CN101402942B - Intestinal cancer cell tumor vaccine modified by PolyLyse-HSP70 fusion protein membrane and preparation method thereof - Google Patents

Abstract

The invention provides a polylysine-heat shock protein 70 (PolyLyse-HSP70) fusion protein membrane-modified intestinal cancer cell tumor vaccine and a preparation method thereof. The intestinal cancer cell tumor vaccine expresses PolyLyse-HSP70 fusion The polyLyse end of the fusion protein is embedded in the intestinal cancer cell membrane, and the HSP70 end is free outside the intestinal cancer cell membrane; the PolyLyse is a 60-100 polylysine peptide segment in length. The novel intestinal cancer vaccine provided by the present invention provides a cell-type tumor vaccine for the treatment of intestinal cancer with stronger immunogenicity and higher anti-tumor effect, and is more effective for metastatic or drug-resistant intestinal cancer cells after chemotherapy , used in the prevention and treatment of colorectal cancer, has great application prospects.

Description

Intestinal cancer cell tumor vaccine modified by PolyLyse-HSP70 fusion protein membrane and preparation method thereof

(1) Technical field

The invention relates to a polylysine-heat shock protein 70 (PolyLyse-HSP70) fusion protein membrane-modified colon cancer cell tumor vaccine which can be used for preventing and treating colorectal cancer and a preparation method thereof.

(2) Background technology

Tumor biological therapy, especially immunotherapy, is an important part of comprehensive tumor treatment. In recent years, significant progress has been made in the clinical immunotherapy of tumors: the combination of certain immune agents such as Herceptin, MabThera and other monoclonal antibodies with chemotherapy can greatly increase the cure rate of tumors and prolong the survival rate of patients. The mechanism is that these antibodies activate host immune cells and antibody/complement-dependent cytotoxicity, promote tumor cell apoptosis, inhibit tumor metastasis and increase tumor cell sensitivity to chemotherapy drugs. These advances indicate that antibodies against tumor cells play an important role in the body's anti-tumor immunity and in improving the sensitivity of chemotherapy drugs. Immunotherapy has become an important aspect of tumor treatment research, and there is a tendency to combine it with chemotherapy to form chemo-immunotherapy.

During the mutation process of tumor cells, especially after chemotherapy, the cell membranes of surviving tumor cells will produce and expose more antigenic determinants, which mostly exist in the form of haptens, and form complete antigens after combining with carriers, which may produce effective anti-tumor Antibody. This has important implications for antitumor chemo-immunotherapy. On the other hand, cytotoxic lymphocyte (CTL) action is known to play an important role in anti-tumor immunity. However, the tumor antigen (CTL epitope) that induces CTL is different in different tumors, and it is difficult to separate, so it is not universal; tumor antigen modulation also affects the CTL effect of tumor vaccines. In recent years, it has been discovered that heat shock proteins (HSPs) can induce specific anti-tumor immunity through a unique mechanism. HSP tumor vaccine can better solve the modulation of tumor antigen, and can induce CTL without isolating tumor antigen, and has no obvious toxic and side effects. In addition, HSP itself has an immune adjuvant effect and has strong applicability. A large number of experiments and clinical treatments have also proved the many advantages of HSP tumor vaccine. HSP anti-tumor application research has become a research hotspot. The HSP whole-cell tumor vaccine has become an important research content because it contains all tumor antigens. However, the anti-tumor effect of current HSP whole-cell tumor vaccines is based on the release of HSP-antigen peptides (antigen components that induce CTL) after tumor cell lysis. This is not conducive to the binding of HSP-antigen peptides to HSP receptors on antigen-presenting cells (APCs), affecting its anti-tumor effect. Studies have found that using genetic engineering to express HSP70 in the cytoplasm on the surface of tumor cells can promote anti-tumor specific and non-specific immunity; tumor vaccines expressing HSP70 on the membrane have stronger anti-tumor effects.

Comprehensive treatment is the development direction of clinical treatment of colorectal cancer, and chemo-immunotherapy has unique advantages.

(3) Contents of the invention

The object of the invention is to provide a tumor vaccine for intestinal cancer cells with strong anti-tumor effect and a preparation method thereof.

The technical scheme adopted in the present invention is:

A polylysine-heat shock protein 70 (PolyLyse-HSP70) fusion protein membrane-modified intestinal cancer cell tumor vaccine, the intestinal cancer cell tumor vaccine is the killing of CT26 intestinal cancer cells transmembrane expressing PolyLyse-HSP70 fusion protein In living cells, the PolyLyse end of the fusion protein is embedded in the intestinal cancer cell membrane, and the HSP70 end is free outside the intestinal cancer cell membrane; the PolyLyse is a polylysine peptide segment with a length of 60--100. The gist of the present invention is to obtain intestinal cancer cell tumor vaccines by expressing the fusion protein across the membrane of intestinal cancer cells. The PolyLyse end of the fusion protein is embedded in the intestinal cancer cell membrane, and the HSP70 end is free outside the intestinal cancer cell membrane. Under the premise of the expression method, those skilled in the art can design the gene sequence, construct the vector, modify the gene of the cell, and screen positive clones according to common knowledge, so as to conveniently obtain the intestinal cancer cell vaccine of the present invention.

Preferably, the PolyLyse is a polylysine peptide segment with a length of 60.

In order to increase spatial mobility and prevent PolyLyse and HSP70 in the fusion protein from interfering with each other, 3-5 alanines may be inserted between the PolyLyse peptide and HSP70.

