CN101584860A - Recombinant human Claudin18.2 tumor vaccine and preparation method thereof - Google Patents
Recombinant human Claudin18.2 tumor vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of medicinal biotechnology, and in particular relates to a recombinant human Claudin18.2 tumor vaccine and a preparation method thereof. The invention aims to solve the problems of immunotherapy for stomach cancer, pancreatic cancer, esophagus cancer and metastatic and non-metastatic ovarian cancer, such as high after-excision recurrence rate, strong chemo-treatment and radiation treatment toxic side effect and high monoclonal antibody therapy cost. The invention adopts a technical proposal that the recombinant human Claudin18.2 tumor vaccine has a sequence of HMKSSQYIKANSKFIGEFDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAV. Animal experiments prove that rhClaudin18.2 fusion protein can induce high-titre neutralizing antibody in the bodies of tumor-bearing mice by over 1:10,000; the antibody can be combined with human KATOIII and PANC-1 tumor cells and mouse stomach cancer MFC and pancreatic cancer MPC-83 cells; and the protein serving as a tumor vaccine can suppress the growth of the stomach cancer MFC and pancreatic cancer MPC-83 cells in the bodies of the mice.
Description
One, technical field
The invention belongs to the medical biotechnology field, be specifically related to the expression in prokaryotic cell of gene clone, gene recombinaton, exogenous gene, destination protein purification, animal immune experiment, induce the sero-fast technology such as tumor experiment that presses down, further relate to recombined human Claudin18.2 tumor vaccine and preparation method thereof.
Two, background technology
20 for many years, and the antigen of expressing about human tumor immunoreation result of study tumor cells showed can cause specific cell and humoral immune reaction, but can be very rare by the case of the spontaneous removing tumor of endogenous immunologic mechanism.2004, Giorgio Parmiani pointed out to use vaccine based on peptide in the clinical I-II phase process of treatment IV phase melanoma patients, only has 12% patient clinical response to occur after treatment.In recent years, compare with said method, in the case of using autologous dendritic cell (DCs) and peptide or tumor lysate together to treat, increase though the patient produces the probability of vaccine specific T cell, relevant clinical response does not significantly improve yet.Immune system can not cause that tumor regression is a great difficulty that is faced in the tumor vaccine research.A large amount of research evidences show that tumor exists the mechanism of many escape immune system recognition and attack, comprising: the down-regulated expression of HLA-I class antigen and immune costimulatory molecules or lose; The down-regulated expression of tumor antigen, lose or suddenly change; Tumor cell secretion inhibitive ability of immunity soluble factor; Express the inhibitive ability of immunity molecule on the tumor cell membrane; Induce regulatory T lymphocyte with immune suppression function etc.Wherein, the inhibition of the immunoreation in the tumor tissues is the key factor of tumor cell escape immune attack.Many evidences show that apoptosis can take place the specific, activated T lymphocytes that is present in the tumor tissues.Behind external activatory tumour-specific T lymphocyte input tumor tissues, its killing activity disappears.These phenomenons all illustrate the immunologic escape mechanism that exists the protection tumor cell in tumor microenvironment.Simultaneously, also have many soluble factor (TGF-β, IL-10, PGE2, Fas, TRAIL and RACS1 etc.) and membrane molecules (CTLA4, B7-H1 etc.) to raise at tumor cells expression with immune suppression function.Therefore, relevant escape mechanism at immune attack is significant for the therapeutic effect that improves tumor vaccine in the research tumor tissues.
1, expression and the present Research thereof of Claudins family protein in tumor
Closely connecting is a kind of of cell adhesion form, mainly is present in the junctional complex between epithelial cell, endotheliocyte.It can keep the stable of organismic internal environment, keeps epithelial polarity, influences motion, propagation, the differentiation of cell, regulates the performance of cell function.Closely link molecule is by Occludin, Claudins be connected adhesion molecule (Junctional adhesion molecules, JAMs) 3 kinds of complete memebrane proteins and closed little cyclase protein (ZO-1, ZO-2 and ZO-3) etc. the periphery plasmosin form, but for they function and to regulate research at present still few.In recent years confirmed that Claudin albumen is to constitute close-connected skelemin, its unconventionality expression can cause epithelial cell, endotheliocyte structural deterioration, function impaired, may play an important role in the pathogenic process of multiple disease.Up to the present, found that the Claudin gene family comprises 24 members, the concordance of its sequence is 12.5%-69.7%, and this result shows that they are the function high conservative on evolving.The molecular mass of Claudin is 22-27kD.Each Claudin molecule all has identical structure, and its peptide chain includes 4 c-terminus, the aminoterminals of striding film district, 2 extracellular annular structures and being arranged in kytoplasm.The the 1st and the 4th aminoacid sequence of striding film district and the 2nd extracellular loop has high conservative.Stride the film district for the 4th locking albumen accurately is positioned closely to connect to play an important role, will cause the proteic carboxyl terminal of the locking ECS that misplaces if lose.Two extracellular loop participate in the combination between of the same race or the xenogenesis Claudin albumen, and tight connection band is formed and the penetrating selectivity of ion has important function.
Claudins is distributed widely in normal structure and different tumor tissues, and the expression of the property of there are differences.Study its effect in tumor generation, growth and transfer process and be a focus in recent years.In recent years, in gastric precancerous lesion and gastric cancer, also find several claudins protein abnormal expression, and relevant with prognosis.In the actual life, gastric cancer, cancer of pancreas, esophageal carcinoma and transfer and not shifting exists excision relapse rate height in the ovarian cancer immunization therapy, the chemotherapy and radiation toxic and side effects is strong and the high problem of monoclonal antibody medical expense, pressing for new technology produces, and the research by claudins is distributed and expresses unusually in gastric cancer, people can better must understand occurrence and development of gastric carcinomas mechanism, thereby help the diagnosis and the treatment of gastric cancer.
Claudins gastric precancerous lesion, each phase of gastric cancer and various in the research of expression be focus in recent years, wherein claudin-4, claudin-7 and ctaudin-18 research is more.
