buffer (250mM imidazole/1 XPBS) and the RGD-anti-P21 Ras single-chain antibody fusion protein in the column was collected, and the concentration of the RGD-anti-P21 Ras single-chain antibody fusion protein was 3.45mg/ml as determined by BCA protein kit (Biyuntian, P0012), and finally 31.6mg of RGD-anti-P21 Ras single-chain antibody fusion protein was purified from 1L bacterial solution (see FIG. 4 of the specification).

3.4.3 dialysis renaturation of RGD-anti-p 21Ras single-chain antibody fusion protein

The target protein eluted needs urea gradient renaturation to remove urea in the urea eluate so as to ensure that the urea eluate is correctly folded. Pretreatment of dialysis bags, cutting dialysis bags to appropriate length according to dialysis bag instructions, containing 2% NaHCO in 500ml₃And 1mmol/L EDTA (pH 8) for 10min, and then the dialysis bag was thoroughly washed with distilled water. Renaturation by dialysis (protein fluid: dialysate: 1:100 by volume) was performed by starting from renaturation solution 1 and dialyzing for 6 hours each time using a magnetic stirrer at 4 ℃ until renaturation by dialysis was completed for renaturation by dialysis of renaturation solution iv, and the formulation of the renaturation buffer by dialysis was shown in table 2. The protein was dialyzed against 0.01M PBS buffer for 6 hours, the protein was recovered, and the protein concentration was measured by BCA methodThe concrete operation steps refer to the instruction, and the frozen storage is carried out at-20 ℃ after filtration sterilization.

TABLE 2 dialysis renaturation liquid

3.4.4 identification of RGD-anti-p 21Ras Single-chain antibody fusion proteins

3.4.4.1 SDS-PAGE identification of purity of RGD-anti-p 21Ras single-chain antibody fusion protein

The concentration of the purified protein of interest was determined by reference to the procedure described in the BCA protein kit (cloudband, P0012). Then, SDS-PAGE was performed according to the concentration of the target protein to determine the purity of the purified target protein. The result shows that the molecular weight of the RGD-anti-p 21 Ras-single-chain antibody fusion protein purified by the nickel ion affinity chromatography column and dialyzed for renaturation is consistent with the theoretical value, compared with that before purification, the hybrid protein band of the recombinant protein purified by the nickel column is obviously reduced, and the purity is about more than 85% (the result is shown in the attached figure 4 of the specification).

3.4.4.2 WB for detecting combining ability of RGD-anti-p 21Ras single-chain antibody fusion protein and p21Ras protein

Firstly, taking the target protein to perform SDS-PAGE gel electrophoresis. Mouse anti-His-tag antibody was diluted at a ratio of 1:4000 and primary antibody was incubated with PVDF membrane overnight at 4 ℃. The secondary HRP-labeled goat anti-mouse IgG antibody was diluted at a ratio of 1:4000 with TBST, and the diluted secondary antibody was incubated with PVDF membrane at 37 ℃ for 1h for DAB color development. WB results show that RGD-anti-p 21Ras single-chain antibody fusion protein can bind to p21Ras protein after dialysis renaturation (see FIG. 5 in the specification for results).

3.4.4.3 ELISA for detecting titer of RGD-anti-p 21Ras single-chain antibody fusion protein

The K-Ras, N-Ras, and H-Ras antigens were diluted to a final concentration of 5ug/ml with a coating solution having a pH of 9.6, and 100ul of the diluted antigens were added to each well of a 96-well plate and plated overnight at 4 ℃. The next day the buffer in the wells was discarded, patted dry using absorbent paper, and the plates were washed three more times with 300ul of ELISA wash (0.5% Tween/0.1M PBS) added to each well. 100ul of 1% BSA-PBS was added to each well of the 96-well plate, and the plate was blocked and incubated at 37 ℃ for about 1 hour. RGD-anti-p 21Ras single-chain antibody fusion protein is added into each hole after washing the plate. Diluting according to different proportions (1: 50, 1:100, 1:200, 1:400, 1:800, 1: 1600, 1: 3200), and incubating at 37 deg.C for 1 h. After washing, 100ul of an anti-His tag antibody which had been diluted at a ratio of 1:4000 was added, followed by incubation in a constant temperature incubator at 37 ℃ for 1 hour. After washing the plate, 100ul of HRP-labeled secondary goat anti-mouse IgG antibody diluted at a ratio of 1:2000 was added to each well, and incubated in an incubator at a constant temperature of 37 ℃ for 40-60 min. 100ul of TMB reagent is added into each well, the 96-well plate is placed in the dark (the step needs to be protected from light) for reaction for 15-20 minutes, and the reaction is stopped when the color in the positive experimental group is changed into blue and the color in the blank and negative control group is not changed obviously. Adding 50ul of stop solution to stop the color reaction, and detecting the light absorption value by an enzyme-linked immunosorbent assay instrument at the wavelength of 450 nm.

