CN100518818C - Vaccine for preventing and/or treating tumour of digestive system - Google Patents

Abstract

The invention discloses a vaccine for preventing and/or treating digestive system tumors. The active ingredient of the vaccine is gastrin G17 cross-linking protein or gastrin G17 fusion protein; the gastrin G17 cross-linking protein is obtained by chemical cross-linking method between P64K protein and gastrin G17 N-terminal 9-peptide sequence; The gastrin G17 fusion protein is a protein with one of the following amino acid residue sequences: 1) sequence 1 in the sequence listing; 2) the amino acid residue sequence of sequence 1 in the sequence listing through one to ten amino acid residues A protein that is substituted, deleted or added and has the effect of inhibiting the growth of digestive system tumors. The vaccine of the present invention can be applied to prevent and/or treat various digestive system tumors.

Description

A vaccine for preventing and/or treating digestive system tumors

technical field

The invention relates to a vaccine, in particular to a vaccine for preventing and/or treating digestive system tumors.

Background technique

Gastrin (gastrin, GAS) is a polypeptide hormone secreted by G cells in gastric antrum and duodenal mucosa. It was first discovered and named by British scholar Edlkine in 1905. Its main physiological function is to promote gastric acid secretion and the growth of digestive tract mucosa. Studies have shown that gastrin is an autocrine growth factor of digestive tract tumors, which promotes tumor growth by promoting tumor cell proliferation, inhibiting apoptosis, stimulating infiltration and synergizing with cyclooxygenase-2 (COX-2).

There are many structural forms of gastrin molecules discovered so far, including gastrin 34 (gastrin 34, G34), gastrin G17 (gastrin 17, G17), gastrin 14 and C-terminal sulfated 6-peptide amide and Glycine-extended gastrin (Gly-gastrin, such as G-G17), in the normal physiological state of the body, mainly G34 and G17. G34 is responsible for stimulating the growth of digestive mucosa and maintaining the basic acidity in the stomach, and is the basic component of gastrin in the interdigestive period; G17 is responsible for the secretion of gastric acid stimulated by food, mainly in the digestive period.

The human gastrin gene is a single-copy gene, and its locus is located in the 17q23 region of the long arm of chromosome 17, with a total length of 4.1kb. G cells transcribe and produce the only gastrin mRNA with a length of 0.7kb, which is translated in the rough endoplasmic reticulum to generate preprogastrin of 101 amino acid residues, which is produced by splicing in different ways Gastrin molecule in various structural forms. Gastrin can act through certain pathways such as endocrine, autocrine or paracrine pathways. The normal and mature gastrin in the human body promotes the growth of digestive mucosal cells: First, endocrine gastrin binds to gastrin receptors on the surface of mucosal cells to generate a signal, and then transmits the signal to the cell, thereby stimulating the cell to start proliferating . The proliferative effect of this mode can be blocked by the corresponding antibody. Studies have shown that colon cancer tumor cells cultured in vitro can be stimulated to proliferate by exogenous gastrin, and gastrin can stimulate the growth of colon cancer grafts, which shows that gastrin works through endocrine.

Studies have shown that human colon cancer tumor cells can also secrete gastrin G17 and gastrin precursor. The expression of gastrin is ubiquitous in tumor cells, and the mode of action of gastrin at this time is different from the endocrine mode in G cells. Tumor cells contain a high proportion of precursor gastrin and low concentrations of mature gastrin. When the basal growth of colon cancer tumor cells was inhibited in vitro by anti-gastrin antibodies and gastrin receptor antagonists, it was demonstrated that endogenously produced gastrin acts in an autocrine form. This conclusion was further verified by Western blot experiments that revealed gastrin mRNA in the same cell line and radioimmunoassays that found gastrin-like immunoreactivity in cell culture supernatants.

Studies have shown that gastrin mediates a series of intracellular signal transduction through its receptors, promotes cell division and DNA synthesis, and has a growth-promoting effect on gastric cancer, colorectal cancer and other digestive tract tumors. The RT-PCR method was used to confirm the expression of gastrin mRNA in human digestive system tumors such as gastric cancer, pancreatic cancer, and colon cancer cell lines.

Gastrin is a growth factor for gastric cancer and has been identified in established cell lines such as murine gastric cancer cell line BV9 and human gastric cancer cell line TMK-1. Moreover, the primary tumor cells cultured from human tumor specimens were more sensitive to the pro-proliferation effect of gastrin G17 than the corresponding normal gastric mucosal cells.

Research on anti-gastrin therapy for gastrointestinal tumors is divided into four aspects: secretion inhibition, receptor antagonism, antisense oligonucleotide inhibition and anti-gastrin antibody, in which the body's own humoral immunity is stimulated to produce anti-gastrin antibody The gastrin therapeutic vaccine is the focus of current research.

The application potential of gastrin receptor antagonists in digestive system tumors, peptic ulcers, and hypergastrinemia has been highly valued. One of the most interesting application prospects is the endocrine therapy of digestive system tumors. The antitumor effects of gastrin receptor antagonists have been confirmed in both animal and clinical experiments. Gastrin receptor antagonists that have been discovered abroad mainly include amino acid derivatives (such as benzotript, D134308/CI-988), gastrin analogs (such as BOC-β-Ala-Trp-leu-Asp-phenylethylster), benzene Diazepine derivatives (such as L-365260), proglumide derivatives (such as Lorglumide/CR1409, loxiglumide/CR1505), quinazolinone derivatives (such as Compound22), Diphenylpyrzo-lidinone derivatives (such as LY262691 pyrazolidine) etc., the latest development of efficient and highly selective CCKB receptor antagonists are YM022 and YF476 and so on. These receptor antagonists inhibited both basal and gastrin-stimulated proproliferative effects in some cell lines and increased survival of tumor-implanted mice. Watson SA et al. found that CR2093 can significantly inhibit the growth-stimulating effect of G17 on tumors. L365260 and CI-988 can effectively neutralize the effect of exogenous gastrin in the growth of gastrointestinal tumors in vivo and in vitro. Some gastrin receptor antagonists such as proglumide have been used clinically, which can reduce the recurrence and prolong the survival time of patients with colorectal cancer.