Claudin-4 is positioned at human chromosomal 7q11.Usefulness SABC methods such as Lee are discovered E-cadherin and claudin-4 equal expression decreased in 69% gastric cancer, and more be common in diffusion-type gastric cancer and the low gastric cancer of differentiation, think that two kinds of albumen effects are similar, may lose relevant with the destruction of gastric gland body structure and the differentiation of gland cell.Research such as Resnick far-end patients with gastric cancer finds that by the COX multivariate analysis expression of high strength claudin-4 significantly descends relevant with patients with gastric cancer life cycle.In addition, Cunningham etc.Find that by the SABC method claudin-4 all has expression fully in 36 routine intestinalization and 14 routine atypical hyperplasia patients, in the metastatic carcinoma tissue of the former bit organization of 98% gastric cancer and 100%, expression is arranged also, and only have 15% normal mucosa tissues to express claudin-4, illustrate that claudin-4 has also played important function in the early stage oncogenic process of gastric cancer.
Johnson etc., in the mouse model of Trefoil factor-1 gene knockout, find by gene sequencing, the claudin-7 gene is the gene of frequent up-regulated in the gastric cancer, use the SABC method to find, when mice and human gastric mucosa body of gland atypical hyperplasia, normal overexpression claudin-7, and normal mucosa tissues is not expressed around it.It reaches positive rate at intestinalization, atypical hyperplasia and gastric cancer invading the exterior and is respectively 30%, 80% and 70%, and how to express in intestinal-type gastric cancer.Park etc. find by SABC and Western blot method, claudin-7 is more normal to be expressed in intestinalization, adenoma and the gastric cancer, and in 47% normal gastric mucosa, do not express, have only 11% intestinal-type gastric cancer not express claudin-7, and diffusion-type gastric cancer not expression rate reach 41%.So research worker thinks that the expression of claudin-7 has participated in gastric cancer evolution takes place in early days.Lioni etc. find that the dysfunction of claudin-7 causes the E-cadherin expression decreased in research esophagus scale cancer, thereby have increased the aggressive of esophagus scale cancer.Make claudin-7 inactivation in the strain of esophagus squamous cell carcinoma with the siRNA method, cause the E-cadherin expression decreased.Because all normal tissue of E-cadherin and claudin-7 and intestinal-type gastric cancer obviously reduce in diffusion-type gastric cancer, infer may also have this mechanism in gastric cancer.
2, Claudin18.2 brief introduction
Humanized Claudin18 gene has 2 first different exons, so can produce two kinds of subtype C laudin18.1 and Claudin18.2.69 amino acid whose structure differences of N end of these two molecules, it is positioned at the circulus of first extracellular region.Two kinds of hypotypes of Claudin18 are carried out transcription amplification respectively in different tissues, wherein, Claudin18.1 mainly is expressed in lung tissue, and Claudin18.2 is then specific expressed in gastric tissue.
2006, Sanada etc. find claudin-18 gene down-regulated expression in 57% gastric cancer, pass through immunohistochemical analysis, express claudin-18 on the after birth of normal gastric mucosa and duodenum paneth's cell, but in the adenoma of stomach of some intestinalization and 90%, the claudin-18 expression decreased, expression decreased is more common than its alloytype gastric cancer in intestinal-type gastric cancer simultaneously, infers that it may participate in the generation of early gastric cancer.Survival analysis shows that the claudin-18 expression decreased is relevant with the poor prognosis of patients with terminal in the gastric cancer, thinks that the claudin-18 expression decreased is the not good factor of patients with gastric cancer prognosis.
2008, Sahin etc. studies confirm that the expression that has Claudin18.2 in the 77% constitutional adenocarcinoma of stomach tissue, and very importantly wherein 56% tissue expression amount reaches more than 60%.Consistent with above-mentioned result of study is the proteic down-regulated expression of Claudin18.2 in the intestinal-type gastric cancer, and the proteic expression of Claudin18.2 is higher in the diffusion-type gastric cancer.But pancreas, esophagus, ovary etc. are organized in does not have the Claudin18.2 protein expression under the normal condition, and can great expression Claudin18.2 albumen in the corresponding tumor tissues.So Claudin18.2 albumen can be used as the target position of clinical tumor diagnosis and treatment.But having not yet to see with this albumen is the tumor vaccine of target position development.
3, therapeutic vaccine progress
In recent years, in the process of the acute and chronic disease of treatment, demonstrated good effect at the proteic monoclonal antibody of mankind itself.But, the costliness of cost and the inconvenience of using have limited the extensive use of monoclonal antibody, therefore, turn to and seek proteic active immunity vaccine by accepting this antibody protein passively at the mankind itself, both the therapeutic modality with active immunity substituted passive immunity, became the developing direction of protein drug.At present, the research of therapeutic vaccine has become a focus, relates to a lot of diseases, as: chronic viral infection, allergy, tumor, Alzheimer thatch disease, diabetes, hypertension, obesity and rheumatic arthritis etc.Therapeutic vaccine makes people's immune system help reaction.Most vaccine can be divided into two big classes: a class is to induce body generation humoral immune reaction, produces antibody; Another kind of is to induce body generation cell immune response, produces cytotoxic T cell (CTLs).Back one class therapeutic vaccine is mainly used in the treatment of tumor and disease of viral infection.
All by inducing production of antibodies to protect body, this just proves by inducing antibody to treat infectious disease is a kind of effective Therapeutic Method to most preventative vaccines.Compare with preventative vaccine, the development of therapeutic vaccine will be many slowly, just see the hope of success up to therapeutic vaccine in recent years.Simultaneously, monoclonal antibody is indicating that in obtained immense success aspect the treatment disease therapeutic vaccine that can induce antibody to produce in vivo has vast potential for future development.In fact, it is possible that the endogenous specific antibody that has had animal experiment to show to induce certain level is treated disease, as: blocking-up TNF-α is with the treatment diseases associated with inflammation.