The intestinal cancer cell vaccine is preferably an inactivated cell of the CT26 intestinal cancer cell expressing the PolyLyse-HSP70 fusion protein across the membrane, because the CT26 intestinal cancer cell vaccine expressing the PolyLyse-HSP70 fusion protein across the membrane is a preserved living cell, However, when it is used as a tumor vaccine, it must be inactivated. The live cells of CT26 intestinal cancer cell lineages expressing PolyLyse-HSP70 fusion protein across the membrane are preserved in the China Center for Type Culture Collection, address: Wuhan, China, Wuhan University, 430072, date of preservation: June 25, 2008, Deposit number CCTCC NO: C200826.

The present invention also relates to a preparation method of the intestinal cancer cell vaccine, the method comprising: modifying the intestinal cancer cell with a transmembrane fusion gene, so that the intestinal cancer cell transmembrane expresses the PolyLyse-HSP70 fusion protein, and the PolyLyse-HSP70 fusion protein of the fusion protein The HSP70 end is embedded in the intestinal cancer cell membrane, the HSP70 end is free outside the intestinal cancer cell membrane, positive clones are screened, and the cells are inactivated to obtain the intestinal cancer cell vaccine.

The present invention modifies intestinal cancer cells with transmembrane fusion genes to express the fusion protein of polylysine (PolyLyse) and heat shock protein 70 (HSP70), and embeds the protein with strong carrier-hapten effect on the cell membrane. PolyLyse, HSP70 is attached to the outside of the cell membrane. Then, PolyLyse can form a complete antigen with various haptens on the cell membrane, and induce a strong anti-tumor humoral immune response and antibody formation; HSP70 can carry tumor antigen peptides from the cell and concentrate on the cell surface to promote the activation of APC and specific T cell immunity; and the combination of HSP70 and receptors on APC can promote PolyLyse to enter APC, thereby promoting anti-tumor humoral immunity and antibody formation. Furthermore, the genetically modified intestinal cancer cells are treated with chemotherapeutic drugs, and the immune effect of the prepared tumor vaccine is stronger, and it has better pertinence to the residual intestinal cancer cells after chemotherapy in vivo.

Alternatively, the method is as follows: modifying intestinal cancer cells with a transmembrane fusion gene, so that intestinal cancer cells express PolyLyse-HSP70 fusion protein across the membrane, the PolyLyse end of the fusion protein is embedded in the intestinal cancer cell membrane, and the HSP70 end is free in the intestinal cancer cells. Outside the cell membrane, the screened positive clones are treated with chemotherapeutic drugs simulating clinical treatment, and the treated surviving cells are inactivated with mitomycin to obtain the intestinal cancer cell tumor vaccine. Described chemotherapeutic drug can adopt the chemotherapeutic drug (for example: fluorouracil, oxaliplatin and irinotecan etc.) that is routinely used for colorectal cancer in this field, and its consumption is the routine dosage of clinical chemotherapy, and the chemotherapeutic drug described in the present invention is preferably Fluorouracil, the dosage is 0.01mg/mL cell suspension.

Preferably, the method is: cloning the full-length cDNA of the HSP70 gene, directional cloning the HSP70 gene into the eukaryotic expression vector pDisplay (other transmembrane, eukaryotic expression vectors are also available), and obtaining the membrane expression vector pDisplay-HSP70 , The HSP70 gene sticky end in the vector pDisplay-HSP70 is ligated with a DNA fragment encoding 4 alanines and 60 lysines to obtain the fusion protein membrane expression vector pDisplay-mPolyLyse-HSP70, and transfect the vector pDisplay-mPolyLyse-HSP70 Intestinal cancer CT26 cells, positive clones screened by G418, were amplified and treated with simulated clinical chemotherapy by adding 0.01 mg of 5-Fu per milliliter of cell suspension. Surviving CT26 cells were inactivated with mitomycin after 5-Fu treatment. The intestinal cancer cell tumor vaccine is obtained.

The cell membrane of intestinal cancer surviving after chemotherapy can produce and expose more and more antigenic haptens; however, there is no report on the use of cell membrane haptens of whole-cell tumor vaccines to induce strong anti-cancer humoral immunity, which is innovative . At the same time, existing studies have also shown that membrane-expressed HSP70 can carry antigenic peptides from the inside of cancer cells to the membrane surface, which can significantly promote the anti-tumor specific CTL effect.

According to the idea of the present invention, a novel intestinal cancer vaccine that can be strengthened through both humoral immunity and cellular immunity. At the same time, treating the tumor vaccine cells with 5-Fu simulated chemotherapy can enhance the immunogenicity of the tumor vaccine and have a stronger pertinence to the remaining cancer cells after chemotherapy in vivo.

Colon cancer is a common malignant tumor, ranking among the top three, and its incidence has continued to rise in recent years. The difficulty of colorectal cancer is chemotherapy resistance and metastatic recurrence of cancer cells; immunotherapy has its unique advantages, and it is more effective and reliable for cancer cells that have metastasized or drug resistance after chemotherapy. The novel intestinal cancer vaccine provided by the present invention is expected to have good application value in the treatment of colorectal cancer and prevention of its recurrence, and produce good economic and social benefits.

The beneficial effects of the present invention are mainly reflected in: providing a tumor vaccine for intestinal cancer cells with stronger immunogenicity and higher anti-tumor effect, which is more effective for metastatic or drug-resistant cancer cells after chemotherapy, and can be used for prevention and treatment Colorectal cancer has great application prospects.