At present, the main problems of gastrin receptor antagonists are: 1) low action intensity, requiring a relatively high action concentration; 2) low specificity, interacting with ligands that block all receptors, such as G34 and CCK; 3) specifically act on CCKB receptors, but have no effect on CCKC receptors; 4) or act on a certain subtype of CCKB receptors, but have no effect on other subtypes; 5) cannot block some Role of newly discovered and undiscovered receptors mediating the proliferative effects of progastrin.

The possible therapeutic effect of neutralizing gastrin with anti-gastrin antibodies was first reported by Hoosein et al. They inhibited gastrin-stimulated basal growth in human colon cancer cell lines cultured in vitro by treatment with an anti-gastrin antibody specific for carboxy-terminal gastrin. This inhibition can be reversed by adding excess G17, showing that the inhibitory effect is antibody specific. A further study was to directly infuse anti-gastrin antibody into experimental animals, and the result was that the growth of human colon cancer transplanted C523 cells was inhibited.

However, direct infusion of anti-gastrin antibody to treat human tumors in clinical practice will cause many problems: first, the antibody has to be injected regularly and needs to be used for a long time; second, the antibody titer needs to exceed the gastrin that neutralizes normal endocrine level to block the role of tumor-associated gastrin; third, some gastrins associated with tumor development require immediate neutralization; fourth, infusion of antibodies produced by other species, whether polyclonal or monoclonal, will have The problem of immunogenicity. And although humanized monoclonal antibodies are feasible, repeated use can lead to adverse consequences.

One way to solve the above problems and the inability to effectively antagonize the action of gastrin due to multiple gastrin receptor subtypes is to prepare a gastrin G17 vaccine to generate antibodies in situ, and when active gastrin binds to the receptor Neutralize them before interacting.

In terms of gastrin G17 vaccine, Watson SA and others in the UK started earlier. They constructed a vaccine called Gastrin Immunogen (Gastrimmune) by cross-linking the gastrin G17 N-terminal 9-peptide sequence through a polypeptide cross-linking bridge (Peptide spacer) and Diphtheria toxoid (DT) , so it is also called G17DT. DT is equivalent to the immune carrier protein. The 9-peptide sequence at the N-terminal of G17 is a B-cell epitope. The polypeptide cross-link bridge enables the space orientation of the gastrin part so that B cells can recognize the entire sequence and produce high-affinity neutralizing antibodies. This B-cell epitope is unique to gastrin G17, and neither G34 nor CCK has this epitope, so cross-reactivity does not occur. Using this Gastrimune to immunize with the traditional method can induce high-affinity antibodies against C-terminal amidated gastrin G17 and glycine extended gastrin (G-G17) in the serum for several months, thus avoiding direct antibodies Problems with infusions. After the first immunization, the antibody titer produced exceeds the level of neutralizing G17 in all serum, so there are enough antibodies to neutralize the tumor-associated gastrin molecule. However, due to the toxicity of DT, the cross-linked protein G17DT has the disadvantage of large local reaction and stimulation.

Gastrimmune-induced antibodies were evaluated in several in vivo and in vitro models of gastrointestinal tumors. Transplanted human colon cancer cells AP5 into nude mice, infusion of Gastrimmune-immunized rabbit antiserum inhibited tumor growth compared with negative control DT-immunized rabbit antiserum. Gast rimmune rabbit antiserum was intravenously injected into mice with early AP5LV tumors. As a result, the antibodies induced by Gas rimmune reduced the growth of early tumors by 30% and inhibited tumor metastasis. The amount of lung nodules was reduced by 70%, and the volume of nodules was only 60% of that of the carrier protein DT control group. Tumor metastasis was reduced by more than 90%. Compared with 5-fluorouracil (5-fluorouracil, 5-FU)/tetrahydrofolate (leucovorin) in the treatment of mice injected with AP5LV cells, the effect of the two on inhibiting early tumors was comparable, but Gastrimune antibody was more effective in inhibiting tumor metastasis. good. This shows that anti-gastrin therapy is more effective than traditional chemotherapy in tumor spontaneous metastasis model.

Aphton of the United States has developed it as a product for the treatment of digestive system tumors, and several indications have entered clinical research, and the progress is very smooth. At present, the indication of using Gastrimune alone in the treatment of pancreatic cancer has completed phase III clinical trials, and new drugs are being applied for in Australia; the indications of using Gastrimune in combination with cisplatin (Gemcitabine) in the treatment of pancreatic cancer have entered phase III clinical trials; combined with 5-FU in the treatment of gastric cancer and Phase II clinical trials for indications of esophageal cancer are about to be completed; phase II clinical trials for indications for the treatment of peptic ulcer and esophageal reflux are recruiting clinical volunteers. The published results of Gastrimmune clinical trials are satisfactory.