It is very effective aspect treatment rheumatoid arthritis and Crohn ' s disease that humanized anti-TNF-alpha monoclonal antibodies has been proved to be.The blocker listing of several TNF-α has been arranged at present, comprise two kinds of monoclonal antibody (infliximab, adalimumab) and a kind of receptor blocking agent (etanercept), they are helping thousands of patient to palliate the agonizing sufferings, and annual income reaches 2,000,000,000 dollars.So the TNF-α of blocking-up overexpression can reach the effect of treatment disease.Verifiedly in animal experiment can specificity induce the neutrality antibody of TNF-α, and inductive antibody titer is enough to treat the scorching model of joint of animal by active immunity.
Other animal experiment shows and can treat following disease by producing high titre antibody in the inductor: the vaccine at angiotensin can be treated hypertension; Can treat Eosinophilia's disease that pathogen causes at the vaccine of IL-9; Vaccine at IL-5 can be treated asthma; Vaccine at N-methyl-D-aspartate receptor-1 (NMDAR1) can be treated apoplexy.In addition, the immunity at some gonadal hormone such as human chorionic gonadotropin (human chorionic gonadotro-pin HCG) can reduce the intravital hormonal readiness of women and reach contraceptive effect; Vaccine at gonadotropin-releasing hormone (GnRH) can be used for the treatment of advanced prostate cancer; Among the cancer of pancreas patient, the antibody that utilizes therapeutic vaccine to induce at gastrin (gastrin) can prolong patient's life late.
So, is there which problem in the research of therapeutic vaccine? as if this problem is answered recently in development process what happens at the therapeutic vaccine of Alzheimer thatch disease.Alzheimer thatch disease is a kind of very suitable disease for the treatment of with therapeutic vaccine that seems, the pathogenic process of this disease reaches several years even many decades.If can induce long-term antibody with therapeutic vaccine, be undoubtedly a kind of ideal Therapeutic Method so, especially this patient often forgets and takes medicine.
The feature of Alzheimer thatch disease is the deposition of speckle in the brain, contains the A beta-peptide in this speckle, and this A beta-peptide is that (amyloid precursor protein APP) derives for precursor protein from amyloid.In the gene of coding APP was undergone mutation the crowd that the generation that causes the A beta-peptide increases, Alzheimer thatch disease was just beginning to have taken place in one's early years.Expressing in the transgenic mice brain of APP of this sudden change has a large amount of plaque deposition, and the speckle of finding in this speckle and the sick patient's brain of Alzheimer thatch is similar.Add that with the A beta-peptide strong immunological adjuvant comes immune this transgenic mice that the speckle in the mouse brain is reduced, the spiritual expression of mice is clearly better.Subsequently, a kind of therapeutic vaccine at Alzheimer thatch disease has begun clinical trial, after it has good tolerability in clinical I phase evidence, the clinical II phase that comprises more than 300 patient has been followed by having begun, but unexpected be 6% subjects owing to produce the aseptic encephalitis and be forced to stop to test.Studies confirm that subsequently it doesn't matter for the titre of this side effect and antibody, in fact, the patient that tried who does not produce antibody response also got the aseptic encephalitis.In addition, in a patient's brain, find to have a large amount of lymphocytic infiltrations.These results of study are consistent with a kind of hypothesis, and that side effect of finding in patient is because A beta-peptide specific T lymphocyte causes, rather than since antibody cause.This just requires to develop the autoimmune second filial generation vaccine that does not cause the T cell mediated.So, how is the effect of this therapeutic vaccine at Alzheimer thatch disease? during the clinical II phase of mentioning in front tests, in the patient who induces anti-A beta-peptide antibody some cognitive power weaken certain postponement, some patients are arranged in addition in this year of receiving treatment state of consciousness improvement has been arranged.If when vaccine design, can solve its safety issue, so in the near future, will occur at the therapeutic vaccine of Alzheimer thatch disease.
Another kind of slightly different vaccine is lower to the requirement of safety, and that is exactly the vaccine at habit-forming medicine.Vaccine at cocaine and nicotine in animal experiment has reduced the level of these medicines in brain, has eliminated their addiction symptom, and experimental animal is being accepted immune then medicine no longer the dependence.In testing in the clinical I phase, the cocaine vaccine has been proved to be good tolerability, and can induce good antibody response, and now, people are longing for its validity result.
The Claudin18.2 limitation is expressed in the minority gastric tissue, gastric cancer, cancer of pancreas, esophageal carcinoma and transfer and do not shift can this albumen of great expression in the ovarian cancer tissue.First extracellular region of this albumen is compared high specificity with other Claudin family member, and the protein structure difference of remainder is not remarkable, so, first extracellular region protein of Claudin18.2 is developed tumor vaccine as target position has very important practical sense.
4, induce the theoretical research of autoantibody
The therapeutic vaccine of only having selected target molecule to make up an oneself protein also needs to induce the antibody of autoimmune system generation at oneself protein.The specific antibody that produces enough high titres is with the treatment relevant disease, and therapeutic vaccine must overcome three obstacles: T cell tolerance, Blymphocyte tolerance, induce antibody under the situation that does not have adjuvant and antigen durative action preparation.As everyone knows, the human immune system mainly starts to attack to external invador, and body itself is not attacked, and this may be because body has the ability that can discern " nonego " and " oneself ".Immune this specific character is commonly called tolerance or anergy.Tolerance occurs in B cell and T cellular level.In general, the T cell tolerance is stricter.Concerning many antigens, when the T cell tolerance took place, normal B cell strain but existed in vivo.In fact, have three kinds of mechanism to cause immunologic tolerance: cell strain is rejected, and promptly specific lymphocyte is thoroughly rejected from lymphocyte populations; Immunity is incompetent, and promptly specific lymphocyte exists, but its function can not be activated; Immunity is ignored, and the lymphocyte that promptly has immunologic function exists, but owing to do not run into the autoantigen that exists with the antigen form, so can not be activated.Concerning the T cell, inducing tolerance and incompetent major organs is thymus (central tolerance), but induces tolerance also can carry out in periphery.Blymphocyte tolerance is mainly induced in bone marrow, but also can induce in periphery.Usually, antigenic immunologic tolerance is easier to be illustrated for enriching of generally expressing.