The ELISA result shows that the combination titer of the RGD-anti-p 21Ras single-chain antibody fusion protein of 1mg/ml and the N-Ras, H-Ras and K-Ras proteins is 1:800, and the RGD-anti-p 21Ras single-chain antibody fusion protein can generate immunoreaction with K-Ras, N-Ras and H-Ras antigens (the result is shown in the attached figure 6 of the specification). The RGD-anti-p 21Ras single-chain antibody fusion protein purified by a nickel column and dialyzed and renatured has immunological activity.

Example 4 in vitro anti-tumor Activity study of RGD-anti-p 21Ras Single-chain antibody fusion protein

4.1 detection of tumor cell membrane penetrating capability by RGD-anti-p 21Ras single-chain antibody fusion protein

4.1.1 immunohistochemical detection of the ability of RGD-anti-p 21Ras single-chain antibody fusion protein to penetrate membranes

The RGD-anti-p 21Ras single-chain antibody fusion protein and SW480 cells are incubated together, and the cell wax block is prepared by centrifugal collection. Cutting a cell wax block into 4um sections by a slicer, then carrying out pathological conventional dewaxing, dehydration and antigen repair, blocking endogenous peroxidase, closing nonspecific binding sites, respectively incubating with a primary antibody of a mouse anti-His label and a secondary antibody of a goat anti-mouse HRP label, then carrying out DAB counterstaining, differentiation and blue marking, and detecting existence of RGD-anti-p 21Ras single-chain antibody fusion protein but not detecting the existence of the single-chain antibody, which indicates that the RGD-anti-p 21Ras single-chain antibody fusion protein can penetrate cell membranes, and the result is shown in the attached figure 7 of the specification.

4.1.2 immunofluorescence assay RGD-anti-p 21Ras single-chain antibody fusion protein transmembrane capability

An SW480 cell slide was prepared, the cells were permeabilized by Triton, and RGD-anti-p 21Ras single-chain antibody fusion protein was added to the slide, incubated at 37 ℃ for 1 hour, and then washed with PBS. The slide was incubated with mouse anti-His-tagged antibody for 1 hour at 37 ℃. After washing, a rhodamine-labeled goat anti-mouse IgG antibody (purchased from China fir Jinqiao) was added to the creeper and treated at 37 ℃ for 40-60 min. After washing, DAPI (purchased from Solarbio) is added to stain the cell nucleus on the slide, and observation under a fluorescent microscope shows that RGD-anti-p 21Ras single-chain antibody fusion protein can enter SW480 cells and is positioned in cell cytoplasm, while the single-chain antibody does not enter the cells, which shows that the RGD-anti-p 21Ras single-chain antibody fusion protein has the capability of penetrating tumor cell membranes, and the result is shown in the attached figure 8 of the specification.

4.2 cell scratch detection of the Effect of RGD-anti-p 21Ras single-chain antibody fusion protein on the migration ability of tumor cells

