CN103520718B - The medicine of Hepatoma therapy and the application of RACO-1 monoclonal antibody - Google Patents
The medicine of Hepatoma therapy and the application of RACO-1 monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a kind of medicine of Hepatoma therapy and the application of RACO-1 monoclonal antibody, the medicine of Hepatoma therapy of the present invention comprises the RACO-1 monoclonal antibody of medicine effective quantity, described RACO-1 monoclonal antibody behaviour source RACO-1 monoclonal antibody, is described people source RACO-1 monoclonal antibody prepared by following steps: extract hepatocellular carcinoma patients peripheral blood lymphocyte total serum IgE, by reverse transcription PCR method clone RACO-1? DNA; Do you adopt RACO-1? DNA carries out phage antibody library structure; Screening phage antibody library obtains object antibacterial; Extract the plasmid DNA in object antibacterial, bacteriophage coat protein III gene in excision plasmid DNA, the plasmid DNA of remainder carried out expressing, purification obtains people source RACO-1 monoclonal antibody, the present invention has the plurality of advantages such as specificity is good, affinity is high, immunogenicity is little, side effect is low.
Description
Technical field
The present invention relates to biomedicine technical field, in particular to a kind of medicine of Hepatoma therapy and the application of RACO-1 monoclonal antibody.
Background technology
Hepatocarcinoma (hepatocellular carcinoma, HCC) is the fifth-largest kinds of tumor of global male, has that prognosis is severe, mortality rate high, and the life of serious threat our people is with healthy.Although in recent years along with the continuous progress of diagnostic imaging and surgical technic and take Sorafenib as the emerging of hepatoma-targeting medicine of representative, for the early diagnosis of hepatocarcinoma and treatment provide may with more and more effective therapeutic choice, make the treatment level of hepatocarcinoma obtain certain raising.But, take a broad view of current hepatocarcinoma clinical treatment present situation, still lack now real effectively, the cancer treatment drug of few side effects, the recurrence of PHC rate of transform still can be in any more, within postoperative 5 years, survival rate is still hovered about 30%, seriously constrain the raising of hepatocarcinoma overall therapeutic level, the improvement obtaining essence cannot be made it.
The research of biotechnology medicine is one of field of enlivening the most in current life science, molecular targeted agents wherein because of its selectivity high, wide spectrum is effective, not easily there is drug resistance, safety simultaneously is also obviously better than cytotoxic chemotherapy agents, various countries are all paid much attention to, and now existing this type of medicine multiple gets the Green Light to go on the market and applies.Recently namely the Sorafenib (Sorafenib) ratified through FDA is a kind of antitumor drug of novel Mutiple Targets.In III clinical trial phase of nearest multicenter, double blinding, placebo, Llovet etc. gave Sorafenib or placebo treatment without the advanced liver cancer patient of systemic treatment in the past to 602 at random, research finds: late in hepatocarcinoma, the median survival time of Sorafenib group patient extends 2.8 months than placebo patients.Therefore, U.S. FDA announce that Sorafenib is applicable to treat cannot the hepatocellular carcinoma of excision.National biological product one kind new medicine " U.S. appropriate former times monoclonal antibody injection (Li Kating) of iodine [131I] " of domestic Chen Zhinan academician independent research is also the specific antibody targeted drug being specifically designed to Hepatoma therapy, now has ratified to go on the market at home.This radioimmunity targeted drug shows and has certain curative effect to advanced liver cancer in the clinical practice in early stage.
In addition, have research report, with anti-human AFP monoclonal antibody be carrier, 131I and mitomycin be " two bullet ", treatment mid and late liver cancer patient, after result display treatment, tumor regression rate is higher than matched group.Also the 131I-Hepama-1 monoclonal antibody targeted therapy advanced liver cancer that report research people Hepatoma m Abs Hepama-1 and 131I coupling are made is had, though some patients cancer signs can be made to alleviate.But these rear two researchs are showed no the report about improving survival rate.
Current monoclonal antibody is mouse mostly, and there are problems when mouse monoclonal antibody is applied to human body therapy: one is can not complement is relevant with Fc receptor in human activin effectively effect system; Two identify by human immune system, human anti-murine antibodies (human antigen mouse antibody, HAMA); Three are disposed very soon in human recycle system.
For HCC in inoperable late period, Drug therapy is its primary treatment regimen.But, system chemotherapy does not still have efficient chemotherapeutics or scheme so far, a large amount of clinical trial statistics displays, the effective percentage of chemotherapeutics single therapy hepatocarcinoma is greatly between 0 ~ 25%, very micro-with non-treatment group Comparative Profits, only appease effect, and the data lacking relevant extensive III clinical trial phase are for reference.Amycin is traditional medicine for liver cancer treatment, but objective remission rate is only 10% ~ 15%, and can not extend the survival of patients phase.The therapeutic effect of targeted drug is also unsatisfactory, and wherein the most successfully the prolongation of BAY 43-9006 drugs on patients life span is also only 3 months.
In sum, the research about the medicine of hepatocarcinoma remains in many problems, still well cannot solve this key issue of survival rate improving hepatocarcinoma patient clinical treatment at present, cannot meet the Treatment need of hepatocarcinoma patient at all.