In the immunoreation at exotic antigen, T cell and B cell are worked in coordination and could be produced antibody effectively: when being subjected to the exotic antigen immunity, specific B cell conjugated antigen produces initial activation signal.In addition, B cell endocytic antigen presents the complex of antigenic peptides and MHC II quasi-molecule on its surface.Usually, the B cell can not activate the Th cell.Activate the Th cell, dendritic cell is absolutely necessary, the dendritic cell antigen uptaking, and, activate the Th cell at the complex of its cell surface antigen-presenting peptide and MHC II quasi-molecule.The antigenic peptides that Th cell recognition B cell surface after the activation presents and the complex of MHC II quasi-molecule cause other conversion of B cell proliferation, production of antibodies and antibody class.If lack the synergism of Th cell because of immunologic tolerance, so just can not produce antibody.In design process at the vaccine of oneself protein, if autoantigen merged with foreign protein or peptide carrier or be coupled at, just might walk around the Th cell tolerance: the specific B cell of autoantigen just can absorb this autoantigen and coupled carrier protein, and present the complex of carrier peptides and MHC II quasi-molecule on its surface, because the Th cell does not have immunologic tolerance to carrier protein, so can be activated, thus collaborative from the specific antibody of the B of antigenic specificity cell generation at autoantigen.
Compare with the T cell tolerance, it is much loose that Blymphocyte tolerance is wanted.In fact, in many cases, autospecific B cell occurs with normal frequency in vivo, will be activated if be subjected to the combined effect of antigen and Th cell.So to many solubility oneself proteins, have its specific b cells strain in the body, especially when this albumen is not very expression more than needed especially like this.For this albuminoid or polypeptide, it is linked to each other with carrier protein, walk around the T cell tolerance, just might produce effective vaccine.
Three, summary of the invention
In sum, the object of the present invention is to provide a kind of recombined human Claudin18.2 tumor vaccine and preparation method thereof, to overcome gastric cancer, cancer of pancreas, esophageal carcinoma and transfer and not shift problem such as the strong and monoclonal antibody medical expense height of excision relapse rate height in the ovarian cancer immunization therapy, chemotherapy and radiation toxic and side effects.
To achieve these goals, the technical solution adopted in the present invention is:
A kind of recombined human Claudin18.2 tumor vaccine, its sequence is: HMKSSQYIKANSKFIGEFDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRG YFTLLGLPAMLQAV
The preparation method of above-mentioned recombined human Claudin18.2 tumor vaccine comprises the steps: successively
(1) clone of rhHis-TT-Claudin18.2 gene and construction of prokaryotic expression vector: adopt the RT-PCR method to obtain first extracellular region gene fragment of Claudin18.2 (the 28th amino acids to the 79 amino acids), with TT
830-843After genetic fragment connects, insert the pQE-30 prokaryotic expression carrier, transformed into escherichia coli; In escherichia coli, obtain to efficiently express, obtain His-TT-Claudin18.2 albumen after purified.
(2) expression of recombinant proteins: with the pQE-30-TT-Claudin18.2 plasmid transformation escherichia coli that builds, in escherichia coli, obtain to efficiently express, behind affinity purification, obtain rhHis-TT-Claudin18.2 albumen.
(3) purification of fusion rotein and renaturation: expression product is through splitting the purification of bacterium, dissolving inclusion body, Ni-NTA affinity column, and purity of protein can reach more than 80%.
In the above-mentioned steps (1): the proteic preparation method of rhHis-TT-Claudin18.2 is: adopt the RT-PCR method to obtain the Claudin18.2 genetic fragment from people's gastric cancer KATOIII cell, this fragment is connected TT
830-843Insert after the genetic fragment and make up the rhHis-TT-Claudin18.2 expression plasmid in the pQE-30 carrier; The construction of prokaryotic expression vector and the expression of recombinant proteins that contain genes of interest rhHis-TT-Claudin18.2; The evaluation of expression product; The purification of recombiant protein; RhHis-TT-Claudin18.2 protein immunization tumor-bearing mice can suppress the growth of interior gastric cancer MFC cell of mice body and cancer of pancreas MPC-83 cell.
Recombined human His-TT-Claudin18.2 tumor vaccine of the present invention (rhHis-TT-Claudin18.2), produce anti-Claudin18.2 antibody by immune tumor-bearing mice, thereby a kind of new pharmaceutical grade protein might be provided for the immunization therapy of gastric cancer and cancer of pancreas.
Application purpose of the present invention is with Claudin18.2 to be that target spot obtains a kind ofly can to impel the tumor vaccine that produces in the tumor-bearing mice body at the neutralizing antibody of Claudin18.2 molecule, thereby research is used the method for active immunity and carried out the immunization therapy of tumor.According to technical scheme of the present invention, adopt the method for RT-PCR to obtain the rhHis-TT-Claudin18.2 gene, make up prokaryotic expression carrier, and in escherichia coli, obtain to efficiently express.Expression product is through the purification of dissolving inclusion body, Ni-NTA affinity column, and purity of protein can reach more than 80%.But not only can produce the antibody of specificity behind the rhHis-TT-Claudin18.2 protein immunization tumor-bearing mice behind the purification, can also suppress the growth of interior gastric cancer MFC of mice body and cancer of pancreas MPC-83 cell simultaneously in conjunction with people's gastric cancer KATOIII and cancer of pancreas PANC-1 cell and Mouse Gastric Cancer MFC and cancer of pancreas MPC-83 cell.
Find by experiment in vitro, use the antibody in the immune serum that obtains of rhHis-TT-Claudin18.2 tumor vaccine to combine with kinds of tumor cells such as gastric cancer and cancer of pancreas by specificity; The antibody that produces after the cell of normal stomach and tissue and this tumor vaccine immunity only has a small amount of the combination; And this antibody does not have obvious the combination with remaining tissue.So, security of products height provided by the present invention.The rhHis-TT-Claudin18.2 tumor vaccine adopts the Ni affinity column chromatography to obtain, and the solution renaturation that the method for employing gradient obtains purification is so preparation method is easily gone.