The normal colon epithelial cells CCD841 and 10 tumor cells driven by ras gene (human lung cancer cell line A549, human pancreatic cancer cell line ASPC-1, human pancreatic cancer cell line PANC-1, human pancreatic cancer cell line MIA PaCa-2, human breast cancer cell line MDA-MB-231, human glioma cell line U251, human liver cancer cell line HepG2, human colon cancer cell line SW480, human colon cancer cell line HCT116, human gastric cancer cell line AGS) respectively contain 5 × 10 per 2ml of culture medium per well⁵The cells were plated in 6-well plates at 37 ℃ with 5% CO₂The cells were cultured overnight in a cell incubator. After 24h, when the cell fusion rate reaches 100%, taking out cells, vertically scratching the cells in a superclean workbench by using a 200ul gun head, dividing each cell into three groups, adding 30uM RGD-anti-p 21Ras single-chain antibody fusion protein into an experimental group, and respectively adding 30uM RGD and PBS with the same volume into a control group. The healing after cell scratching was observed under a microscope at 0h, 24h, and 48h respectively and photographed. The results show that the recombinant protein RGD-p21Ras-scFv does not affectThe migration rate of the normal colon epithelial cell CCD841 has no obvious difference between the migration area of the experimental group and the migration area of the control group, the cell migration rate of the RGD-anti-p 21Ras single-chain antibody fusion protein group at 48h is 43.353 +/-0.422, the cell migration rate of the RGD group is 44.967 +/-1.644, and the cell migration rate of the PBS group is 42.770 +/-0.728. However, in the human lung cancer cell A549, the RGD-anti-p 21Ras single-chain antibody fusion protein group obviously inhibits the migration of tumor cells, and the cell migration area of the RGD and PBS control group is found to be larger than that of the RGD-anti-p 21Ras single-chain antibody fusion protein group by photographing and sampling at different time points. And the other 9 tumor cells all show that the cell migration area of the RGD group and the PBS group is obviously larger than that of the RGD-anti-p 21Ras single-chain antibody fusion protein group, and the migration area of the RGD-anti-p 21Ras single-chain antibody fusion protein group is slowly increased, and the result shows that the RGD-anti-p 21Ras single-chain antibody fusion protein can inhibit the migration rate of different tumor cells, and the result is shown in the attached figure 9 of the specification.

4.3 plate cloning detection of the Effect of RGD-anti-p 21Ras Single-chain antibody fusion protein on the proliferation ability of tumor cells

To examine whether RGD-anti-p 21Ras single-chain antibody fusion protein could inhibit proliferation of Ras-driven tumor cells, normal colonic epithelial cells CCD841 and the above 10 tumor cells driven by Ras were plated in 6-well plates containing 100 cells per 2ml of medium per well, and placed at 37 ℃ with 5% CO₂The cells were cultured overnight in a cell incubator. After the cells are attached to the wall, each cell is divided into three groups, the experimental group is added with 30uM RGD-anti-p 21Ras single-chain antibody fusion protein, and the control group is respectively added with 30uM RGD and PBS with the same volume. After 2 weeks of culture, macroscopic colonies appeared in the plates, at which time the culture was terminated, and then the culture solution of 6-well plate species was discarded, washed 2 times with PBS (0.01mol/L, pH 7.4), and then fixed for 15min to 30min by adding 3ml of methanol. Removing the fixing solution, adding a proper amount of the Giemsa working solution for staining for 30min, then slowly washing away the staining solution by PBS, and air-drying. Clones larger than 50 cells were counted under a microscope. The clone formation ratio (%) × 100% (number of clones/number of seeded cells).

The results show that the RGD-anti-p 21Ras single-chain antibody fusion protein has no influence on the proliferation capacity of the normal colon epithelial cell CCD841, and the cell clone formation numbers of the experimental group and the control group have no obvious difference. However, the cloning formation number of the RGD-anti-p 21Ras single-chain antibody fusion protein group of 10 tumor cells with Ras gene mutation is obviously lower than that of RGD and PBS control groups, which indicates that the recombinant protein RGD-p21Ras-scFv can inhibit the proliferation of tumor cells, wherein the inhibition effect on 3 tumor cells, namely human colon cancer cells HCT116, human lung cancer cells A549 and human pancreatic cancer cells MIA PaCa-2, is stronger, and the result is shown in the attached figure 10 of the specification.