Summary of the invention
The present invention aims to provide a kind of medicine of Hepatoma therapy and the application of RACO-1 monoclonal antibody, to solve in prior art the technical problem that well cannot solve the survival rate improving hepatocarcinoma patient clinical treatment.
To achieve these goals, according to an aspect of the present invention, a kind of medicine of Hepatoma therapy is provided.This medicine comprises the RACO-1 monoclonal antibody of medicine effective quantity.
Further, RACO-1 monoclonal antibody behaviour source RACO-1 monoclonal antibody.
Further, people source RACO-1 monoclonal antibody is prepared by following steps: extract hepatocellular carcinoma patients peripheral blood lymphocyte total serum IgE, by reverse transcription PCR method clone RACO-1 DNA; RACO-1 DNA is adopted to carry out phage antibody library structure; Screening phage antibody library obtains object antibacterial; Extract the plasmid DNA in object antibacterial, bacteriophage coat protein III gene in excision plasmid DNA, the plasmid DNA of remainder is carried out express, purification obtains people source RACO-1 monoclonal antibody.
Further, the sequence of RACO-1 DNA forward primer used is cloned as SEQ ID NO:1; The sequence of downstream primer is as SEQ ID NO:2.
Further, the sequence of RACO-1 DNA forward primer used is cloned as SEQ ID NO:3; The sequence of downstream primer is as SEQ ID NO:4.
According to another aspect of the present invention, a kind of RACO-1 monoclonal antibody is provided to prepare the application in Hepatoma therapy medicine.
Further, RACO-1 monoclonal antibody behaviour source RACO-1 monoclonal antibody.
According to a further aspect of the invention, the application that a kind of RACO-1 monoclonal antibody suppresses in hepatoma carcinoma cell is in vitro provided, comprises and RACO-1 monoclonal antibody is contacted with hepatoma carcinoma cell with effective inhibition concentration.
Apply technical scheme of the present invention, by the medicine of RACO-1 monoclonal antibody for the preparation of hepatocarcinoma, RACO-1 monoclonal antibody can suppress the expression of RACO-1 in human body specifically, thus effectively reduces hepatocarcinoma invasive ability, improves the five year survival rate of liver cancer patient.Preferably, what RACO-1 monoclonal antibody adopted is people source RACO-1 monoclonal antibody, and it has the plurality of advantages such as specificity is good, affinity is high, immunogenicity is little, side effect is low.
Accompanying drawing explanation
The Figure of description forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows RACO-1 mRNA and albumen high expressed in liver cancer tissue; A:Real-time PCR detects RACO-1mRNA level in 25 pairs of liver cancer tissues and corresponding cancer beside organism, and result display RACO-1mRNA is significance high expressed in liver cancer tissue; T, liver cancer tissue; ANLT, contiguous non-tumor hepatic tissue; NL, normal liver tissue; B:Western-blot detects the expression of RACO-1 albumen in liver cancer tissue and contiguous non-tumor hepatic tissue, and result shows, RACO-1 albumen at Expression In Hepatocellular Carcinoma apparently higher than contiguous non-tumor hepatic tissue and normal liver tissue, *,
p<0.01;
Fig. 2 shows Real-time PCR and Western-blot and detects the expression of RACO-1 in 5 kinds of hepatic cell lines; A:Real-time PCR detects RACO-1mRNA level in 5 kinds of hepatic cell lines, and result display RACO-1mRNA level raises with cell line Invasion and Metastasis potential and increases; B:Western-blot detects RACO-1 protein level in 5 kinds of hepatic cell lines, and result display RACO-1 protein level raises with cell line Invasion and Metastasis potential and increases;
Fig. 3 shows the immunostaining (× 400) of RACO-1 at liver cancer tissue, and result display RACO-1 is mainly distributed in cytoplasm: A:Shimizu scoring for-, negative to express; B:Shimizu scoring is 1+; C:Shimizu scoring is 2+; D:Shimizu scoring is 3+; The prognostic analysis of E:RACO-1 immunostaining intensity and hepatocarcinoma, high expressed group Overall survival be starkly lower than low expression group (
p=0.004); F:RACO-1 immunostaining intensity and liver cancer recurrence transfer analysis;
Fig. 4 shows anti-hepatocarcinoma humanized RACO-1 monoclonal antibody and suppresses hepatoma carcinoma cell Invasion and Metastasis in vitro; A: cut Healing Experiments Effect of Anti hepatocarcinoma humanized RACO-1 monoclonal antibody is on the impact of hepatoma carcinoma cell HCCLM3 travel motion ability; B:Transwell attacks cell experimentation anti-hepatocarcinoma humanized RACO-1 monoclonal antibody to the impact of hepatoma cell line HCCLM3 invasive ability; C: cut Healing Experiments Effect of Anti hepatocarcinoma humanized RACO-1 monoclonal antibody is on the impact of hepatoma carcinoma cell MHCC97-H travel motion ability; D:Transwell attacks cell experimentation anti-hepatocarcinoma humanized RACO-1 monoclonal antibody to the impact of hepatoma carcinoma cell MHCC97-H invasive ability;
Fig. 5 shows anti-hepatocarcinoma humanized RACO-1 monoclonal antibody and suppresses hepatocarcinoma Invasion and Metastasis in vivo; A: utilize HCCLM3 cell construction hepatocellular carcinoma in nude mice metastasis model; Red arrow refers to primary tumor, and black arrow refers to metastatic tumor; B: show metastasis under mirror; C: anti-hepatocarcinoma humanized RACO-1 monoclonal antibody processed group compares with matched group metastasis number; And
Fig. 6 shows anti-hepatocarcinoma humanized RACO-1 monoclonal antibody and suppresses hepatoma carcinoma cell Invasion and Metastasis A in vivo and in vitro: cut Healing Experiments Effect of Anti hepatocarcinoma humanized RACO-1 monoclonal antibody is on the impact of hepatoma cell line HCCLM3 and MHCC97-H travel motion ability.B:Transwell attacks the impact of cell experimentation anti-hepatocarcinoma humanized RACO-1 monoclonal antibody on hepatoma cell line HCCLM3 and MHCC97-H invasive ability.C: anti-hepatocarcinoma humanized RACO-1 monoclonal antibody group compares with nude mice of control group Intrahepatic metastasis stove number.