Four, description of drawings
Fig. 1 is that the enzyme action of recombinant expression plasmid pQE-30-TT-Claudin18.2 is identified the about 220bp of purpose clip size (TT+Claudin18.2);
Fig. 2 is the expression of rhClaudin18.2, wherein 1: (bottom one: 10000Da) of molecular weight protein matter standard, 2: do not induce rhClaudin18.2,3: the rhClaudin18.2 after inducing, 4: split the bacterium precipitation, 5: split the bacterium supernatant, 6: rhClaudin18.2 behind the purification, 7: the rhClaudin18.2 albumen after the renaturation;
The immunoblotting of Fig. 3 rhClaudin18.2 protein expression and purification identifies, wherein 1: do not induce rhClaudin18.2,2: the rhClaudin18.2 after inducing, 3: split the bacterium precipitation, 4: split the bacterium supernatant, 5: rhClaudin18.2 behind the purification, 6: the rhClaudin18.2 albumen after the renaturation;
Fig. 4 rhClaudin18.2 immune serum combines with tumor cell, 1:MFC wherein, 2:MPC-83,3: islet cells cancerous tissue lysate, 4:KATOIII, 5:PANC-1;
Fig. 5 tumor-bearing mice tumour inhibiting rate, wherein MFC is gastric cancer group (black is the Claudin18.2 immune group, and white is physiology saline control group), MPC-83 is cancer of pancreas group (black is the Claudin18.2 immune group, and white is physiology saline control group).
Five, the specific embodiment
For a more clear understanding of the present invention, the present invention is described in further detail for the embodiment that finishes below in conjunction with the inventor.
The present invention is described in further detail below in conjunction with drawings and Examples.
One, recombined human Claudin18.2 tumor vaccine provided by the present invention (rhHis-TT-Claudin18.2), its aminoacid sequence is:
HMKSSQYIKANSKFIGEFDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAV
Two, above-mentioned recombined human Claudin18.2 tumor vaccine (rhHis-TT-Claudin18.2) preparation method may further comprise the steps successively:
(1) clone of rhHis-TT-Claudin18.2 gene and construction of prokaryotic expression vector:
According to first extracellular region gene order of people Claudin18.2 among the GeneBank, adopt the RT-PCR conventional method, the EcoRI restriction enzyme site is introduced in the upstream, and the downstream is introduced the SalI restriction enzyme site and is obtained Claudin18.2
28aa-79aaWith the tetanus toxoid epi-position (be TT
830-843Epi-position: aminoacid sequence is QYIKANS KFIGITE, with cell immune response stronger in the primosome, thereby promotes that tumor vaccine better plays a role) gene connection (TT
830-8435 ' end of genetic fragment is BamHI, and 3 ' end is EcoRI), insert the pQE-30 carrier.Connect product transformed competence colibacillus cell DH5 α, picking monoclonal overnight incubation is extracted plasmid, identifies the insertion fragment (Fig. 1) that obtains the expection size through enzyme action, checks order.The plasmid called after pQE-30-rhHis-TT-Claudin18.2 that checks order correct.Engineering bacteria called after pQE-30-rhHis-TT-Claudin18.2/DH5 α.
(2) expression of recombinant proteins:
Picking engineering bacteria list colony inoculation in 5mLLB culture fluid (containing Amp100mg/L), 37 ℃ of shaking table overnight incubation, next day, the ratio with 1: 100 was transferred in the LB culture fluid that contains Amp, cultivated 3h to logarithmic growth (OD in mid-term at 37 ℃ of shaking tables
600Be 0.4～0.6), adding final concentration is the IPTG of 1mmol/L, continues to cultivate 4h.Centrifugal collection thalline.
Above-mentioned expression product is identified:
①SDS-PAGE
Get 30 μ L and split the bacterium supernatant, add 30 μ L, 2 * load sample buffer, mixing.Other gets other samples and adds 30 μ L water, adds 30 μ L, 2 * load sample buffer mixing behind the suspendible again.Boil 5min in the boiling water, the centrifugal 5min of 12000rpm, get 10 μ L supernatant application of samples and concentrate glue in 18% separation gel 6%, voltage is the about 20min of 60V behind the last sample, voltage rose to the about 4-5h of 100V after sample entered separation gel, with 0.5% Coomassie brilliant blue R-250 vibration dyeing 2-3h, decolouring is preserved (referring to Fig. 2) until the dried glue of the clear back preparation of background.
2. immunoblotting reaction
After SDS-PAGE finishes, press the Bio-Rad description of product, gel is near negative electrode one side, nitrocellulose membrane (NC film) is near anode one side, put (25mmol/L Tris in the transfering buffering liquid, 192mmol/LGlycine, 20% methanol), 100V constant voltage 1h, with albumen from the gel electrotransfer to the NC film, after electrotransfer finishes, take out the NC film, (contain 0.02mol/L pH7.4TBS with cleaning mixture TBST, 0.4%Tween20) room temperature is washed 3 times, immerse in the confining liquid (TBST that contains 2%BSA) 37 ℃, 1h, cleaning mixture (TBST) room temperature is washed 3 times, add the anti-people Claudin18.2 of goat antibody (Santa cruze company), hatch 1h for 37 ℃, the TBST room temperature is washed film 3 times, adds the anti-goat IgG-HRP of rabbit two anti-(middle China fir company), hatch 1h for 37 ℃, the TBST room temperature is washed film 3 times, and reuse TBS washes 3 times, and the NC film immerses in the OPD colour developing liquid, room temperature lucifuge colour developing 10min, distilled water flushing cessation reaction (referring to Fig. 3).