4.4MTT detection of the killing ability of RGD-anti-p 21Ras single-chain antibody fusion protein to tumor cells

The 10 tumor cells driven by normal human colonic epithelial cells CCD841 and ras are prepared into single cell suspension by using complete culture solution containing 10% fetal calf serum, the single cell suspension is inoculated into a 96-well plate according to the cell number of 1000-10000 per well, the volume is 100ul, the 96-well plate is placed at 37 ℃, and 5% CO is added₂The cells were cultured overnight in a cell incubator. When the cell attachment is about 50%, the experimental group is added with 30uM RGD-anti-p 21Ras single-chain antibody fusion protein, and the control group is added with 30uM RGD protein and PBS with the same volume. After continuous culture for three days, 3 wells per group of cells were added with 20. mu.l of MTT solution to each well every day, and incubation was continued at 37 ℃ for 4 hours. The culture was terminated and the culture supernatant in the wells was carefully aspirated off. Add 150. mu.l DMSO into each well, and shake for 10min to fully melt the crystals. Measured after 15-20min (mauve solution) at room temperature. Measuring the light absorption value of each pore on an enzyme-linked immunosorbent instrument at 490nm wavelength, and recording the result to draw a cell growth curve.

The result shows that the RGD-anti-p 21Ras single-chain antibody fusion protein has no killing effect on normal colon epithelial cells CCD841, but has killing effect on different tumor cells, and can inhibit the growth of 10 tumor cells, and the result is shown in the attached figure 11 of the specification. 4.5TUNEL detection of the Effect of RGD-anti-p 21Ras single-chain antibody fusion protein on tumor cell apoptosis

The 10 tumor cells driven by normal colon epithelial cells CCD841 and Ras are respectively prepared into cell climbing sheets, and are respectively co-cultured with 30uM RGD-anti-p 21Ras single-chain antibody fusion protein, RGD protein and PBS, and then the condition that the recombinant protein induces tumor cell apoptosis (roche) is detected. The TUNEL result shows that the RGD-anti-p 21Ras single-chain antibody fusion protein can promote the apoptosis of tumor cells, and the apoptosis quantity of the tumor cells is obviously higher than that of RGD protein group and PBS group. However, there is no obvious difference in the apoptosis quantity of the cells of the experimental group and the control group for the normal cell CCD841, which shows that the RGD-anti-p 21Ras single-chain antibody fusion protein can promote the apoptosis of the tumor cells, but can not cause the apoptosis of the normal cells, and the result is shown in the attached figure 12 of the specification.

In conclusion, the results of example 4 indicate that the RGD-anti-p 21Ras single-chain antibody fusion protein has better in vitro tumor inhibition activity on Ras-driven tumor cells, and has no obvious toxic effect on normal cells.

Sequence listing

<110> China people liberation military joint work support army ninth two good quality hospital

<120> RGD-anti-p 21Ras single-chain antibody fusion protein capable of entering tumor cells and preparation method thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 822