Detailed description of the invention
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
It is known that the present inventor adopts Real-time PCR method to detect RACO-1(RACO-1, gene order can be downloaded in gene bank on the internet, such as NCBI) expression in liver cancer tissue and hepatoma cell line, found that RACO-1 mRNA in liver cancer tissue in high expressed, and its expression in the hepatoma cell line HCCLM3 of high invasive ability also strengthens apparently higher than the hepatoma cell line HepG2 of low invasive ability, RACO-1 mrna expression level successively along with hepatoma cell line invasive ability grows from weak to strong.The invasive ability of RACO-1 and hepatocarcinoma is in close relations as can be seen here.Simultaneously, Western-bolt method is adopted to detect the expression of RACO-1 albumen in liver cancer tissue and hepatoma cell line, result shows RACO-1 albumen in liver cancer tissue equally in high expressed, and its expression strengthens successively along with hepatoma cell line invasive ability grows from weak to strong, completely the same with Real-time PCR result, as depicted in figs. 1 and 2.
Fig. 1 shows RACO-1 mRNA and albumen high expressed in liver cancer tissue.A in Fig. 1 shows Real-time PCR and detects RACO-1mRNA level in 25 pairs of liver cancer tissues and corresponding cancer beside organism's (numbering is respectively N129, N190, N109, N185, N122, N124, N116, N191, N160, N106, N107, N126, N143, N152, N128, N132, N144, N114, N103, N163, N013, N033, N056, N098, N121).Wherein, RACO-1 PCR detection primer is as follows: forward primer sequence is SEQ ID NO:1(5 '-aacaaggggtctgtggaaatc-3 '); Downstream primer sequence is SEQ ID NO:2:(5 '-tgtagtcggtcagtgccttct-3 ').GAPDH(glyceraldehyde-3-phosphate dehydrogenase) as internal reference primer: forward primer: 5 '-GCACCGTCAAGGCTGAGAAC-3 ', downstream primer: 5 '-TGGTGAAGACGCCAGTGGA-3 '.All primers are all for subsequent use by 100 μMs of concentration dilutions.By TOYOBO company PCR test kit description, set up 50 μ l PCR reaction systems as follows: SYBR Green Realtime PCR MasterMix-Plus-25 μ l; Plus Solution 5 μ l; Forward primer (10 μMs) 2 μ l; Downstream primer (10 μMs) 2 μ l; Detect sample cDNA solution 5 μ l; DH
2o 11 μ l.PCR course of reaction at ABI Prism 7300 Real-Time PCR System(Applied Biosystems, Foster City, CA, USA) on complete.Reaction condition is 95 DEG C of denaturation 60 sec; 95 DEG C of 15 sec, 60 DEG C of 15 sec, 72 DEG C of 45 sec.Found that RACO-1 mRNA in liver cancer tissue in high expressed, and its expression in the hepatoma cell line HCCLM3 of high invasive ability also strengthens apparently higher than the hepatoma cell line HepG2 of low invasive ability, RACO-1 mrna expression level successively along with hepatoma cell line invasive ability grows from weak to strong.
B in Fig. 1 shows the expression that Western-blot detects RACO-1 albumen in liver cancer tissue and contiguous non-tumor hepatic tissue.Its operating procedure is as follows: get the 100 μ g that the add loading buffer in advance total protein from tissue or cell extraction.Degeneration about ten minutes in boiling water, then by sample with 12% polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), first 80V electrophoresis is about 30min, protein sample goes to 120V electrophoresis and wets robin to pvdf membrane (Millipore through electrophoresis tank after protein isolate on earth after running out of spacer gel, Bedford, MA, USA.After after being cut by required banded zone glue, under 12V, transferring film is about 80-90min, 5% defatted milk powder is dissolved in PBST to close and to insert after about one hour in certain density primary antibodie solution (antibody concentration is diluted by antibody description), 4 DEG C of overnight incubation, PBST washs pvdf membrane 45 min, about one hour is hatched under inserting the anti-room temperature of rabbit anti goat igg two of the HRP labelling of 1:3000 dilution again, pvdf membrane is washed ten minutes with PBST, after three times, development after mixing with high sensitivity chemistry luminescence reagent box one to.The primary antibodie that this test uses is antihuman RACO-1 antibody(Sigma-Aldrich, St. Louis, MO), internal reference primary antibodie selects mouse-anti people β-actin antibody (Santa Cruz Biotechnology), rabbit anti-mouse igg antibody (the KPL of two anti-selection HRP labellings, Gaithersburg, MD).Digital camera is clapped and is got result, analyzes, represent the expression of protein level with the gray value of band with Bandscan 5.0 pairs of histogram pictures.Each sample duplicate detection 3 times, result gets its meansigma methods.Result shows RACO-1 albumen in liver cancer tissue equally in high expressed.