(3) purification of fusion rotein and renaturation
Get the bacterial strain that contains the pQE-30-TT-Claudin18.2 recombiant plasmid and induce bacterium liquid in a large number, the centrifugal 10min of 5000rpm receives bacterium, ultrasonicly splits bacterium; 4 ℃, 12000rpm, centrifugal 20min collects supernatant and precipitation respectively.To precipitate with the 6mol/L urea liquid resuspendedly, leave standstill dissolving under 4 ℃.Dissolved precipitation and supernatant taken a sample respectively carry out the SDS-PAGE electrophoresis, the expression-form of destination protein is analyzed.After confirming the formal representation of destination protein, with Ni-NTA post affinitive layer purification dissolved precipitation (inclusion body) with inclusion body.Adding 10mL in the 1g thalline, to split the ratio of bacterium buffer resuspended with thalline, carries out the ultrasonic bacterium of splitting under the condition of ice bath.12000rpm, centrifugal 15min abandons supernatant, and the 1g precipitation adds 10mL 6M carbamide, 0.1MNaH
2PO
4, 0.01M Tris (pH 8.0).4 ℃ are stirred 2h, 12000rpm, and centrifugal 15min, centrifugal 2 times altogether, it is stand-by to collect supernatant.Use 6M carbamide, 0.1M NaH
2PO
4, 0.01M Tris (pH 8.0) is balance Ni post fully, earlier with the 6M carbamide that contains the 10mmol imidazoles, 0.1M NaH
2PO
4, 0.01M Tris (pH 8.0) eluting foreign protein, reuse contains the 6M carbamide of 250mmol imidazoles, 0.1M NaH
2PO
4, 0.01M Tris (pH 8.0) eluting destination protein.Collect eluting peak, purified product is dialysed to 2M carbamide, 0.1M NaH
2PO
4, 0.01MTris (pH 6.0) renaturation, final concentration 1mg/ml.Dialysis is at last gone among the 0.01M Tris (pH 6.0), after PEG8000 concentrates, measures protein concentration (referring to Fig. 2) with the Bradford method.
Three, animal model experiment:
(1) mice group and immunization protocol
Grouping: 24 BALB/c mouse are divided into 4 groups, subcutaneous in mouse back according to 1 * 10
7Individual/as only, to inoculate each 2 groups in gastric cancer MFC and cancer of pancreas MPC-83 cell respectively for 6 every group.2 groups of tumor-bearing mices of each tumor cell inoculation, treat into tumor after, inject rhClaudin18.2 or normal saline respectively according to the routine immunization method.Pluck the eyeball blood sampling in back 10 days in the 4th immunity, measure anti-Claudin18.2 antibody titer in the serum.Immunization protocol: the subcutaneous multi-point injection in back; RhClaudin18.2:40 μ g/+normal saline cumulative volume is 200 μ L/, respectively injection every other week; The 4th is only used g/+normal saline 200 μ L/ of rhClaudin18.2:40 μ, lumbar injection.
(2) the ELISA method is measured the anti-Claudin18.2 antibody titer of serum
In the 4th immunity blood sampling in back 10 days, collect antiserum, measure the anti-Claudin18.2 antibody titer of serum by indirect ELISA.Each sample is all established 6 holes.The ELISA experimental technique is as follows:
The preparation of required solution: a. bag is cushioned liquid: carbonate buffer solution is got Na
2CO
3: 0.32g (final concentration 0.015mol/L), NaHCO
3: 0.59g (final concentration 0.035mol/L), pure water are settled to 200mL (pH 9.6), effect duration: two weeks.B. cleaning mixture: the PBS (pH 7.4) that contains 0.5%Tween-20.C. confining liquid: the cleaning mixture that contains 1%BSA.D. diluent: the cleaning mixture that contains 0.5%BSA.E. substrate buffer solution: Na
2HPO
412H
2O:1.84g, citric acid: the 0.51g pure water is settled to 100mL (pH5.0).F. substrate colour developing liquid: OPD:8mg, 3%H
2O
2: 30 μ L.H. stop buffer: H
2SO
4: 1mol/L.
Operational approach:
The bag quilt: get 96 hole elisa plates earlier, the Claudin18.2 protein dissolution is cushioned (0.5 μ g/ml) in the liquid in bag, add in the plate hole by 100 μ L/ holes, 4 ℃ of bags are spent the night; Outwell coating buffer, add confining liquid, seal 2h up for safekeeping under the room temperature; Discard confining liquid, wash 6 times, each 2min with cleaning mixture; Add the anti-people Claudin18.2 of goat (500 μ g/ml) and the antiserum of doubling dilution respectively, 1h is hatched under the room temperature in 100 μ L/ holes; Discard antibody and antiserum, wash 6 times, each 3min with cleaning mixture; The anti-goat two that adds the HRP labelling is anti-, 100 μ L/ holes, and room temperature breeds 1h; Discard two and resist, wash 6 times, each 3min with cleaning mixture; Add substrate colour developing liquid, 100 μ L/ holes, room temperature, colour developing 10-20min; Add stop buffer, 50 μ L/ holes, cessation reaction; The microplate reader reading, mensuration there is not the light absorption value of hole at 490nm.
(3) immunoblotting reaction is measured the combination activity of the anti-Claudin18.2 antibody of serum
Get people's gastric cancer KATOIII and cancer of pancreas PANC-1 cell and Mouse Gastric Cancer MFC respectively and cancer of pancreas MPC-83 cell carries out the SDS-PAGE electrophoresis according to 3.1 method, after the end, press the Bio-Rad description of product, gel is near negative electrode one side, nitrocellulose membrane (NC film) is near anode one side, put (25mmol/LTris in the transfering buffering liquid, 192mmol/L Glycine, 20% methanol), 100V constant voltage 1h, with albumen from the gel electrotransfer to the NC film, after electrotransfer finishes, take out the NC film, (contain 0.02mol/L pH7.4TBS with cleaning mixture TBST, 0.4%Tween20) room temperature is washed 3 times, immerses in the confining liquid (TBST that contains 2%BSA) 37 ℃, 1h, cleaning mixture (TBST) room temperature is washed 3 times, and loading tumor mouse resisting anteserum is hatched 1h for 37 ℃, the TBST room temperature is washed film 3 times, add the anti-goat IgG-HRP of rabbit two anti-(middle China fir company), hatch 1h for 37 ℃, the TBST room temperature is washed film 3 times, reuse TBS washes 3 times, the NC film immerses in the OPD colour developing liquid, room temperature lucifuge colour developing 10min, distilled water flushing cessation reaction (referring to Fig. 4).
(4) press down the tumor experiment
Observe mice internal antibody titre weekly and change, wait to observe and detect gross tumor volume and mice body weight every other day after forming tumor tissue.Take out tumor tissues during dead mouse and weigh, calculate tumour inhibiting rate (referring to Fig. 5).
(5) in sum, effect of the present invention is as follows:
1. design and made up the rhClaudin18.2 amalgamation protein vaccine.