In 2002, the FDA designated G17DT immunogen, which is used in combination with cisplatin and 5-FU to treat stage IV gastric cancer, to improve the average survival rate, as a fast-track product, and the indication for the treatment of pancreatic cancer and gastric cancer is "orphandrug". In the same year, Australia approved Aphton's Gastrimmune as an "orphan drug" for the treatment of gastric cancer and pancreatic cancer. In July 2003, the European Union approved Gastrimmune as an "orphan drug" for the treatment of pancreatic cancer and gastric cancer.

Yu Wen from China Pharmaceutical University in China conducted research on anti-gastrin polyepitope vaccines. The B-cell epitope of the N-terminal 9-peptide of G17 was selected to be fused with the human T helper epitope of the 13-peptide from tetanus toxin, and then a cysteine (for coupling to other proteins) and a sperm were introduced at the C-terminal Amino acid (trypsin site), five repeated fusion protein genes were constructed by rail-laying PCR. The fusion protein was expressed and purified, and a rat model was used to evaluate the immunogenicity of the recombinant protein, but the experimental results were not reported.

P64K is an outer membrane protein from Neisseria meningococcus, found in the serum of meningitis convalescent patients and individuals vaccinated with the Cuban VA-MENGOC-BC vaccine. The gene encoding P64K protein is 1pdA, and its coding sequence is 1782bp long. P64K protein contains 593 amino acids and consists of two domains, namely N-terminal 81 amino acids and C-terminal 482 amino acids (Kacko Tozawa, R.William Broadhurt, Andrew R.C.Raine, etc.Solution structure of the lipoyl domain of the chimericdihydrolipoyl dehydrogenase P64K from Neisseria meningitidis. Eur. J. Biochem., 2001, 268: 4908-4917). The two domains of the P64K protein are separated by a 30 amino acid long linker region (rich in Ala and Pro), which appears to be structurally "flexible". P64K protein has dihydrolipoic acid amidoacyl dehydrogenase activity.

Because of the high molecular weight of P64K protein and its immunogenicity in different animal models, P64K is considered as a promising antigen carrier for weak antigens. The purified recombinant P64K protein was used to immunize mice, rabbits and monkeys, and it was found that the P64K protein was immunogenic to these animals. Studies in recent years have shown that P64K has a better immune enhancement effect than traditional carrier proteins such as BSA and TT.

Contents of the invention

The object of the present invention is to provide a vaccine for preventing and/or treating digestive system tumors.

The vaccine for preventing and/or treating digestive system tumors provided by the present invention has an active ingredient of gastrin G17 cross-linking protein or gastrin G17 fusion protein; the gastrin G17 cross-linking protein is a combination of P64K protein and gastric The nine-peptide sequence at the N-terminal of G17 is obtained by chemical cross-linking; the G17 fusion protein is a protein with one of the following amino acid residue sequences:

1) Sequence 1 in the sequence listing;

2) The amino acid residue sequence of Sequence 1 in the sequence listing has undergone one to ten amino acid residue substitutions, deletions or additions and has the effect of treating digestive system tumors.

The amino acid residue sequence of Sequence 1 in the sequence listing is a protein consisting of 609 amino acid residues, named G17P64K, the 2nd-10th amino acid residues from the amino terminal are gastrin G17 N-terminal 9-peptide sequence, and the amino acid residues from the amino terminal 1 The 11-16 amino acid residues are the connecting peptide sequence, and the 17-609 amino acid residues from the amino terminal are the P64K protein sequence,

The nine-peptide sequence of the gastrin G17 N-terminal has the amino acid residue sequence of sequence 2 in the sequence listing.

The P64K protein is derived from meningococcus.

The chemical cross-linking method is MBS method or EMCS method.

In order to facilitate cross-linking by the MBS method, a 6-peptide or 7-peptide is tightly connected to the carboxy-terminus of the 9-peptide sequence at the N-terminal of gastrin G17, and the carboxy-terminal amino acid residue of the 6-peptide or 7-peptide is cysteine .

In chemical cross-linking, the 6-peptide has the amino acid residue sequence of sequence 3 in the sequence listing; the 7-peptide has the amino acid residue sequence of sequence 7 in the sequence listing.

When the 6-peptide has the amino acid residue sequence of sequence 3 in the sequence table, the preparation method of the gastrin G17 cross-linked protein comprises the following steps:

1) Synthesizing 15 peptides having the amino acid residue sequence of sequence 4 in the sequence listing;

2) Cross-linking the P64K protein with a mass ratio of 3-5:7-10 and the 15 peptide in step 1) by the MBS method to obtain a gastrin G17 cross-linked protein.

Both the gastrin G17 fusion protein coding gene and the P64K coding gene belong to the protection scope of the present invention.

The gastrin G17 fusion protein encoding gene, G17P64K gene, has one of the following nucleotide sequences:

1) the polynucleotide sequence of sequence 5 in the sequence listing;

2) A nucleotide sequence that can hybridize to the DNA sequence defined in Sequence 5 in the Sequence Listing under high stringency conditions.

The highly stringent condition is to wash the membrane at 65°C with a solution containing 0.1×SSPE (or 0.1×SSC), 0.1% SDS after hybridization.

Sequence 5 consists of 1830 bases, and its coding sequence is from the 1st base to the 1830th base at the 5' end.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the above vaccine. The carrier includes conventional diluents, absorption enhancers and the like in the field of pharmacy.

The vaccine of the present invention can be made into injection solution, and the vaccine in this dosage form can be prepared according to the conventional methods in the field of pharmacy.