(4) Description of drawings

Fig. 1 is the schematic diagram of the structure of the transmembrane PolyLyse-HSP70 fusion protein gene expression vector constructed;

Figure 2 is the DNA electrophoresis results of pDisplay-polyLyse-HSP70 expression; Lane 1 and Lane 1': CT26 with primers for HSP70 and β-actin, respectively; Lane 2 and Lane 2': mock-CT26 with primers for HSP70 and β-actin, respectively Primers; Lane 3 and Lane 3': CT26-pDisplay-polyLyse-HSP70 with primers for HSP70 and β-actin respectively;

Figure 3 shows the results of confocal laser microscopy of tumor cells after the membrane expresses and binds to the fusion protein PolyLyse-HSP70; A cell membrane surface expresses and binds to HSP70 protein, and its membrane surface shows green fluorescence (FITC labeling); B cell membrane surface expresses and binds to cancer cells For c-myc epitope of gene, its membrane surface shows red fluorescence (TRITC marker); for C cell membrane surface bound to HSP70 protein and oncogene c-myc epitope, its cell membrane surface shows yellow fluorescence (red and green superimposed color), indicating The second segment protein (polypeptide) appeared on the same cell membrane at the same time; the D isotype-matched antibody (Ig2b) control showed no secondary antibody binding on the cell membrane surface (black background)

Figure 4 is the result of HSP70 amplification;

Figure 5 is the result of PolyLys1 amplification;

Figure 6 is the PolyLys2 amplification result;

Figure 7 shows the splicing results of HSP70-PolyLys1;

Figure 8 is the splicing result of HSP70-PolyLys1-PolyLys2;

Fig. 9 is pdisplay-HSP70-PolyLys1-PolyLys2 plasmid;

Fig. 10 is a pdisplay-HSP70-PolyLys1-PolyLys2 plasmid digestion identification diagram;

Figure 11 is the pdisplay-HSP70-PolyLys1-PolyLys2 plasmid sequencing results;

Figure 12 is the NK activity in the immunotherapy effect of each tumor vaccine;

Figure 13 is the NK activity in the immune protection of each tumor vaccine;

Figure 14 is the CTL activity in the immunotherapy effect of each tumor vaccine;

Figure 15 is the CTL activity in the immune protection of each tumor vaccine;

Figure 16 is the level of IL-2 in the immune protection experiment of each tumor vaccine;

Figure 17 is the level of INF-γ in the immune protection experiment of each tumor vaccine;

Figure 18 is the level of IL-4 in each tumor vaccine immune protection experiment;

Figure 19 is the level of IL-10 in the immune protection experiment of each tumor vaccine;

Figure 20 is the tumor-inhibitory effect of each group of tumor vaccines on tumor-bearing mice;

Figure 21 is the survival prolongation effect of each group of tumor vaccines on tumor-bearing mice;

Fig. 22 is the tumor body growth of each group of tumor vaccine immunized mice (tumor vaccine anti-tumor attack effect);

Fig. 23 shows the survival of mice immunized with tumor vaccines in each group after tumor challenge (anti-tumor challenge effect of tumor vaccines).

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Embodiment 1: HSP70 full-length gene cDNA clone

1.1 Extraction of total RNA from rat liver tissue

100 mg of fresh liver tissue from BALB/c mice was cut into pieces, heat-treated at 42°C for 20 minutes, then placed in a mortar with a little PBS and ground into a slurry. Transfer to an Eppendorf tube, add 1ml Trisol reagent (Gibco, BRL) and mix well, then transfer to a dedicated Eppendorf tube, put in a water bath at 25°C for 5 min. Add 0.2ml of chloroform (Shanghai Shenggong Company) and mix well, then warm bath at 15-30°C for 2min. Centrifuge at 12000r/min×15min at 4°C, and stratification will appear. Pipet 500ul of the upper aqueous phase into a new Eppendorf tube, add an equal volume of isopropanol, and mix gently. 25 ℃ warm bath, 10min. Centrifuge at 12000r/min×15min at 4°C, suck off the supernatant, add 1ml of 70% ethanol to the bottom of the tube, shake and mix, then warm bath at 25°C for 5min. Then centrifuge at 10000r/min×10min at 4°C, suck off the supernatant, and dry the pellet in air for several minutes. Add DEPC-treated water to dissolve the RNA pellet. Transfer the RNA solution to a dedicated Eppendorf tube. Keep 5 μl and measure the concentration with a UV spectrophotometer to be 300 μg/ml.

1.2 RT-PCR method to obtain HSP gene cDNA

According to the instructions of the kit M-MuLV (Gibco, BRL), take 10 μl (3 μg) of the RNA template, add 1 μl of oligo(dT)18primer (included in the kit) and 10 μl of deionized water, and incubate at 70°C for 5 minutes. Take it out and place it on ice, and add in order: 5×RT buffer (included in the kit) 4 μl; RNasin (included in the kit) 1 μl (20U); 10Mm dNTP mix (included in the kit) 2 μl. Centrifuge briefly and incubate at 37°C for 5 minutes. Add 200 U of reverse transcriptase (included in the kit), incubate at 42°C for 1 hour, then incubate at 70°C for 10 minutes. The reaction was terminated, centrifuged briefly, and the reverse transcription product was obtained, which was used as a template for the next PCR.

Embodiment 2: Construction of the eukaryotic expression vector of membrane expression HSP70

2.1 Selection of carrier

The vector pDisplay (Invitrogen, USA), which can be expressed in eukaryotic cells, has a mouse κ chain signal peptide sequence upstream of the polyclonal restriction site, and a platelet-derived growth factor receptor downstream of the polyclonal restriction site. body (PDGFR) transmembrane sequence. Therefore, it is suitable for constructing a vector expressing HSP70 protein chimeric on the cell membrane surface.