Fig. 2 shows Real-time PCR and Western-blot and detects the expression of RACO-1 in 5 kinds of hepatic cell lines (step is the same), and these 5 kinds of hepatic cell lines are respectively LO2, HepG2, MHCC97-L, MHCC97-H, HCCLM3.Wherein, the A in Fig. 2 shows Real-time PCR and detects RACO-1mRNA level in 5 kinds of hepatic cell lines, and result display RACO-1mRNA level raises with cell line Invasion and Metastasis potential and increases; B in Fig. 2 shows Western-blot and detects RACO-1 protein level in above-mentioned 5 kinds of hepatic cell lines, and result display RACO-1 protein level raises with cell line Invasion and Metastasis potential and increases.
Further employing Immunohistochemical Method have detected the expression of RACO-1 albumen in liver cancer tissue, Fig. 3 result shows, RACO-1 albumen high expressed in liver cancer tissue, obvious expression is then had no in Para-cancerous tissue, and after the operation in patients of RACO-1 high expressed group without tumor life span and total life span lower than the low expression group of RACO-1, confirm that the prognosis of its high expressed and hepatocarcinoma difference is closely related.Fig. 3 shows the immunostaining (× 400) of RACO-1 at liver cancer tissue, and result display RACO-1 is mainly distributed in cytoplasm: A Shimizu wherein in Fig. 3 mark for-, negatively to express; B Shimizu in Fig. 3 marks as 1+; C Shimizu in Fig. 3 marks as 2+; D Shimizu in Fig. 3 marks as 3+; The prognostic analysis of E:RACO-1 immunostaining intensity and hepatocarcinoma, high expressed group Overall survival be starkly lower than low expression group (
p=0.004); F:RACO-1 immunostaining intensity and liver cancer recurrence transfer analysis.Laboratory operating procedures is as follows: all paraffin specimens all carry out serial section with 3 ~ 5 μm of thickness, by paraffin section 60 DEG C of roasting agreement that contracts a film or TV play to an actor or actress 30min before carrying out SABC, then slice, thin piece is immersed in dimethylbenzene and dewaxes.Take out after about 20 minutes, use 100% respectively, 95%, 80%, 70% ethanol and distilled water serial dehydration successively, under room temperature, 3 min, take out microscope slide rapidly, are immersed in PBS(Phosphate Buffered Saline) middle 5min.By slide with being put in fragmentation frame, being immersed in the EDTA repair liquid (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, Beijing) of 1:50 dilution, putting into the high fire of microwave oven and boil 10 minutes.Then use the universal two step method immunohistochemical kit of Polink-2 plus (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, Beijing) to dye, with after PBS rinsing 3 times after dyeing, the slice, thin piece after rinsing drips 3%H
2o
2hatch.PBS rinsing 3 times after about 10 minutes.The antihuman RACO-1 antibody (Sigma-Aldrich, St. Louis, MO) 37 DEG C dripping 1:400 dilution hatches 1 hour, after PBS rinsing 3 times, drip poly-HRP anti-Goat IgG, hatch 30 minutes at 37 DEG C, PBS rinsing 3 times.The DAB(diaminobenzidine of fresh configuration is dripped, Diaminobenzidine after rinsing) nitrite ion colour developing.Dyeing is stopped rapidly when there is obvious brownish discoloration in Microscopic observation to cytoplasm.After tap water, immerse haematoxylin and redye after liquid redyes, about about 1 minute, after completing, use 60 DEG C of baking boxs dried section, resinene mounting.
Inventor's result of study shows, RACO-1 has played key effect in the Invasion and Metastasis process of hepatocarcinoma.RACO-1 regulates and controls the epithelial cell mesenchymal transformation process of hepatocarcinoma by AP-1 signal path thus promotes hepatocarcinoma Invasion and Metastasis, and mechanism of action is clear and definite, and powerful, high specificity, is expected to become liver cancer treatment target spot.