The dosage of the above-mentioned vaccines is generally 20-200 μg gastrin G17 cross-linked protein or gastrin G17 fusion protein/kg body weight/time, one basic immunization, one booster immunization 14 days later, a total of 2-4 immunizations are required.

The vector containing the gene encoding the gastrin G17 fusion protein, engineering bacteria and cell lines also belong to the protection scope of the present invention.

The P64K protein coding gene has the nucleotide sequence of sequence 6 in the sequence listing.

Sequence 6 consists of 1785 bases, and its coding sequence is from the 1st base at the 5' end to the 1785th base.

Compared with DT, the P64K protein has extremely high safety and immunogenicity in the human body. The vaccine for preventing and/or treating digestive system tumors using the P64 protein of the present invention has good immune effect and high stability. The treatment of gastrointestinal tumors has high safety and good curative effect; the preparation process of the vaccine is simple, the production cycle is short, the production scale is large, the quality control is simple, the output is high, and the cost is low, and it has important application in medicine prospect.

Description of drawings

Figure 1a is a 1% agarose gel electrophoresis profile of the PCR-amplified P64K gene.

Figure 1b is a 1% agarose gel electrophoresis profile of the G17P64K gene amplification product.

Figure 1c is a 1% agarose gel electrophoresis pattern of the digested product of plasmid pET28a-G17P64K after Nco I and EcoRI double enzyme digestion.

Figure 2 is a 15% SDS-PAGE map of the expression product of pET28a-P64KG17/BL21(DE3).

Fig. 3 is a 15% SDS-PAGE map of the purified expression product of pET28a-P64KG17/BL21 (DE3).

Fig. 4 is a high-density growth curve of engineering bacteria G17P64K/BL21 (DE3) cultured in a 5L fermenter.

Fig. 5 is the SDS-PAGE pattern of the fusion protein G17P64K expressed by culturing the engineered bacteria G17P64K/BL21 (DE3) in a 5L fermenter.

Fig. 6 is the HPLC identification chart of the synthesized G17 polypeptide.

Figure 7 is the electrophoresis diagram of the purification of rabbit anti-gastrin antibody.

Figure 8 is the growth curve of SW480 cells inhibited by rabbit anti-gastrin antibody.

Detailed ways

Marker

Nucleic acid molecular weight standard DL2000 (2000, 1000, 750, 500, 250, 100bp) (purchased from Dalian Bao Biological Engineering Company)

Protein low molecular weight standards (14, 20, 30, 45, 66, 97 kDa) (purchased from Pharmacia).

Embodiment 1, the preparation of fusion protein G17P64K

1. Acquisition of G17P64K fusion protein gene

1) Acquisition of P64K gene

The primer sequences are as follows:

Primer 1: 5'CATG CCATGG CTTTAGTTGAATTGAA3' (the underlined base is Nco I) Primer 2: 5'GG GAATTC TTATTTTTTCTTTTGCGGAG3' (the underlined base is EcoR I)

(The synthesis of primers was completed by Shanghai Sangon Bioengineering Co., Ltd.)

The thallus of Neisseria meningitids (Neisseria meningitids, Hebei type B, number 050089, purchased from the strain room of the Institute of Epidemiology, Academy of Military Medical Sciences) was lysed by boiling, and the obtained thalline lysate was PCR amplification. Template for the P64K gene.

Using the cell lysate obtained above as a template, under the guidance of primers 1 and 2, the P64K gene was amplified by PCR. The reaction system of PCR is as follows:

ddH ₂ O 39.0μl

10×Buffer 5.0μl

4×dNTP 1.5μl

Primer 1 2.0μl

Primer 2 2.0μl

Template 0.2μl

Pyrobest DNA polymerase (purchased from Invitrogen) 0.3 μl

Total volume 50.0μl

PCR cycling conditions were: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 sec, 50°C annealing for 30 sec, 72°C extension for 2 min, 30 cycles; 72°C extension for 10 min. Carry out 1% agarose gel electrophoresis to PCR amplified product, the result is as shown in Figure 1a (swimming lane M is Marker DL2000, swimming lane 2 and swimming lane 3 are PCR amplified product), shows that PCR product size is about 1.8Kb, shows that correct The P64K gene was amplified. A DNA fragment GENECLEAN II glass milk recovery kit (purchased from Q Biogene) was used to recover a gene fragment of about 1.8Kb in size, cloned into the pGEM-T vector, and carried out gene sequence analysis. The results showed that the amplified P64K gene had the The nucleotide sequence of SEQ ID NO: 6, its coding sequence is from the 1st to the 1785th base at the 5' end.

2) PCR amplification of the G17P64K fusion protein gene

The primer sequences are as follows:

Primer 3: 5'CATG CCATGG AAGGCCCTTGGCTTGAAGAGGAAGAATCTTCACCCCCTCCGCCGGCTTTAGTTGAATTGAAAGTG3' (the underlined base is Nco I)

Primer 4: 5'GG GAATTC TTATTTTTTCTTTTGCGGAG3' (the underlined base is EcoR I)

(The synthesis of primers was completed by Shanghai Sangon Bioengineering Co., Ltd.)