2. confirm that by zoopery the rhClaudin18.2 fusion rotein can induce high titre neutralizing antibody more than 1: 10000 in the tumor-bearing mice body.
3. this antibody (anti-rhClaudin18.2 polyclonal antibody) can combine with people KATOIII and PANC-1 tumor cell and Mouse Gastric Cancer MFC and cancer of pancreas MPC-83 cell.
4. this albumen can suppress the growth of gastric cancer MFC and cancer of pancreas MPC-83 cell in the mice body as tumor vaccine.
Claims (3)
1. recombined human Claudin18.2 tumor vaccine, its sequence is:
HMKSSQYIKANSKFIGEFDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGF
TECRGYFTLLGLPAMLQAV。
2. obtain the preparation method of recombined human Claudin18.2 tumor vaccine as claimed in claim 1, it is characterized in that, comprise the steps: successively
(1) clone of rhHis-TT-Claudin18.2 gene and construction of prokaryotic expression vector: adopt the RT-PCR method to obtain first extracellular region gene fragment of Claudin18.2 (the 28th amino acids to the 79 amino acids), with TT
830-843After genetic fragment connects, insert the pQE-30 prokaryotic expression carrier, transformed into escherichia coli; In escherichia coli, obtain to efficiently express, obtain His-TT-Claudin18.2 albumen after purified.
(2) expression of recombinant proteins: with the pQE-30-TT-Claudin18.2 plasmid transformation escherichia coli that builds, in escherichia coli, obtain to efficiently express, behind affinity purification, obtain rhHis-TT-Claudin18.2 albumen.
(3) purification of fusion rotein and renaturation: expression product is through splitting the purification of bacterium, dissolving inclusion body, Ni-NTA affinity column, and purity of protein can reach more than 80%.
3. the preparation method of recombined human Claudin18.2 tumor vaccine as claimed in claim 2 is characterized in that, comprises the steps: successively
(1) clone of rhHis-TT-Claudin18.2 gene and construction of prokaryotic expression vector:
According to first extracellular region gene order of people Claudin18.2 among the GeneBank, adopt the RT-PCR conventional method, the EcoRI restriction enzyme site is introduced in the upstream, and the downstream is introduced the SalI restriction enzyme site and is obtained Claudin18.2
28aa-79aaWith the tetanus toxoid epi-position (be TT
830-843Epi-position: aminoacid sequence is QYIKANS KFIGITE, with cell immune response stronger in the primosome, thereby promotes that tumor vaccine better plays a role) gene connection (TT
830-8435 ' end of genetic fragment is BamHI, and 3 ' end is EcoRI), insert the pQE-30 carrier.Connect product transformed competence colibacillus cell DH5 α, picking monoclonal overnight incubation is extracted plasmid, identifies the insertion fragment that obtains the expection size through enzyme action, checks order;
The plasmid called after pQE-30-rhHis-TT-Claudin18.2 that checks order correct, engineering bacteria called after pQE-30-rhHis-TT-Claudin18.2/DH5 α
Its specific amino acid is:
HMKSSQYIKANSKFIGEFDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGF
TECRGYFTLLGLPAMLQAV
(2) expression of recombinant proteins:
Picking engineering bacteria list colony inoculation in 5mLLB culture fluid (containing Amp100mg/L), 37 ℃ of shaking table overnight incubation, next day, the ratio with 1: 100 was transferred in the LB culture fluid that contains Amp, cultivated 3h to logarithmic growth (OD in mid-term at 37 ℃ of shaking tables
600Be 0.4～0.6), adding final concentration is the IPTG of 1mmol/L, continues to cultivate 4h, centrifugal collection thalline;
The evaluation of expression product
SDS-PAGE
Get 30 μ L and split the bacterium supernatant, add 30 μ L, 2 * load sample buffer, mixing.Other gets other samples and adds 30 μ L water, adds 30 μ L, 2 * load sample buffer mixing behind the suspendible again.Boil 5min in the boiling water, the centrifugal 5min of 12000rpm, get 10 μ L supernatant application of samples and concentrate glue in 18% separation gel 6%, voltage is the about 20min of 60V behind the last sample, voltage rose to the about 4-5h of 100V after sample entered separation gel, with 0.5% Coomassie brilliant blue R-250 vibration dyeing 2-3h, decolouring is preserved until the dried glue of the clear back preparation of background;
Immunoblotting reaction
After SDS-PAGE finishes, press the Bio-Rad description of product, gel is near negative electrode one side, and nitrocellulose membrane (NC film) is put (25mmol/L Tris in the transfering buffering liquid near anode one side, 192mmol/LGlycine, 20% methanol), 100V constant voltage 1h, with albumen from the gel electrotransfer to the NC film, after electrotransfer finishes, take out the NC film, (contain 0.02mol/LpH7.4 TBS with cleaning mixture TBST, 0.4%Tween20) room temperature is washed 3 times, immerse in the confining liquid (TBST that contains 2%BSA) 37 ℃, 1h, cleaning mixture (TBST) room temperature is washed 3 times, add the anti-people Claudin18.2 of goat, hatch 1h for 37 ℃, the TBST room temperature is washed film 3 times, adds the anti-goat IgG-HRP two of rabbit and resists, hatch 1h for 37 ℃, the TBST room temperature is washed film 3 times, and reuse TBS washes 3 times, and the NC film immerses in the OPD colour developing liquid, room temperature lucifuge colour developing 10min, the distilled water flushing cessation reaction.