The present inventor, on the basis of above-mentioned discovery, prepares corresponding human monoclonal antibody, verifies its antineoplastic pharmacodynamic action by vitro and experiments in vivo, final acquisition liver cancer treatment drug candidate.Phage antibody library technique is taked to prepare RACO-1 human monoclonal antibody, phage antibody library (phage antibody library) technology is the great progress in the 90's of 20th century initial stage antibody engineering field, it combines phage display and Antibody Combination library technology, the DNA of external source is inserted in the gene of phage-coded coat protein p III or p VIII, expression product corresponding to foreign DNA fragment is merged in bacteriophage coat protein, forms fusion rotein.The method comprise the generation of phage library, the displaying antibody of conjugated antigen screening, show there is the Phage amplification of high-affinity antibody, has the separation of the antibody of high-affinity and concrete steps qualitatively.Its concrete operation step is as follows:
Its basic skills is from human peripheral lymphocyte, extract RNA or genomic DNA, design nucleotide primer, be assembled in phage expression vector with PCR method human cloning repertoire antibody heavy chain and chain variable region gene, merge with phage coat protein gene, ehec infection also makes antibody fragment be expressed in phage.Then utilize Ag-Ab specific binding and filter out required antibody, and carrying out Clonal expansion, obtaining soluble antibody fragment (as Fab, scFV and dsFv etc.), i.e. phage antibody.
According to a kind of typical embodiment of the present invention, provide a kind of medicine of Hepatoma therapy.The medicine of this Hepatoma therapy comprises RACO-1 monoclonal antibody.RACO-1 monoclonal antibody can be the monoclonal antibody that those skilled in the art are prepared according to RACO-1 complete genome sequence by conventional technological means.Antibody preparation is for being aseptic during vivo medicine-feeding, and this point is easy to realize, and such as, uses degerming membrane filtration.Route of antibody administration is consistent with known method, as passed through in vein, peritoneum, brain, muscle, ophthalmic, tremulous pulse, sheath, sucking or the injection of damage zone approach or transfusion etc.
The effective dose for the treatment of antibody used depends on, such as, and the approach of therapeutic goal, administration and the state of an illness of patient.Therefore, therapist's preferred titration determination dosage also revises route of administration, as requested to obtain optimum therapeuticing effect.Generally, clinicist by administered antibody until reach the dosage realizing required effect.The carrying out of this Therapeutic Method is easy to be monitored by common detection methods." effective dose " mentioned in the present invention common with this area " effective dose " implication is identical; Equally, in the present invention " effective inhibition concentration " of indication be clinicist by administered antibody until reach the concentration realizing required effect.
Monoclonal antibody of the present invention, can be prepared into mixture with medicinal carrier.This pharmaceutical composition, by vein, nose or lung administration, is preferably liquid or powder aerosol (lyophilizing).Compositions can also parenteral or subcutaneous administration as required.When administered systemically, treatment group and thing should be aseptic, without thermal source, and to can be used in parenteral solution, and with due regard to arrive pH value, isotonicity and stability.These conditions are known in those skilled in the art.In brief, by the monoclonal antibody with required purity with physiology can the mixing of carrier, excipient or stabilizing agent.The use amount of these materials and concentration are nontoxic to receiver, comprise buffer as phosphate, citrate, acetate and other organic salt; Antioxidant is as ascorbic acid; Peptide is as poly arginine for small-molecular-weight (being less than 10 amino acid residues), and albumen is as serum albumin, gelatin or immunoglobulin etc.
Humanized antibody refers to that the variable region portion (i.e. Vh and Vl district) of antibody or antibody are all coded by human antibody gene.Humanized antibody can greatly reduce the immune side reaction that heterologous antibody causes human body.Preferably, RACO-1 monoclonal antibody behaviour source RACO-1 monoclonal antibody.Because it is good that human monoclonal antibody has specificity, and affinity is high, and immunogenicity is little, the feature that side effect is low.
Preferably, people source RACO-1 monoclonal antibody is prepared by following steps: extract hepatocellular carcinoma patients peripheral blood lymphocyte total serum IgE, by reverse transcription PCR method clone RACO-1 DNA; The RACO-1 DNA adopting step 1) clone to obtain carries out phage antibody library structure; Screening phage antibody library obtains object antibacterial; Extract the plasmid DNA in object antibacterial, bacteriophage coat protein III gene in excision plasmid DNA, the plasmid DNA of remainder is carried out express, purification obtains people source RACO-1 monoclonal antibody.
According to a kind of typical embodiment of the present invention, the sequence of the forward primer that clone RACO-1 DNA is used is as SEQ ID NO:1(5 '-aacaaggggtctgtggaaatc-3 '); The sequence of downstream primer is as SEQ ID NO:2(5 '-tgtagtcggtcagtgccttct-3 ').
According to the another kind of typical embodiment of the present invention, the sequence of the forward primer that clone RACO-1 DNA is used is as SEQ ID NO:3(5 '-TCAGGGAGAGAAAGGAAGACTG-3 '); The sequence of downstream primer is as SEQ ID NO:4(5 '-GTGGACAGAGCGACTTGGATAA-3 ').
In the present invention, provide a kind of RACO-1 monoclonal antibody preparing the application in Hepatoma therapy medicine, wherein, RACO-1 monoclonal antibody behaviour source RACO-1 monoclonal antibody, is about to a kind of method of expression as Hepatoma therapy of interference RACO-1 gene.
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1
1) extraction of total serum IgE, reverse transcription PCR method clone RACO-1 gene.