Using the P64K gene fragment with a size of about 1.8 Kb purified and recovered in step 1) as a template, and under the guidance of primers 3 and 4, the G17P64K gene was amplified by PCR. The reaction system of PCR is as follows:

ddH ₂ O 39.0μl

10×amplification buffer 5.0μl

4×dNTP 1.5μl

Primer 3 2.0μl

Primer 4 2.0μl

Template 0.2μl

Pyrobest DNA Polymerase 0.3μl

Total volume 50.0μl

PCR cycling conditions were: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 sec, 50°C annealing for 30 sec, 72°C extension for 2 min, 30 cycles; 72°C extension for 10 min. The PCR amplification product was subjected to 1% agarose gel electrophoresis, and the results were as shown in Figure 1b (swimming lane M is Marker DL2000, swimming lane 2 and swimming lane 3 are PCR amplification products), indicating a band of about 1800bp, the size and prediction The results were consistent. A DNA fragment GENECLEAN II glass milk recovery kit (purchased from Q Biogene) was used to recover a fragment of about 1800bp in size, cloned into the pGEM-T vector, and subjected to gene sequence analysis. The results showed that the amplified G17P64K gene had the SEQ ID in the sequence table The nucleotide sequence of NO: 5, its coding sequence is from the 1st to the 1830th base at the 5' end, and the coding sequence is the amino acid residue sequence of Sequence 1 in the sequence listing.

2. Construction and identification of recombinant expression vector pET28a-G17P64K

Both the G17P64K fusion protein gene obtained above and the vector pET28a (purchased from Novagen) were double-digested with Nco I and EcoR I restriction endonucleases (NcoI and EcoRI were purchased from TaKaRa Co.), and then T ₁ DNA ligase (purchased from Shanghai Huamei Bioengineering Co., Ltd.) by linking the G17P64K fusion protein gene into pET28a to obtain the recombinant expression vector pET28a-G17P64K containing the G17P64K fusion protein gene. The recombinant expression vector pET28a-G17P64K was transformed into Escherichia coli DH5α (purchased from Beijing Broadtech Co., Ltd.) by CaCl _method , and then screened on resistant LB medium (penicillin 60 μg/ml); under the guidance of primer 3 and primer 4 , positive clones were screened by the method of colony PCR; positive clones were placed in LB liquid medium at 37°C and 150rpm for shaking culture, until the bacterial density _OD600 was about 1.0, the SV Minipreps plasmid recovery kit (purchased from Promega company ) to extract the plasmid; the plasmid was identified by double digestion with Nco I and EcoR I restriction endonucleases, and the digested product was subjected to 1% agarose gel electrophoresis, the results are shown in Figure 1c (swimming lane 1 is the G17P64K gene, Lane 2 is pET28a-G17P64K double digestion product, and lane M is Marker DL2000), indicating that the double digestion product has a large and small fragment, the small fragment should be a G17P64K gene fragment, about 1800bp, and the large fragment is a carrier fragment, proving that The strain is a positive clone containing the recombinant expression vector pET28a-G17P64K. The positive cloned strain was sent to Boya Company for sequencing. The results showed that the G17P64K gene has the nucleotide sequence of sequence 5 in the sequence listing and encodes the amino acid residue of sequence 1 in the sequence listing. sequence. The sequencing results were compared with the predicted sequence, which further proved that the constructed recombinant expression vector pET28a-G17P64K was correct.

3. Expression of fusion protein G17P64K

The sequenced correct recombinant expression vector pET28a-G17P64K was transformed into Escherichia coli BL21(DE3) (purchased from Beijing Broadtech Co., Ltd.) by CaCl ₂ method to obtain the recombinant bacteria, which was named as G17P64K/BL21(DE3). (containing penicillin 60 μg/ml) for screening; select a single colony and inoculate it in 20ml LB medium (containing penicillin 60 μg/ml) at a rate of 1% to prepare a seed solution at 37°C, and inoculate the seed solution at a rate of 10% in Ferment and cultivate in LB liquid medium at 37°C until _OD600 is about 0.8, add IPTG (final concentration: 1mM) and continue to cultivate for 5h; collect the fermented bacteria after ultrasonic disruption, centrifuge at 5000rpm for 10min, divide into supernatant and precipitate, and separate the precipitate Wash twice with PBS buffer, take 20 μl each of the bacterial liquid after induction of expression, the supernatant of the bacterial liquid after centrifugation, the supernatant of bacterial cell ultrasonic lysis, the bacterial liquid of bacterial cell after ultrasonic lysis, and the bacterial liquid without IPTG-induced expression, and add 10 ×SDS-PAGE After 2 μl of sample buffer was boiled for 5 minutes, 15% SDS-PAGE was performed. The results are shown in Figure 2, indicating that the G17P64K fusion protein is expressed soluble in the cell, and the expression level of the G17P64K fusion protein is determined to be about 100% of the whole bacterial protein by scanning. 40% of. In Fig. 2, M is the low molecular weight protein standard, 1 is the bacterium liquid after IPTG induction, 2 is the centrifugation supernatant after the bacterial cell ultrasonic cracking after the IPTG induction, 3 is the supernatant after the centrifugation of the bacterium liquid after the IPTG induction (being culture medium supernatant ), 4 is the bacterial liquid induced by IPTG and then centrifuged after ultrasonic lysis, and 5 is the bacterial liquid without IPTG-induced expression.