2.2 Amplification of the target gene

According to the gene sequence of HSP70 in GeneBank, a pair of primers P1 and P2 (Shanghai Sangon Co., Ltd.) were designed and synthesized, respectively containing Sac II and Sal I restriction sites. Upstream primer P1: 5′-G CCGCGG CATGGCCAAAGCCGCTGCAGTCGGCAT-3′ (the underlined part is the Sac II restriction site, and the 24th italic T is the mutated base); downstream primer P2: 5′-CG GTCGAC ATCTACCTCCTCAATGGTG-3′ (The underlined part is the Sal I restriction site). Using this as a primer, the full-length cDNA of HSP70 obtained by RT-PCR was used as a template to amplify HSP70 cDNA by PCR. Using high-fidelity enzyme Pfu, heat denaturation at 95°C for 3min, cycle conditions: denaturation at 94°C for 45s; annealing at 58°C for 45s; extension at 72°C for 3min50s. After 28 cycles an extension was performed at 72°C for 10 min. The product was stored at 4°C for later use, and a small amount was taken for gel electrophoresis to observe the results.

2.3 Target gene directional cloning

The construction strategy is: step-by-step digestion and directional cloning. HSP70 DNA and vector pDisplay (purchased from Invitrogen, USA) were subjected to step-by-step enzyme digestion: first, the Sac II site was cut with Sac II enzyme (Fermentas, USA), and the DNA gel was recovered and purified with Sal I enzyme (Fermentas, USA) Then carry out the Sal I site digestion. After digestion, 4.5 μl each of HSP70 DNA with two sticky ends and the vector pDisplay, add T4 DNA ligase (Promega), 2× buffer, and construct the vector after overnight at 4°C.

2.4 Confirmation of construction vector

The constructed vector was transformed into Escherichia coli JM109 (preserved by the Institute of Immunology, Zhejiang University), positive colonies were screened on ampicillin-resistant culture dishes, amplified, and plasmids were extracted. The completion of the construction of the vector pDisplay-mbHSP70 was confirmed by electrophoresis of the digested product of the plasmid and sequencing of the plasmid DNA.

Example 3: Construction of membrane-type polyLyse-HSP70 fusion protein eukaryotic expression vector

Connect 3 to 5 alanines and 60 lysines to the sticky end of HSP70 of the constructed pDisplay-HSP70 vector. The construction strategy: amplify the partial sequence of hsp70 and splice the synthesized fragments, and then use the enzyme digestion of hsp70 itself The site SmlI is linked to the rest of it and the vector pDisplay.

The specific process is as follows:

The sequence of the amino acid to be synthesized: 208bp

GGAGGTAGAT gcc gcg gcg gcc aag aag aaa aag aag aag aag aaa aag aag aag aag aag aag aaa aag

Hsp70 sticky end 4 alanines 60 lysines

aag aag aag aag aag aag aag aag aag aag aag aaa aag aag aag aag aag aag aag aag

aag aaa aag aag aag aag aag aag aag aaa aag aag aag aag aag aag aag aaa aag aag

aag aag aag aag aag aag aag gtcgac

Acc I

Because the sequence repeats too much, it cannot be synthesized, and now the poly-lysine is inserted into the restriction site to synthesize two sequences:

(1) This sequence has a total of 118bp, which contains 4 alanine and 30 lysine sequences, that is, a 90bp sequence: (PolyLys1)

GGAGGTAGAT gcc gcg gcg gcc aag aag aaa aag aag aag aag aaa aag aag aag aag aag aag aaa aag

4 alanines 30 lysines

aag aag aag aag aag aag aag aag aag aag aag aaa aag aag aag aag gaattc

EcoRI

(2) This sequence is 102bp in total, including 90bp of 30 lysine sequences: (PolyLys2)

gaa ttc aag aag aag aag aag aaa aag aag aag aag aag aag aaa aag aag aag aag aag aag aag aaa

aag aag aag aag aag aag aag aag aag aag gtcgac

Acc I

Experimental steps:

1. PCR amplification:

① Amplify part of the HSP70 sequence from the pDisplay-HSP70 vector:

Primer sequence: Length: 615bp

Primer F: HSP70-4: 5′-GGTGCTGATCCAGGTGTAC-3′

Primer R: HSP70-2: 5′-ATCTACCTCCTCAATGGTGG-3′

PCR reaction system: 40ul

          10×Buffer 4.0ul

Mg ²⁺ (25mmol/L) 3.2ul

dNTPs (each 10mmol/l) 0.8ul

                                                                                               

                                                                                                           

                                                                                               

H ₂ O 28.7ul

Template (100ug/ml) 2.0ul

Reaction conditions:

② Amplify the first sequence (PolyLys1) from the vector:

Primer sequence: Length: 121bp

Primer F: PolyLys1-1: 5′-GGAGGTAGATGCCGCGGC-3′

Primer R: PolyLys1-3: 5′-ccgGAATTCCTTCTTCTTCTTTTTC-3′

PCR reaction system: 40ul

                                                                                                                                

Mg ²⁺ (25mmol/L) 3.2ul

                                                                  

                                                                 

                                                                          

                                                                                         

H ₂ O 28.7ul

Template (100ug/ml) 2.0ul

Reaction conditions:

③ Amplify the second sequence (PolyLys2):

Primer sequence: Length: 179bp

Primer F: PolyLys2-1: 5′-CCGCTCTAGAACTAGTGGATC-3′

Primer R: PolyLys2-2: 5′-CGACTCACTATAGGGCGAAT-3′

PCR reaction system: 40ul

                                                                                                               

Mg ²⁺ (25mmol/L) 3.2ul

                                                                                        

                                                                                               

                                                                                                           

                                                                                               

H ₂ O 28.7ul

Template (100ug/ml) 2.0ul

Reaction conditions:

2. PCR product purification (purification kit, purchased from QIAGEN, USA):

① Use agarose gel electrophoresis to cut off the target DNA fragment and put it into a 1.5ml centrifuge tube.