RACO-1 high expressed hepatocellular carcinoma patients (hepatocellular carcinoma patients, women, 41 years old) peripheral blood lymphocyte total serum IgE is extracted by guanidine thiocyanate-phenol-chloroform one-step method.Get about 20 μ g total serum IgE, add 1 μ gOligodT, 5ul 250mmol/LdNTP and 200UM-MLV reverse transcriptase (GIBCOLBRL), carry out cDNA synthesis by product description.Pcr amplification RACO-1 gene, wherein, amplimer is as follows: forward primer sequence is SEQ ID NO:1 NO:1(5 '-aacaaggggtctgtggaaatc-3 '); The sequence of downstream primer is as SEQ ID NO:2(5 '-tgtagtcggtcagtgccttct-3 ').The condition of pcr amplification is: 94 DEG C of degeneration 20min, 52 DEG C of annealing 50s, 72 DEG C extend 1.5min, carry out 35 altogether and take turns circulation, last 72 DEG C extend 10min.
2) colibacillary Electroporation Transformation
The XL1-Blue bacterium liquid of 2.5ml incubated overnight is inoculated in 500ml SB culture medium, and 37 DEG C are cultured to A600=0.7 ~ 0.8, ice bath 15min, 4 DEG C centrifugal, abandons supernatant, after antibacterial being washed 2 times with 10% cold glycerol, be suspended in 2ml 10% glycerol ,-70 DEG C frozen for subsequent use.Taken out by antibacterial during electroporation and melt, use the Genepulsertransfection system of Bio-Rad company, by specification is bored a hole.
3) structure of phage antibody library
The RACO-1 DNA of pcr amplification is digested through SacI+XbaI, after Purified in electrophoresis in insertion vector pComb3H, Electroporation Transformation XL1-Blue cell, then in XL1-Blue, 3ml SOC culture fluid is added, add containing ampicillin 50 μ g/ml after 30 DEG C of cultivation 1h, the SB culture medium 20ml of tetracycline 10 μ g/ml, 37 DEG C of shaken cultivation 2h, add the helper phage VCSM13 of about 1012 plaque forming units (PFU), be inoculated into 100ml containing identical (i.e. ampicillin 50 μ g/ml, tetracycline 10 μ g/ml) in antibiotic SB culture medium, 37 DEG C of shaken cultivation 2h, add kanamycin to final concentration 70 μ g/ml, 30 ~ 37 DEG C of shaken cultivation are spent the night, collected by centrifugation supernatant, add PEG-8000 to 4%, NaCl is to 3%, after ice bath 30min, centrifugally abandon supernatant, with the TBS dissolution precipitation of 2ml containing 1% BSA, collected by centrifugation supernatant is phage antibody library.
4) mensuration of phage antibody library titre
Phage antibody library solution is pressed the dilution proportion of 1:100 in SB culture medium, getting 10ml is added in 100ml XL1-Blue (A600=1.0), paving ampicillin medium, 37 DEG C of overnight incubation, next day, colonies, calculated colony forming unit (CFU).
5) bacterium colony Direct PCR identification method
The single bacteria colonies of picking, is suspended from 200ml water, boils 2min, gets the suitable primer of 2ml (forward primer sequence is SEQ ID NO:1(5 '-aacaaggggtctgtggaaatc-3 '); Downstream primer sequence is SEQ ID NO:2:(5 '-tgtagtcggtcagtgccttct-3 ') carry out PCR reaction, agarose gel electrophoresis detects corresponding amplified band.
6) screening (Panning) of phage antibody library
Phage antibody library solution is diluted to 40 μ g/ml with 0.05% carbonate buffer solution, every hole 25 μ l(1 μ g RACO-1) wrap by elisa plate, suck coating buffer, with containing 3% BSA(bovine serum albumin) PBS buffer fill it up with aperture, 37 DEG C of closed 1h, suck confining liquid, add 50 μ l phage antibody library liquid (about 1011 ~ 1012 CFU), cultivate 2h, suck phage antibody liquid for 37 DEG C, aperture is filled it up with, firmly piping and druming with the TBS containing 0.05% tween.Inhale after 5min and abandon TBS(the 2nd and take turns and wash 5 times, 3rd take turns after wash 10 times), add eluent (the 0.1mol/L HCl of 50 μ l, pH2.2 is adjusted to glycine, add BSA to 0.1%), room temperature leaves standstill 10min, eluent is blown and beaten rear sucking-off a little, add 3 μ l 2mol/L Tris immediately to neutralize, add 2ml XL1-Blue (A600=1), room temperature leaves standstill 15min and infects, be inoculated into 10ml SB culture medium (containing ampicillin 20 μ g/ml, tetracycline 10 μ g/ml) in, get 1.0 μ l and 10 μ l pavings dish mensuration CFU, all the other bacterium liquid 30 DEG C of shaken cultivation 1h, add ampicillin to final concentration 50 μ g/ml, 37 DEG C are continued to cultivate 1h, add helper phage (VCSM13) 1012 PFU, be inoculated into 100ml SB culture medium (containing ampicillin 20 μ g/ml, tetracycline 10 μ g/ml) in, cultivate 2h for 37 DEG C, add kanamycin to final concentration 70 μ g/ml, 30 ~ 37 DEG C of shaken cultivation are spent the night, centrifugal, supernatant carries out PEG(Polyethylene Glycol) precipitation.