<212> DNA

<213> Artificial sequence

<400> 1

gaagaagaag aagaagaaga agaaggtccg ctgggtctgg caggtcgtcg tcgtcgtcgc 60

cgtcgtcgtc gtatggcaca ggtgaaactg caggaaagcg gtgaaggtct ggttaaaccg 120

ggtggtagcc tgaaactgag ctgtgccgcc agtggtttta catttagcga ttattatatg 180

tattgggtgc gtcagacacc ggaaaaacgt ctggaatggg ttgcaattat tagcgatggt 240

ggtagctata cctattatcc ggatagtgtt aaaggtcgct ttaccatttc tcgtgataat 300

accaaaaaga atctgtatct gcagatgagc agcctgcgta gcgaagatac agcaatgtat 360

tattgtgcac gtgatcctca ttatagcggt agtagtcgtc tgtttgtgaa ttggggtcag 420

ggtacaaccg ttaccgtttc aagcggtggt ggtggtagtg gtggtggtgg ctccggtggt 480

ggtggttcag atattgaact gacgcagtca ccggcgagtc tggcggtgag tctgggtcag 540

cgtgcaacca ttagttatcg tgcgagtaaa agtgttagca cgagtggtta tagttatatg 600

cattggaacc agcagaaacc gggtcagcct ccgcgcctgc tgatttatct ggttagtaat 660

ctggaaagcg gtgtgccggc acgctttagc ggtagcggta gcggcaccga ttttacgctg 720

aatattcatc cggtggaaga agaagatgca gcaacctatt attgtcagca tattcgtgaa 780

ctgacacgta gtgaaggtgg tccgtcatgg cagattaaac gt 822

<210> 2

<211> 822

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 2

Glu Glu Glu Glu Glu Glu Glu Glu Gly Pro Leu Gly Leu Ala Gly Arg

1 5 10 15

Arg Arg Arg Arg Arg Arg Arg Arg Met Ala Gln Val Lys Leu Gln Glu

20 25 30

Ser Gly Glu Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys

35 40 45

Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Val Arg

50 55 60

Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ile Ile Ser Asp Gly

65 70 75 80

Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile

85 90 95

Ser Arg Asp Asn Thr Lys Lys Asn Leu Tyr Leu Gln Met Ser Ser Leu

100 105 110

Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Asp Pro His Tyr

115 120 125

Ser Gly Ser Ser Arg Leu Phe Val Asn Trp Gly Gln Gly Thr Thr Val

130 135 140

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val

165 170 175

Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val

180 185 190

Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly

195 200 205

Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly

210 215 220

Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

225 230 235 240

Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln

245 250 255

His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Gln Ile

260 265 270

Lys Arg

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<400> 3

caacgtgaaa aaattattat tcgc 24

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<400> 4

gtaaatgaat tttctgtatg a 21

<210> 5

<211> 750

<212> DNA

<213> Balb/c mice (Mus musculus)