② Add 500ul Solution SN, place in a 65°C water bath to completely melt the finger glue, and mix well several times in the middle. After the glue is melted, add 100ul Solution B and mix well.

③Put the 3S column into a 2ml collection tube, transfer the melted gel solution to the 3S column, let it stand at room temperature for 2 minutes, and centrifuge at 10,000 rpm for 1 minute.

④ Remove the 3S column, pour off the waste liquid in the collection tube, put the 3S column into the same collection tube, add 600ul Wash Solution, and centrifuge at 10000rpm for 1min.

⑤Repeat step ④

⑥Remove the 3S column, discard the waste liquid in the collection tube, put the 3S column into the same collection tube, and centrifuge at 10000rpm for 2min.

⑦Put the 3S column into a new 1.5ml centrifuge tube, add 30ul of water to the center of the 3S column membrane, place at room temperature for 2min, and centrifuge at 10,000rpm for 1min. The liquid in the centrifuge tube is the recovered DNA fragment.

3. PCR splicing: splicing HSP70 and PolyLys1:

Primer sequence: Length: 726bp

Primer F: HSP70-4: 5′-GGTGCTGATCCAGGTGTAC-3′

Primer R: PolyLys1-3: 5′-ccgGAATTCCTTCTTCTTCTTTTTC-3

PCR reaction system: 40ul

                                                                                                                                        

Mg ²⁺ (25mmol/L) 3.2ul

                                                                          

                                                             

                                                                   

                                                                                                           

H ₂ O 26.7ul

Template 1 (100ug/ml) 2.0ul

Template 2 (100ug/ml) 2.0ul

Reaction conditions:

4. Digestion: EcoRI digestion of HSP70-PolyLys1 splicing product and PolyLys2

5. Ligation: Ligate the digested HSP70-PolyLys1 splicing product and PolyLys2 with T4DNA ligase:

25ul connection system:

6. PCR amplification of HSP70-PolyLys1-PolyLys2: using the ligation product as a template, amplify with HSP70-4 upstream primers and PolyLys2 downstream primers.

Primer sequence: Length: 862bp

Primer F: HSP70-4: 5′-GGTGCTGATCCAGGTGTAC-3′

Primer R: PolyLys2-2: 5′-CGACTCACTATAGGGCGAAT-3′

PCR reaction system: 40ul

                                                                                                                                       

Mg ²⁺ (25mmol/L) 3.2ul

                                                                     

                                                       

                                                                       

                                                                                                        

H ₂ O 26.7ul

Template 1 (100ug/ml) 2.0ul

Template 2 (100ug/ml) 2.0ul

Reaction conditions:

7. Enzyme digestion:

The spliced product of pDisplay-HSP70 vector, HSP70 and the first sequence was digested with BlpI enzyme and ACCI enzyme:

8. Connection Transformation

Connection: 25ul connection system

Conversion:

    Take 10ul of the ligation product and add it to the competent cells

                ↓

Ice bath for 30 minutes

                ↓

                                                                                               

                ↓

Ice bath for 3 minutes

                ↓

Add 1ml LB medium

                ↓

                                                                                               

                ↓

Spread evenly on LB Petri dishes, place at 37°C for 3h

                ↓

    Blot the residual liquid, invert the plate, and leave overnight at 37°C

                ↓

 Pick the clones and add them to the medium with 1‰ antibiotics, shake the bacteria overnight

9. Plasmid extraction (plasmid extraction kit, purchased from QIAGEN, USA)

①Centrifuge 2ml of the bacteria cultured overnight at high speed for 1min, and remove the supernatant completely.

②Add 100ul Solution I and fully suspend the bacteria with the pipette tip.

③Add 200ul Solution II, mix upside down immediately to lyse the bacteria, and place at room temperature for 2min until the solution becomes clear.

④ Add 400ul SolutionIII, immediately turn it upside down 5-10 times to fully neutralize it, and place it at room temperature for 2 minutes.

⑤15000rpm, centrifuge for 10min.

⑥Put the 3S column into a 2ml collection tube, transfer the supernatant to the 3S column, place at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.

⑦Remove the 3S column, discard the waste liquid in the collection tube, put the 3S column into the same collection tube, add 700ul Wash Solution, and centrifuge at 12000rpm for 1min.

⑧Repeat step ⑦

⑨Remove the 3S column, discard the waste liquid in the collection tube, put the 3S column into the same collection tube, and centrifuge at 12000rpm for 2min.

⑩Put the 3S column into a new 1.5ml centrifuge tube, add 50ul of water to the center of the 3S column membrane, place at room temperature for 2min, centrifuge at 12000rpm for 1min, the liquid in the centrifuge tube is the plasmid.

Agarose电泳检测。

Agarose electrophoresis detection.

10. Sequencing

Experimental results:

1. PCR amplification results: Figures 4 to 6 are the PCR electrophoresis detection images of HSP70, PolyLys1 and PolyLys2, respectively.