4. Purification and purity analysis of fusion protein G17P64K

Buffer A: 20mmol/L Tris-HCl, 1mol/L (NH4), 1mmol/L EDTA, pH7.2

Buffer B: 20mmol/L Tris-HCl, 1mmol/L EDTA, pH7.2

Buffer C: 20mmol/L phosphate, 1mmol/L EDTA, pH6.0

Buffer D: 20mmol/L phosphate, 500mmol/L NaCl, 1mmol/L EDTA, pH6.0

After the IPTG-induced bacterial cells in step 3 were ultrasonically lysed, the centrifuged supernatant was precipitated with 30% saturated ammonium sulfate, and then subjected to three-step chromatography of hydrophobicity, molecular sieve and anion. The specific steps are as follows: take 20 g of wet cells induced by IPTG (final concentration: 1 mM) in step 3 for 5 h, wash once with buffer B, centrifuge, discard the supernatant, resuspend the cells with 150 ml of buffer A, and ultrasonically After crushing for 30 min, take the supernatant after centrifugation, add (NH ₄ ) ₂ SO ₁ to a final concentration of 1 mol/L, adjust the pH to 7.2, filter with a 0.45 μm membrane, and perform Butyl Sepharose 4F.F (purchased from Pharmacia) hydrophobic layer analysis. Elute in stages at a flow rate of 3ml/min (15% buffer B5×column bed volume, 20% buffer B3×column bed volume, 100% buffer B5×column bed volume), collect 20% buffer B eluted samples solution. Then the sample was concentrated and passed through a G200 Sephadex gel column (purchased from Pharmacia Company), eluted with PBS solution, and the eluted peaks of the sample were collected. After the collected samples were diluted with 5×column bed volume of 20mmol/L buffer C and adjusted to pH 6.0, ion-exchange chromatography was carried out on Q Sepharose High Performance 16/100 (filler purchased from Pharmacia). Use buffer D for stage elution (20% buffer D5×column bed volume, 30% buffer D3×column bed volume, 100% buffer D5×column bed volume), and collect 30% gradient elution peaks.

Add 20 μl of each of the above-mentioned purified protein samples to 10×SDS-PAGE sample buffer, boil 2 μl for 5 minutes, and then perform 15% SDS-PAGE. The purity of the fusion protein G17P64K sample can reach more than 90%. In Figure 3, 1 is the protein low molecular weight standard; 2 is the bacterial liquid after IPTG induction; 3 is the centrifuged supernatant after IPTG induction of the bacterium after ultrasonic lysis; 4 is the hydrophobic chromatography sample; 5 is the molecular sieve chromatography sample; 6 is anion Chromatography samples.

5. Analysis of N-terminal amino acid residues of fusion protein G17P64K

After performing 15% SDS-PAGE reduction electrophoresis on the G17P64K protein sample purified in step 4, the protein was transferred to a PVDF (polyvinyl difluoride) membrane by electrotransfer. After the PVDF membrane was stained and decolorized with Coomassie Brilliant Blue R-250, it was sent to the Central Laboratory of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences for N-terminal amino acid residue sequence analysis. The N-terminal sequencing results showed that the N-terminal amino acid sequence of the expressed fusion protein G17P64K was MEGPW, which was consistent with expectations.

6. Cultivate engineering bacteria with 5L bioreactor

Inoculate a single colony of a high-expression strain of G17P64K fusion protein into 5ml LB (containing 30 μg/ml kanamycin) medium, cultivate until the _OD600 value is about 0.8, and transfer to 200ml LB (containing 30 μg/ml kanamycin) ) to continue culturing until the OD ₆₀₀ value is about 0.6-1.0, as a fermented seed liquid. Put all 200ml of seed bacteria into 3L high-density medium, culture at 37°C until the _OD600 value is about 20-25, add IPTG (final concentration: 1mmol/L) to induce expression for 7h, then stop the culture, and collect the bacteria by centrifugation . During the cultivation process in tanks, samples (fermentation broth) were taken every 1 hour to measure the bacterial density. The results are shown in Figure 4, indicating that the biomass can reach OD ₆₀₀ =34. After the induction, a sample (fermentation broth) was taken every 1h to the 7th hour, and 1 μl of the fermentation broth of each sampling was taken for 15% SDS-PAGE analysis. The results are shown in Figure 5, indicating that the expression level of the fusion protein G17P64K reached 5h after induction. maximum value. In Fig. 5, M is the low molecular weight protein standard, 0 is the sample before induction, 1 is the sample of induction 1 hour, 2 is the sample of induction 2 hours, 3 is the sample of induction 3 hours, 4 is the sample of induction 4 hours, 5 is the sample of induction 5 hours, 6 Induction 6 hours samples, 7 induction 7 hours samples.

Embodiment 2, the preparation of P64-G17 cross-linked protein

1. Synthesize G17 polypeptide by chemical synthesis

Using 9-fluoromethoxycarbonyl (9-fluoromethyloxycarbonyl, FMOC) chemical solid-phase method to synthesize G17 polypeptide, the synthesized G17 polypeptide is cross-linked with a B-cell epitope of 9 amino acids at the N-terminal of gastrin G17 and a polypeptide of 5 amino acids Bridge, C-terminal plus 1 Cys for cross-linking with carrier protein, the sequence is as follows: N-terminal pyro (pyrophosphorylation)-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Gly-Gly- Gly-Gly-Ser-Cys C-terminus, and then the synthesized G17 polypeptide was desalted and purified by gel chromatography column and reverse high-performance liquid chromatography according to conventional methods, and finally sampled and identified by HPLC method, the results are shown in Figure 6, It shows that the synthesized amount of the G17 polypeptide sample is 60 mg, and the purity is 60% (the retention time of the G17 polypeptide is 19.857 minutes).