<400> 5

atggcccagg tgaagctgca ggagtctggg gaaggcttag tgaagcctgg agggtccctg 60

aaactctcct gtgcagcctc tggattcact ttcagtgact attacatgta ttgggttcgc 120

cagactccgg aaaagaggct ggagtgggtc gcaatcatta gtgatggtgg tagttacacc 180

tactatccag acagtgtgaa ggggcgattc accatctcca gagacaatac caagaaaaac 240

ctgtacctgc aaatgagcag tctgaggtct gaggacacag ccatgtatta ctgtgcaaga 300

gatccccatt actccggtag tagccgcctg tttgttaact ggggccaagg caccacggtc 360

accgtctcct caggtggagg cggttcaggc ggaggtggct ctggcggtgg cggatcggac 420

atcgagctca ctcagtctcc agcttcctta gctgtatctc tggggcagag ggccaccatc 480

tcatacaggg ccagcaaaag tgtcagtaca tctggctata gttatatgca ctggaaccaa 540

cagaaaccag gacagccacc cagactcctc atctatcttg tatccaacct agaatctggg 600

gtccctgcca ggttcagtgg cagtgggtct gggacagact tcaccctcaa catccatcct 660

gtggaggagg aggatgctgc aacctattac tgtcagcaca ttagagagct tacacgttcg 720

gaggggggac caagctggca aatcaaacgg 750

<210> 6

<211> 72

<212> DNA

<213> Artificial sequence

<400> 6

gaagaagaag aagaagaaga agaaggtccg ctgggtctgg ctggtcgtcg tcgtcgtcgt 60

cgtcgtcgtc gt 72

<210> 7

<211> 822

<212> DNA

<213> Artificial sequence

<400> 7

gaagaagaag aagaagaaga agaaggtccg ctgggtctgg ctggtcgtcg tcgtcgtcgt 60

cgtcgtcgtc gtatggccca ggtgaagctg caggagtctg gggaaggctt agtgaagcct 120

ggagggtccc tgaaactctc ctgtgcagcc tctggattca ctttcagtga ctattacatg 180

tattgggttc gccagactcc ggaaaagagg ctggagtggg tcgcaatcat tagtgatggt 240

ggtagttaca cctactatcc agacagtgtg aaggggcgat tcaccatctc cagagacaat 300

accaagaaaa acctgtacct gcaaatgagc agtctgaggt ctgaggacac agccatgtat 360

tactgtgcaa gagatcccca ttactccggt agtagccgcc tgtttgttaa ctggggccaa 420

ggcaccacgg tcaccgtctc ctcaggtgga ggcggttcag gcggaggtgg ctctggcggt 480

ggcggatcgg acatcgagct cactcagtct ccagcttcct tagctgtatc tctggggcag 540

agggccacca tctcatacag ggccagcaaa agtgtcagta catctggcta tagttatatg 600

cactggaacc aacagaaacc aggacagcca cccagactcc tcatctatct tgtatccaac 660

ctagaatctg gggtccctgc caggttcagt ggcagtgggt ctgggacaga cttcaccctc 720

aacatccatc ctgtggagga ggaggatgct gcaacctatt actgtcagca cattagagag 780

cttacacgtt cggagggggg accaagctgg caaatcaaac gg 822

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence

<400> 8

gaagaagaag aagaagaaga ag 22

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

acgtttaatc tgccatgacg 20

Claims

1. A DNA molecule or gene encoding an RGD-anti-p 21Ras single-chain antibody fusion protein, characterized in that: the nucleotide sequence of the fusion protein of the RGD-anti-p 21Ras single-chain antibody is shown as SEQ ID NO: 1 is shown.

2. A prokaryotic recombinant expression vector characterized by: contains the DNA molecule or gene fragment of claim 1, and clones the gene fragment encoding RGD-anti-p 21Ras single-chain antibody fusion protein between Kpn I and Hind III restriction enzyme recognition sites of prokaryotic expression vector pET-32 a.

3. A prokaryotic recombinant expression strain, which is characterized in that: it is formed by chemical transformation or electric transformation of the prokaryotic recombinant expression vector of claim 2 into the competence of Escherichia coli Origami (DE3), and can express RGD-anti-p 21Ras single-chain antibody fusion protein.

4. A method for preparing RGD-anti-p 21Ras single-chain antibody fusion protein, which comprises the following steps: a) carrying out codon optimization on the RGD-anti-p 21Ras single-chain antibody fusion protein gene of claim 1 according to the codon preference of an escherichia coli host cell, cloning the protein gene into a prokaryotic expression vector pET-32a, and constructing a recombinant prokaryotic expression vector; b) transforming the recombinant prokaryotic expression vector of the step a) into a host cell Origami (DE3) competence; c) the RGD-anti-p 21Ras single-chain antibody fusion protein is expressed by inducing the recombinant prokaryotic expression strain in a fermentation tank through a self-induction culture medium, and the fermentation conditions are as follows: inoculating pET32a-RGD-p21Ras scfv/Origami (DE3) secondary seed liquid into 60L self-induction culture medium according to the proportion of 1:20, culturing at 37 ℃ and 200rpm for 8h to complete thallus amplification, and performing induction expression at 20 ℃, pH7.4 and 200rpm for 20h, wherein during the period, lactose, peptone and yeast powder are fed; d) centrifugally collecting thalli, resuspending the thalli by using an ultrasonic buffer solution, ultrasonically treating the thalli on ice for 30min, adding pure TritonX-100 into an ultrasonic lysate to a final concentration of 1% (V/V), incubating the thalli on ice for 10min, centrifugally collecting an inclusion body precipitate, washing the inclusion body to remove impurities, dissolving the inclusion body precipitate by using a balance buffer solution, and centrifugally collecting a supernatant; e) separating and purifying RGD-anti-p 21Ras single-chain antibody fusion protein from the inclusion body dissolving supernatant in the last step by using an AKTA chromatography system and adopting nickel ion affinity chromatography, wherein the purification conditions are as follows: an Xk30/20 chromatographic column with an inner diameter of 2cm and a height of 30cm was used, 25ml of Ni Sepharose 6FF/HP chromatography packing was loaded into the column, a flow rate was set at 10ml/min, after the nickel column was equilibrated with 3 column volumes of equilibration buffer, the equilibration buffer containing the inclusion body proteins was loaded onto the nickel column, the pressure was maintained at less than 0.4MPa, after loading was completed, the hybrid proteins were washed with 20 column volumes of washing buffer (25mM imidazole/1 XPBS), when the effluent OD value was less than 0.01, the target protein was eluted with 5 column volumes of elution buffer (250mM imidazole/1 XPBS) and the RGD-anti-p 21Ras single-chain antibody fusion protein in the column was collected, and the fusion protein was restored to immunological activity by gradient dialysis and renaturation.

The application of RGD-anti-p 21Ras single-chain antibody fusion protein in preparing a medicinal preparation for targeting and killing tumor cells is characterized in that: the tumor cells are pancreatic cancer cells, lung cancer cells and brain glioma cells.