2. PCR splicing results:

HSP70 and PolyLys1 were spliced, and the target fragment was about 726bp. The splicing result of HSP70-PolyLys1 was shown in Figure 7;

3. Plasmid extraction and enzyme digestion identification:

After constructing the HSP70-PolyLys1-PolyLys2 splicing product into the pdisplay plasmid, the integrity of the extracted plasmid was detected by electrophoresis. The electrophoresis results of the pdisplay-HSP70-PolyLys1-PolyLys2 plasmid are shown in Figure 10. M is a 1kb marker; 1 is the extracted plasmid.

In order to identify whether the HSP70-PolyLys1-PolyLys2 splicing product was successfully constructed into the pdisplay plasmid, first use BlpI and ACCI enzyme digestion to identify, pdisplay-HSP70-PolyLys1-PolyLys2 plasmid enzyme digestion identification diagram is shown in Figure 11, M is Marker; 1, 2, 3 are plasmid double-digestion, non-digestion and single-digestion respectively. In the double-enzyme-digested plasmid, there is a target band above 750bp.

4. Sequencing results:

The extracted plasmids were sequenced and identified. The pdisplay-HSP70-PolyLys1-PolyLys2 plasmid sequencing results are shown in Figure 11. The splicing is basically correct. There are 58 lysines in total, and one of the first 30 lysines is mutated to arginine (AGG ), the deletion of 3 bases GAA resulted in the deletion of 1 lysine.

Example 4: Intestinal cancer CT26 cells transfected with fusion gene expression vector and identification of positive clones

The constructed vector pDisplay and empty pDisplay expressing the membrane-type fusion gene were transfected into mouse intestinal cancer cell CT26. After G418 resistance screening, positive clones were obtained and amplified. The specific process is as follows:

Transplant CT26 cells into 3 wells of a 24-well plate and culture them routinely. The next day, 1 μg of plasmid DNA to be transfected was diluted to 60 μl with RPMI-1640 cell culture medium without serum, protein or antibiotics. Mix for a few seconds. Add 5 μl SuperFect-Transfection-Reagent (QIAGEN, USA) to the DNA solution and mix. Incubate at room temperature for 10 min to form transfection complexes. At the same time, the cells in the culture plate were washed once with PBS. Add 350 μl of RPMI-1640 cell culture medium (with serum, protein or antibiotics) to the tube containing the transfection complex. Pipette twice to transfer to cells in a 24-well plate. After routine incubation for 2--3 hours, wash the cells once with PBS, add freshly prepared RPMI-1640 culture medium (containing serum, protein or antibiotics) and incubate. After 48h, the transfected gene began to be expressed. Digest and transplant cells, add 0.5ml of medium containing G418 700, 800, 900, 1000, 1100, 1200 μg/ml respectively, set up a control, and screen G418-resistant clones. The medium was changed once every 2-4 days. When most of the cells in the control group died, the concentration of G418 was reduced to 200 μg/ml for maintenance. After 4 weeks, it was found that in the culture wells containing 1200 μg/ml G418, all CT26 cells died, while in the culture wells containing 1100 μg/ml G418, a small number of CT26 cells survived, and the limited dilution resistant clones were transferred to 96-well plates. detection.

Add 2 μl each of mouse anti-HSP70 monoclonal antibody and mouse anti-hemagglutinin A monoclonal antibody at the concentration (200 μg/ml) to 100 μl suspension of positive clones, that is, CT26 tumor cells expressing polylyse-HSP70 on the cell membrane surface, and incubate at room temperature for 1 h ; After centrifugation and washing with PBS, the secondary antibody Goatanti-mouse IgG/FITC was added. Incubate at 4°C for 1 hour; wash with PBS centrifugation, adjust the cell concentration to 1×10 ⁶ /ml, and leave 200 μl for flow cytometry detection. Take 1 drop on a glass slide, lightly cover the cover glass and seal it with nail polish. Make 3 slides for immunofluorescence confocal microscope observation. In each of the above groups, unmodified B16 cells were set up, and PBS was used instead of the primary antibody as blank antibody controls.

The surface modification of cells was confirmed by RT-PCR, laser confocal microscopy and flow cytometry detection: membrane expression and binding fusion protein PolyLyse-HSP70. The results are shown in Figure 2 and Figure 3.

Example 5: 5-Fu Treatment of Intestinal Cancer Cells

After amplification of the positive clones of CT-26 cells transfected with resistance-selected genes or empty vectors, fluorouracil (5-Fu), a commonly used chemotherapeutic drug for colon cancer, was added to the cell culture medium to make each ml of cell suspension contain 0.01 mg 5-Fu, for 5 days of routine culture, washed to remove 5-Fu in the cell suspension and cultured, and repeated the above process once a month later. Treat the cells with mitomycin 100 μg/ml at 37°C for 0.5 h to inactivate the cells to form two groups of intestinal cancer vaccines: CT26 of Polyse-HSP70 (that is, the inactivated cells of CCTCC NO: C200826) and mock-CT26 each 1×10 ⁷ cells.

Example 6: Immune Effects of the Novel Intestinal Cancer Vaccine

1) Cell proliferation stimulating effect of tumor vaccine:

The stimulant was 1×10 ⁶ /ml inactivated various modified cells and control cells; the proliferating cells were 1×10 ⁶ /ml normal BALB/c mouse splenocytes. 50 μl of each was mixed and added to a 96-well plate for routine culture for 3 days, the absorbance (A) was measured by MTT colorimetry, and the cell proliferation effect was expressed by the proliferation index PI; PI=A value of the experimental group/A value of the control group. The results are as follows:

Table 1: In vitro cell proliferation stimulating effect of tumor vaccines

Co-culture with lymphocytes: P.I. 1. PBS 0.52 2. AntiHSP+CT-26-polylys-HSP70 0.40 3. CT-26 0.76 4. CT-26-polylysHSP70 1.06

Conclusion: The in vitro lymphocyte proliferation stimulation experiment shows that the developed tumor vaccine: CT26 tumor vaccine modified by fusion protein of heat shock protein 70 and polylysine (CT-26-polylysHSP70) has a strong lymphocyte proliferation stimulation effect. This effect depends on the interaction between heat shock protein 70 and receptors.