2. Chemical cross-linking of synthetic G17 polypeptide with carrier protein P64K and DT

The cross-linking of the synthesized G17 polypeptide with the carrier protein P64K and DT adopts the MBS method. MBS is a heterobifunctional cross-linking agent. The first step is to link MBS with carrier protein to form MBS/P64K or MBS/DT linker. After chromatographic purification, the conjugate was cross-linked with a Cys-containing G17 synthetic peptide. During the reaction, the Cys residue of the synthetic peptide provides the sulfhydryl group, and the N-terminal of the carrier protein and the Lys side chain provide the amino group. The specific method is as follows:

2.0 mg of carrier protein DT (purchased from Sigma, product number D2189) and 3.4 mg of G17 polypeptide with a purity of 60% prepared in step 1 were cross-linked by conventional MBS method and 5 mg of P64K synthesized and purified by conventional methods were mixed with step 1 After the prepared 8.3 mg G17 polypeptide with a purity of 66% was cross-linked by the conventional MBS method, the obtained two cross-linked protein products were desalted by dialysis, and then concentrated by centrifugation at 12,000 rpm for 15 min in an ultrafiltration centrifuge tube with a molecular weight cut-off of 50,000. Finally, the protein concentration of the concentrate was determined by the Lowry method, and the results showed that 8.0 mg of P64K-G17 cross-linked protein and 3.0 mg of DT-G17 cross-linked protein were finally obtained.

Example 3, Fusion protein G17P64K, P64K-G17 cross-linked protein, DT-G17 cross-linked protein as antigen to prepare gastrin G17 antibody and determination of its titer

1. Preparation of rabbit anti-gastrin G17 antibody

Experimental animals: Rabbits, the strain is big-eared white, the grade is second-class, the number is 10, half male and half female, weighing about 2Kg, strong and strong, and the time is three months.

Adjuvant: CFA Freund's complete adjuvant, for the first immunization; IFA Freund's incomplete adjuvant, for booster immunization.

The animal experiments were divided into five groups (2 rabbits in each group), all at week 0 (day 0, first immunization), week 3 (day 21, booster immunization), week 6 (day 42, booster immunization) ) for three times of immunization, each subcutaneously injecting the following substances at a dose of 0.5ml/body/time: Group A solution (polypeptide G17)+physiological saline+adjuvant; group B solution (G17-DT cross-linked protein)+physiological Saline + adjuvant; group C solution (G17-P64K cross-linked protein) + normal saline + adjuvant; group D solution (G17P64K fusion protein) + normal saline + adjuvant; group E solution P64K protein + normal saline + adjuvant. The doses of immunization injections are shown in Table 1:

Table 1. Immunization dose list

Rabbits treated by the above method were used to draw blood from the conventional ear vein at 0, 2, 5, and 8 weeks (ie, 0, 14, 35, and 56 days) after the first immunization, 2 ml each time. At the 12th week (that is, the 84th day), blood was collected by carotid artery stripping and catheterization.

Place the obtained blood sample in a sterilized container at 4°C, place the container at an angle to allow the serum to precipitate naturally, then carefully suck the precipitated serum with a gun (or pipette), and place the obtained serum in another sterile container. Store in bacteria-free containers at -20°C.

2. Determination of antibody titer of rabbit G17 antiserum

Use the G17 synthetic peptide solution (5 μg/μl) as the antigen; use the antisera on days 0, 14, 35, 56, and 84 after the first immunization in step 1 as the primary antibody, and use the serum of the same rabbit before immunization as negative As a control, PBS was used as the blank control; HRP-labeled goat anti-mouse antibody was used as the enzyme-labeled secondary antibody (purchased from Beijing Dingguo Bioengineering Company); the serum antibody titers of different samples at different times before and after immunization were measured by conventional indirect ELISA. Spend. Among them, the sample OD ₄₉₂ value ≥ 2 times the negative control OD ₄₉₂ value was judged as positive. The titer was judged according to the reciprocal of the maximum dilution multiple of the antiserum solution showing a positive reaction. The experimental results are shown in Table 2:

Table 2. Rabbit anti-gastrin G17 antiserum antibody titer table

Table 2 shows that the immunization effects of the DT-G17 cross-linked protein group and the P64K-G17 cross-linked protein group were similar, and both produced high-titer antiserum antibodies. However, the immune effect of fusion protein G17P64K group was poor, and the antiserum antibody titer was not as good as that of synthetic G17 polypeptide alone.

Embodiment 4, in vitro activity evaluation of rabbit G17 antiserum antibody

1. Culture of SW480 cells

SW480 cells (purchased from the Cell Bank of Peking Union Medical College) were inoculated in L15 medium (Leibovitz's L-15 Medium, powder, manufacturer IVGN) or IMDM medium (Iscove's Modified Dulbecco's Medium, powder, manufacturer IVGN), in the medium containing 5% CO ₂ cell culture incubator, 37 ° C, cultured in saturated air humidity for 48 hours for subculture (1/4-1/2 cell volume).

2. Purification of rabbit G17 antiserum antibody

The antibody was purified by saturated ammonium sulfate precipitation method, first extracted three times with saturated ammonium sulfate solutions with concentrations of 50%, 40% and 33%, and then dialyzed. Get the purified samples of each step and do 15% SDS-PAGE electrophoresis analysis, and the purification results are as shown in Figure 7 (swimming lane M is a low molecular weight protein standard, swimming lane 1 is serum, swimming lane 2 is a 50% ammonium sulfate precipitation sample, and swimming lane 3 is a 40% Ammonium sulfate precipitated sample, lane 4 is 33% ammonium sulfate precipitated sample, lane 5 is the sample after dialysis), indicating that the antibody obtained after dialysis has the highest purity.