7) expression of solubility Fab molecule
The phage antibody library of screening amplification is extracted Double stranded plasmids DNA, digest with SpeI/NheI, the encoding gene of excision bacteriophage coat protein III, linked enzyme transforms XL1-Blue antibacterial after connecting recirculation, paving ampicillin dish (20 μ g/ml), next day random picking list colony, be inoculated into 10ml SB culture medium (containing 50 μ g/ml ampicillin, 20mmol/LMgCl
2), 37 DEG C of shaken cultivation 6h, add IPTG(isopropyl-beta D-thio galactopyranoside) to 1mmol/L, abduction delivering solubility Fab molecule, then cultivation temperature to 30 DEG C is reduced, shaken cultivation is spent the night, and collected by centrifugation supernatant is used for ELISA and detects (supernatant is the monoclonal antibody that can be used for Hepatoma therapy).
Embodiment 2
Pcr amplification RACO-1 gene (from hepatocellular carcinoma patients 1 example, women, 48 years old), wherein, amplimer is as follows: the sequence of forward primer is as SEQ ID NO:3(5 '-TCAGGGAGAGAAAGGAAGACTG-3 '); The sequence of downstream primer is as SEQ ID NO:4(5 '-GTGGACAGAGCGACTTGGATAA-3 ').All the other experiment conditions are all identical with embodiment 1.
In following experiment, RACO-1 monoclonal antibody processes cell by adding in cell culture fluid.
It is as follows that monoclonal antibody embodiment 1 obtained carries out in vitro tests operation:
First cell (HCCLM3 needed for flow process culture experiment routinely
controland HCCLM3
anti-RACO-1and MHCC97-H
controland MHCC97-H
anti-RACO-1), the equal sterilization of apparatus of all energy sterilizing, ruler and marker pen ultra-vioket radiation 30min before operation.First with 0.25% trypsin digestion cell, neutralize with the DMEM culture fluid containing 10%FBS, and single cell suspension is made in slight piping and druming, little culture dish or 6 orifice plates are inoculated in by 5 × 105/porocyte density, when cell attachment grows to 95%-100% fusion, with marker pen in 6 orifice plates behinds, compare with ruler, evenly horizontal line must be drawn, approximately every 0.5-1cm together, ruler is compared with 10 μ l pipettor heads, hang down as horizontal line cut behind as far as possible, rifle head wants vertical, cell is washed 3 times with aseptic PBS after cut, clean floating broken cell, then serum-free medium is added at 5% CO
2, continue to cultivate 24-48 hour in 37 DEG C of incubators.Took pictures to cell respectively at after cut 48 hours, observe cut healing state, often organize cell and do 3 parallel holes, repeat 3 times, calculate cut healing rate (B and D in Fig. 4, wherein, illustrated in B that Transwell attacks cell experimentation anti-hepatocarcinoma humanized RACO-1 monoclonal antibody to the impact of hepatoma cell line HCCLM3 invasive ability; Illustrate in D that Transwell attacks cell experimentation anti-hepatocarcinoma humanized RACO-1 monoclonal antibody to the impact of hepatoma carcinoma cell MHCC97-H invasive ability).Experimental result shows that the fucosylation motor capacity after the process of RACO-1 monoclonal antibody obviously declines.By invasion and attack cell laboratory observation anti-hepatocarcinoma humanized RACO-1 monoclonal antibody on the impact of Invasive Ability of Hepatocellular Carcinoma.Required detection cell (HCCLM3
controland HCCLM3
anti-RACO-1and MHCC97-H
controland MHCC97-H
anti-RACO-1) with 2 × 10
4total cellular score adds each cell, add 300 μ l DMEM serum-free mediums again to cultivate, add the DMEM culture medium of 500 μ l containing 10% FBS in lower room simultaneously, cell is placed on supporting lower room, we adopt aperture to be the Transwell plate (Costar overlaying Matrigel glue of 8 μm, Cambridge, USA) detect Invasive Ability of Hepatocellular Carcinoma.Every plate is 16 holes, to add a small amount of serum-free DMEM allow its aquation before using in little indoor.Complete after aforesaid operations at 5% CO
2, cultivate 12-24 hour under 37 DEG C of conditions.Take out Transwell cell, wipe Matrigel glue and the cell of going up to the greatest extent face, room with cotton swab, after fixing, dye with 1% crystal violet solution in lower face, room, microscopic examination.Get 3 high power lens visuals field at random, under counting cell, the cell number in face, room is the cell number penetrating Matrigel glue, experiment repetition 3 times, (A and C in Fig. 4, has wherein illustrated in A that cut Healing Experiments Effect of Anti hepatocarcinoma humanized RACO-1 monoclonal antibody is on the impact of hepatoma carcinoma cell HCCLM3 travel motion ability to results averaged; Illustrate in C that cut Healing Experiments Effect of Anti hepatocarcinoma humanized RACO-1 monoclonal antibody is on the impact of hepatoma carcinoma cell MHCC97-H travel motion ability), confirm that RACO-1 monoclonal anti physical ability obviously reduces the motion invasive ability of hepatoma cell line.