2) Detection of NK cell activity:

YAC-1 cells were used as target cells; immunized mice were sacrificed, and lymphocytes isolated from spleen were routinely taken as effector cells. Lactate dehydrogenase release method (LDH method) to detect NK cell activity: according to the requirements of the kit, add target cells, effector cells, 1640 and NP-40 to 100 μl of each well (both target cells and effector cells are 2× ¹⁰⁴ ) , conventional culture. Centrifuge at 1000 rpm and transfer the supernatant to a new well, then add the enzyme substrate. Place at 37°C for 10 minutes, add stop solution, and measure its OD value (A) with a microplate reader. Killing activity=(A killing hole-A target cell release hole)/(A target cell maximum release hole-A target cell release hole)×100%. The results are shown in Figure 12 and Figure 13.

Conclusion: The detection experiment of NK cell activity shows that the developed tumor vaccine: CT26 tumor vaccine modified by the fusion protein of heat shock protein 70 and polylysine (CT-26-polylysHSP70) has a strong immunity to induce NK lymphocyte activity. learning effect.

3) Determination of CTL killing activity

Splenocytes and tumor vaccine cells of the immunized mice were added to a 96-well plate at a cell number ratio of 1:2 (the number of cells in each well was 1×10 ⁶ , 100 μl). The number of cells in each well was the same, and incubated for 7 days to collect the suspension cells as CTL effector cells. Live CT26 cells were used as target cells for 4-h ⁵¹ Cr release killing experiments: CT26 cells were labeled with 200uCi Na ₅₁ CrO ₄ for 2 hours, and then the cells were washed 3 times with serum-free medium, and the target cells were selected according to different effect: target ratios (E : T) Co-cultivation with effector cells. Take the supernatant and measure the cpm value on a gamma counter. BALB/c mouse B lymphoma A20 cells were used as control target cells. The maximum release group was target cells plus 1% SDS; the spontaneous release group was target cells plus culture medium. Killing activity=(experimental group cpm-spontaneous release group cpm)/(maximum release group cpm-spontaneous release group cpm)×100%;

The CTL activity in the immunotherapy effect of each tumor vaccine is shown in Figure 14, and the CTL activity in the immune protection effect of each tumor vaccine is shown in Figure 15.

Conclusion: The detection experiment of CTL cell activity shows that the developed tumor vaccine: CT26 tumor vaccine modified by the fusion protein of heat shock protein 70 and polylysine (CT-26-polylysHSP70) has a strong immunity to induce CTL lymphocyte activity. learning effect. That is, it has the effect of inducing specific cellular immunity.

4) Detection of cytokines:

Mouse splenocytes in each group were co-incubated with tumor seedling cells for 48 hours, the culture supernatant was collected, and the levels of IFN-γ, IL-2, IL-4 and IL-10 in the supernatant were detected according to the instructions of the ELISA kit.

The levels of IL-2 in the immune protection experiment of each tumor vaccine are shown in Figure 16; See Figure 19 for the level of IL-10 in the vaccine immune protection experiment.

Conclusion: The detection experiment of cytokine activity shows that the developed tumor vaccine: CT26 tumor vaccine modified by the fusion protein of heat shock protein 70 and polylysine (CT-26-polylysHSP70) has a strong ability to induce IFN-γ, IL -2. Immunological effects of cytokine activity such as IL-4.

5) Deletion experiments in T cell subpopulations:

Five days before the tumor vaccine treatment, the mice were intraperitoneally injected with the corresponding blocking antibody, 1 time/2 days, a total of 5 times. Mice were divided into 4 groups, IgG2b anti-CD4 group; IgG2b anti-CD8 group; rat IgG control group; PBS control group. This process was confirmed by flow cytometry, confirming >98% deletion of specific subsets in peripheral blood and spleen of mice.

6) Heat shock protein function blocking experiment:

Before immunizing mice with tumor vaccine, add mouse anti-HSP70 MAb to 4 μg/ml, incubate at room temperature for 1 hour, centrifuge and wash, and inoculate experimental mice in the treatment model to set up control. For the specific immunological effect results, see the corresponding tumor vaccine groups in the above-mentioned Figures 12 to 19.

Conclusion: Various immunological experiments show that the developed tumor vaccine: CT26 tumor vaccine modified by fusion protein of heat shock protein 70 and polylysine (CT-26-polylysHSP70) has strong specific and non-specific immunostimulatory effects. This effect depends on the action of heat shock protein 70; blocking the action of heat shock protein 70 and receptors can block the immunostimulatory effect of tumor vaccines.