The concentration of the purified antibody was determined by the Lowry method, and the titer was determined after dilution to 0.5 mg/ml respectively. The results are shown in Table 3:

Table 3. Rabbit anti-gastrin G17 purified antibody titer table

It shows that both the DT-G17 cross-linked protein group and the P64K-G17 cross-linked protein group have produced high-titer antiserum antibodies, and the immune effect is better.

3. In vitro activity evaluation of rabbit G17 antibody

The antibody IgG purified in step 2 was serially diluted to quantitatively determine its inhibitory effect on the growth of SW480 cells (purchased from the Cell Bank of Peking Union Medical College, China). The experiment was divided into 3 groups, namely: experimental group 1 (DT-G17 polypeptide cross-linking protein group), experimental group 2 (P64K-G17 polypeptide cross-linking protein group) and control group (P64K protein group), the specific steps are as follows:

1) Use 0.25% trypsin to digest SW480 cells cultured in monolayer adherence, and use IMDM culture medium containing 10% HyClone fetal bovine serum: 10% HyClone fetal bovine serum, 100 units/ml penicillin and 100 units/ml streptomycin (Penicillin is purchased from Ameresco Company, product number 0339; streptomycin is purchased from Ameresco Company, product number 0382), 15mM HEPES (3.57465g/L; molecular weight 238.31), 2.93g/L L-glutamine, 2g/L sodium bicarbonate, 5×10 ^-5 M mercaptoethanol (4 μl/L; molecular weight 78.13) to prepare a single cell suspension with a concentration of 5×10 ⁴ cells/ml;

2) Add 100 μl of single-cell suspension to each well of a 96-well cell culture plate, move the culture plate into a _CO2 incubator, and incubate for 2-3 hours at 37°C, 5% _CO2 and saturated air humidity to make the cells stick to each other. wall;

3) Prepare the antibody IgG purified in step 2 to a standard protein concentration of 0.5 mg/ml, dilute it 2 times with IMDM culture medium, add diluted samples or purified antibody IgG (control) to each well, and do 4 parallels for each dilution hole. 8 blank control wells and 8 positive control wells were cultured for 48 hours in a CO ₂ incubator at 37°C, 5% CO ₂ and saturated air humidity;

4) Add 10 μl of MTT staining solution to each well, and continue culturing for 4-6 hours in a CO ₂ incubator at 37° C., 5% CO ₂ and saturated air humidity;

5) Add 50-70 μl of 10mmol/L HCl containing 10% SDS to each well and let it stand for 12-24 hours until the reduced product is completely dissolved.

Finally, it was placed on an enzyme-linked detector to measure the OD ₅₇₀ value, and the logarithm of the OD ₅₇₀ logarithm was plotted against the logarithm of the sample dilution, indicating that the rabbits of the DT-G17 polypeptide protein cross-linking group and the P64K-G17 polypeptide protein cross-linking group Anti-gastrin G17 antibody can inhibit the growth of tumor cells, and the effects of the two groups are comparable.

sequence listing

<160>7

<210>1

<211>609

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>1

<210>2

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>2

<210>3

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>3

<210>4

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>4

<210>5

<211>1830

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>5

<210>6

<211>1785

<212>DNA

<213> Neisseria meningitids

<400>6

<210>7

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>7

Claims (11)

1, a kind of vaccine that prevents and/or treats digestive system tumor, its active component are gastrin G17 crosslinking protein or gastrin G17 fusion rotein; Described gastrin G17 crosslinking protein is that 9 peptide sequences of P64K albumen and gastrin G17N end obtain by chemical crosslink technique; The amino acid residue sequence of described gastrin G17 fusion rotein is shown in the sequence in the sequence table 1.

2, vaccine according to claim 1 is characterized in that: described P64K dietary protein origin is in meningococcus.

3, vaccine according to claim 1 and 2 is characterized in that: described chemical crosslink technique is MBS method or EMCS method.

4, vaccine according to claim 3 is characterized in that: the carboxyl terminal of 9 peptide sequences of described gastrin G17N end closely is connected with one 6 peptide or 7 peptides, and the carboxyl terminal amino acid residue of described 6 peptides or 7 peptides is a cysteine.

5, vaccine according to claim 4 is characterized in that: the amino acid residue sequence of described 6 peptides is shown in sequence in the sequence table 3; The amino acid residue sequence of described 7 peptides is shown in sequence in the sequence table 7.

6, vaccine according to claim 5 is characterized in that: the preparation method of described gastrin G17 crosslinking protein may further comprise the steps:

1) synthetic 15 peptide ammino acid residue sequence shown in sequence in the sequence table 4;

2) be that the P64K albumen of 3-5:7-10 and 15 peptides in the step 1) are crosslinked by the MBS method with mass ratio, obtain gastrin G17 crosslinking protein.

7, vaccine according to claim 1 is characterized in that: described gastrin G17 fusion rotein encoding gene, its nucleotide sequence is shown in sequence in the sequence table 5.

8, vaccine according to claim 1 is characterized in that: the nucleotide sequence of described P64K protein coding gene is shown in sequence in the sequence table 6.

9, the carrier that contains gastrin G17 fusion rotein encoding gene; The amino acid residue sequence of described gastrin G17 fusion rotein is shown in the sequence in the sequence table 1.

10, the engineering bacteria that contains gastrin G17 fusion rotein encoding gene; The amino acid residue sequence of described gastrin G17 fusion rotein is shown in the sequence in the sequence table 1.

11, the cell line that contains gastrin G17 fusion rotein encoding gene; The amino acid residue sequence of described gastrin G17 fusion rotein is shown in the sequence in the sequence table 1.

Images