(3) purification of fusion rotein and renaturation:
Get the bacterial strain that contains the pQE-30-TT-Claudin18.2 recombiant plasmid and induce bacterium liquid in a large number, the centrifugal 10min of 5000rpm receives bacterium, ultrasonicly splits bacterium; 4 ℃, 12000rpm, centrifugal 20min collects supernatant and precipitation respectively.To precipitate with the 6mol/L urea liquid resuspendedly, leave standstill dissolving under 4 ℃.Dissolved precipitation and supernatant taken a sample respectively carry out the SDS-PAGE electrophoresis, the expression-form of destination protein is analyzed.After confirming the formal representation of destination protein, with Ni-NTA post affinitive layer purification dissolved precipitation (inclusion body) with inclusion body.Adding 10mL in the 1g thalline, to split the ratio of bacterium buffer resuspended with thalline, carries out the ultrasonic bacterium of splitting under the condition of ice bath.12000rpm, centrifugal 15min abandons supernatant, and the 1g precipitation adds 10mL 6M carbamide, 0.1M NaH
2PO
4, 0.01M Tris (pH 8.0).4 ℃ are stirred 2h, 12000rpm, and centrifugal 15min, centrifugal 2 times altogether, it is stand-by to collect supernatant.Use 6M carbamide, 0.1M NaH
2PO
4, 0.01M Tris (pH 8.0) is balance Ni post fully, earlier with the 6M carbamide that contains the 10mmol imidazoles, 0.1MNaH
2PO
4, 0.01MTris (pH 8.0) eluting foreign protein, reuse contains the 6M carbamide of 250mmol imidazoles, 0.1M NaH
2PO
4, 0.01M Tris (pH 8.0) eluting destination protein.Collect eluting peak, purified product is dialysed to 2M carbamide, 0.1M NaH
2PO
4, 0.01MTris (pH 6.0) renaturation, final concentration 1mg/ml.Dialysis is at last gone among the 0.01M Tris (pH 6.0), after PEG8000 concentrates, measures protein concentration with the Bradford method.
Animal model experiment
Mice group and immunization protocol
Grouping: 24 BALB/c mouse are divided into 4 groups, subcutaneous in mouse back according to 1 * 10
7Individual/as only, to inoculate each 2 groups in gastric cancer MFC and cancer of pancreas MPC-83 cell respectively for 6 every group.2 groups of tumor-bearing mices of each tumor cell inoculation, treat into tumor after, inject rhClaudin18.2 or normal saline respectively according to the routine immunization method.Pluck the eyeball blood sampling in back 10 days in the 4th immunity, measure anti-Claudin18.2 antibody titer in the serum.Immunization protocol: the subcutaneous multi-point injection in back; RhClaudin18.2:40 μ g/+normal saline cumulative volume is 200 μ L/, respectively injection every other week; The 4th is only used g/+normal saline 200 μ L/ of rhClaudin18.2:40 μ, lumbar injection.
The ELISA method is measured the anti-Claudin18.2 antibody titer of serum
In the 4th immunity blood sampling in back 10 days, collect antiserum, measure the anti-Claudin18.2 antibody titer of serum by indirect ELISA.Each sample is all established 6 holes.The ELISA experimental technique is as follows:
The preparation of required solution: a. bag is cushioned liquid: carbonate buffer solution is got Na
2CO
3: 0.32g (final concentration 0.015mol/L), NaHCO
3: 0.59g (final concentration 0.035mol/L), pure water are settled to 200mL (pH 9.6), effect duration: two weeks.B. cleaning mixture: the PBS (pH 7.4) that contains 0.5%Tween-20.C. confining liquid: the cleaning mixture that contains 1%BSA.D. diluent: the cleaning mixture that contains 0.5%BSA.E. substrate buffer solution: Na
2HPO
412H
2O:1.84g, citric acid: the 0.51g pure water is settled to 100mL (pH5.0).F. substrate colour developing liquid: OPD:8mg, 3%H
2O
2: 30 μ L.H. stop buffer: H
2SO
4: 1mol/L.
Operational approach:
The bag quilt: get 96 hole elisa plates earlier, the Claudin18.2 protein dissolution is cushioned (0.5 μ g/ml) in the liquid in bag, add in the plate hole by 100 μ L/ holes, 4 ℃ of bags are spent the night; Outwell coating buffer, add confining liquid, seal 2h up for safekeeping under the room temperature; Discard confining liquid, wash 6 times, each 2min with cleaning mixture; Add the anti-people Claudin18.2 of goat (500 μ g/ml) and the antiserum of doubling dilution respectively, 1h is hatched under the room temperature in 100 μ L/ holes; Discard antibody and antiserum, wash 6 times, each 3min with cleaning mixture; The anti-goat two that adds the HRP labelling is anti-, 100 μ L/ holes, and room temperature breeds 1h; Discard two and resist, wash 6 times, each 3mim with cleaning mixture; Add substrate colour developing liquid, 100 μ L/ holes, room temperature, colour developing 10-20min; Add stop buffer, 50 μ L/ holes, cessation reaction; The microplate reader reading, mensuration there is not the light absorption value of hole at 490nm.
Immunoblotting reaction is measured the combination activity of the anti-Claudin18.2 antibody of serum
Get people's gastric cancer KATOIII and cancer of pancreas PANC-1 cell and Mouse Gastric Cancer MFC respectively and cancer of pancreas MPC-83 cell carries out the SDS-PAGE electrophoresis according to 3.1 method, after the end, press the Bio-Rad description of product, gel is near negative electrode one side, nitrocellulose membrane (NC film) is near anode one side, put (25mmol/L Tris in the transfering buffering liquid, 192mmol/L Glycine, 20% methanol), 100V constant voltage 1h, with albumen from the gel electrotransfer to the NC film, after electrotransfer finishes, take out the NC film, (contain 0.02mol/L pH7.4TBS with cleaning mixture TBST, 0.4%Tween20) room temperature is washed 3 times, immerses in the confining liquid (TBST that contains 2%BSA) 37 ℃, 1h, cleaning mixture (TBST) room temperature is washed 3 times, and loading tumor mouse resisting anteserum is hatched 1h for 37 ℃, the TBST room temperature is washed film 3 times, add the anti-goat IgG-HRP of rabbit two anti-(middle China fir company), hatch 1h for 37 ℃, the TBST room temperature is washed film 3 times, reuse TBS washes 3 times, the NC film immerses in the OPD colour developing liquid, room temperature lucifuge colour developing 10min, distilled water flushing cessation reaction.
Press down the tumor experiment
Observe mice internal antibody titre weekly and change, wait to observe and detect gross tumor volume and mice body weight every other day after forming tumor tissue.Take out tumor tissues during dead mouse and weigh, calculate tumour inhibiting rate.
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