Embodiment 7: The animal experiment of tumor vaccine anti-tumor

The xenograft tumor model was established from CT26 intestinal cancer cells that survived treatment with chemotherapeutic drug 5-Fu. Tumor vaccine immunization was given before and after implantation of transplanted tumors. The tumor growth and the average survival period of tumor-bearing mice were observed. To study the therapeutic effect of tumor vaccine on tumor-bearing mice and the protective effect of anti-tumor attack. specific method:

1) Immunoprotective effect against tumor attack:

The mice were divided into 4 groups, and the following inactivated cancer cells or PBS were injected subcutaneously, once a week, 3 times in total: 1. CT26-mPolyLys-HSP70 (expressing CT26 cells expressing the fusion protein in the membrane, and analogously for other groups), 2. Empty pDisplay-CT26 (CT26 cells transfected with empty pDisplay), 3. PBS, 4. The same group 1 (for unrelated tumor A20 attack). After immunization, group 4 was challenged with A20 lymphoma cells and the rest were challenged with CT26 (wild type, surviving cells after 5-Fu treatment). One week later, 3 rats in each group were sacrificed to take spleens to measure NK and CTL activities. The tumor size of the remaining 5 mice was detected and the survival period of the mice was observed. The tumor-inhibiting effect of each group of tumor vaccines on tumor-bearing mice is shown in Figure 20, and the survival-prolonging effect of each group of tumor vaccines on tumor-bearing mice is shown in Figure 21.

Conclusion: Compared with the unmodified tumor vaccine, the tumor vaccine studied has a stronger protective effect against tumor attack, that is, it has a stronger anti-tumor recurrence effect. This effect is specific (only to the same tumor cells).

2) Immunotherapy effect on tumor-bearing mice:

After subcutaneous injection of 5×10 ⁶ surviving CT26 cells treated with 5-Fu in the right hind limb of the mice, the tumor-bearing mice were divided into 4 groups on day 5. The above 4 groups of preparations were injected respectively. 1 time in 3 days, 4 times in total. One week later, 3 mice in each group were sacrificed to take spleens, NK activity was measured by LDH release method, and CTL activity was measured by ⁵¹ Cr release method. The tumor size of the remaining 5 mice was detected and the survival period of the mice was observed. See Figure 22 for the tumor growth of mice immunized with tumor vaccines in each group (anti-tumor attack effect of tumor vaccines), and Figure 23 for the survival of mice immunized with tumor vaccines in each group after tumor challenge (anti-tumor attack effect of tumor vaccines).

Conclusion: Compared with the unmodified tumor vaccine, the studied tumor vaccine has a stronger therapeutic effect on tumor-bearing mice, and this effect is specific (only effective on the same kind of tumor cells).

Sequence Listing_ST25

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Claims (8)

1. the fusion protein film modified intestinal cancer cytoma vaccine of a polylysine-heat shock protein 70 (PolyLyse-HSP70), described intestinal cancer cytoma vaccine is the as killed cells of striding the CT26 colon-cancer cell of film expression PolyLyse-HSP70 fusion rotein, and the PolyLyse end of described fusion rotein embeds colon-cancer cell cytolemma, HSP70 end dissociative outside the colon-cancer cell film; Described PolyLyse is that length is 60～100 poly-lysine peptide sections.

2. intestinal cancer cytoma vaccine as claimed in claim 1 is characterized in that described PolyLyse is that length is 60 poly-lysine peptide section.

3. intestinal cancer cytoma vaccine as claimed in claim 2 is characterized in that being inserted with between described PolyLyse peptide section and the HSP70 3～5 L-Ala.

4. intestinal cancer cytoma vaccine as claimed in claim 1, it is characterized in that described intestinal cancer cytoma vaccine is the as killed cells of striding the CT26 colon-cancer cell of film expression PolyLyse-HSP70 fusion rotein, described CT26 colon-cancer cell of striding film expression PolyLyse-HSP70 fusion rotein, be preserved in Chinese typical culture collection center, address: China, Wuhan, Wuhan University, 430072, preservation date: on June 25th, 2008, deposit number CCTCC NO:C200826.

5. preparation is as the method for intestinal cancer cytoma vaccine as described in one of claim 1～3, described method comprises: modify colon-cancer cell with the transmembrane fusion gene, make colon-cancer cell stride film expression PolyLyse-HSP70 fusion rotein, the PolyLyse end of described fusion rotein embeds colon-cancer cell cytolemma, HSP70 end dissociative outside the colon-cancer cell film, screening positive clone, as killed cells obtains described intestinal cancer cytoma vaccine.

6. method as claimed in claim 5, it is characterized in that described method is: modify colon-cancer cell with the transmembrane fusion gene, make colon-cancer cell stride film expression PolyLyse-HSP70 fusion rotein, the PolyLyse end of described fusion rotein embeds colon-cancer cell cytolemma, HSP70 end dissociative outside the colon-cancer cell film, screening positive clone is handled with chemotherapeutic simulation clinical treatment, the mitomycin deactivation of the cell of the survival after the processing obtains described intestinal cancer cytoma vaccine.

7. method as claimed in claim 6 is characterized in that described chemotherapeutic is a Fluracil, and consumption is the 0.01mg/mL cell suspension.

8. method as claimed in claim 5, it is characterized in that described method is: the full-length cDNA of clone HSP70 gene, HSP70 gene directed cloning is inserted among the carrier for expression of eukaryon pDisplay, obtain film expression carrier pDisplay-HSP70, HSP70 gene cohesive end in carrier pDisplay-HSP70 connects the dna fragmentation of 4 L-Ala of coding and 60 Methionins, obtain fusion protein film expression vector pDisplay-mPolyLyse-HSP70, with carrier pDisplay-mPolyLyse-HSP70 transfection intestinal cancer CT26 cell, the G418 screening positive clone, after the amplification, the amount that adds the 0.01mg5-Fluracil with every ml cells suspension is simulated the clinical chemotherapy processing, 5 FU 5 fluorouracil is handled the CT26 cell of back survival with the mitomycin deactivation, obtains described intestinal cancer cytoma vaccine.

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