It is as follows that monoclonal antibody embodiment 1 obtained carries out the operation of tumor formation in nude mice in body: to the raising of animal and the pertinent regulations of drawing materials all in accordance with management of laboratory animal and protection in experimentation.Rearing conditions is as follows: constant temperature (25 DEG C-27 DEG C), constant humidity (45%-50%), fresh air height dust and bacteria removing, without under special mycoplasma environment raise about one week.Buy male BALB/C-nu/nu nude mice 8 in 4-6 age in week (often organizing 4), raise in the aseptic receptacle of SPF level.Animal is placed in dedicated incubator, is placed in the ultra-clean frame of laminar-flow type.To experimental group cell (adding GFP fluorescence) and the cellular control unit (without GFP) of exponential phase be in, when being cultured to q.s, (reach 1 × 10
7/ only) time carries out nude mice hepatocyte in situ and plants into tumor experiment.
Prepare before cell experiment: first experimental group cell and cellular control unit 1:1 are mixed (HCCLM3
controland HCCLM3
anti-RACO-1and MHCC97-H
controland MHCC97-H
anti-RACO-1), with 1mlTIP head cell blown and beaten repeatedly and becomes cell suspension, cell is transferred to centrifuge tube centrifugal after, continue to add culture medium and cell concentration be adjusted to 5 × 10
7/ ml.Test with nude mice 1% pentobarbital sodium intraperitoneal anesthesia, every nude mice consumption is about 0.2-0.5ml, first injects 0.2ml, observes about 2 minutes, as injection volume adds 0.1ml not again, till nude mice reaches anaesthetic effect.Nude mice right upper quadrant of the abdomen skin has been anaesthetized with chelated iodine sterilization, scope is about about 3cm, 1cm angular cut is about under upper right abdominal rib edge, appear liver lobus dexter, ready cell suspension 0.2ml is drawn with syringe, after liver tunicle hemostasis 0.2ml amount is made in right lobe of liver region, observe liver bleeding, stop blooding if desired.Abdomen is closed in careful stitching, and average stitch 2 pin is only every.Experimental result is observed: when about 40 days, dislocation of cervical vertebra method is adopted to put to death nude mice, global solution takes out liver Implanted, perusal liver Implanted is also taken pictures, lung tissue is soaked in the neutral formalin of 10% and spends the night, row serial section after paraffin embedding, the conventional quantity making counted under microscope liver Pulmonary metastasis focuses after immunohistochemical staining of anti-GFP antibody.And do statistical analysis.
In vivo in tumor formation in nude mice, prove that anti-hepatocarcinoma humanized RACO-1 monoclonal antibody is lowered RACO-1 and obviously can be suppressed the Invasion and Metastasis of HCCLM3 cell (as shown in Figure 5, in Fig. 5, A shows and utilizes HCCLM3 cell construction hepatocellular carcinoma in nude mice metastasis model result; Red arrow refers to primary tumor, and black arrow refers to metastatic tumor; B shows metastasis under mirror; C shows anti-hepatocarcinoma humanized RACO-1 monoclonal antibody processed group and compares with matched group metastasis number).
The monoclonal antibody that embodiment 2 obtains tested, experimental procedure is the same, has also drawn close result, as shown in Figure 6.Fig. 6 shows anti-hepatocarcinoma humanized RACO-1 monoclonal antibody and suppresses hepatoma carcinoma cell Invasion and Metastasis in vivo and in vitro.Wherein, the A in Fig. 6: cut Healing Experiments Effect of Anti hepatocarcinoma humanized RACO-1 monoclonal antibody is on the impact of hepatoma cell line HCCLM3 and MHCC97-H travel motion ability.B:Transwell attacks the impact of cell experimentation anti-hepatocarcinoma humanized RACO-1 monoclonal antibody on hepatoma cell line HCCLM3 and MHCC97-H invasive ability.C: anti-hepatocarcinoma humanized RACO-1 monoclonal antibody group compares with nude mice of control group Intrahepatic metastasis stove number
RACO-1 has played key effect in the Invasion and Metastasis process of hepatocarcinoma.RACO-1 is by regulating and controlling AP-1 signal path with the interaction of AP-1 member c-JunD, and this is very crucial to the activation of AP-1 signal path.Research finds that the cell proliferation knocking out RACO-1 obviously slows down, and meanwhile, in AP-1 path, the expression of proliferation-associated protein cdc2, cyclinD1 and hb-egf obviously weakens.The more important thing is that process LAN RACO-1 can promote the formation of mice digestive tract tumor.In addition, also there is important regulating and controlling effect to other tumor signal paths such as Wnt and Ras signal path in RACO-1.Our research has confirmed that RACO-1 passes through regulation and control epithelial cell mesenchymal transformation and promotes the Invasion and Metastasis of hepatocarcinoma, the Invasion and Metastasis of hepatocarcinoma significantly can be suppressed in vivo and in vitro by anti-hepatocarcinoma humanized RACO-1 monoclonal antibody, be expected to the important drugs becoming clinical treatment hepatocarcinoma, thus promote the raising of liver cancer treatment level.
Apply technical scheme of the present invention, by the medicine of RACO-1 monoclonal antibody for the preparation of hepatocarcinoma, RACO-1 monoclonal antibody can the expression of specific suppression RACO-1 in human body, thus effectively reduce hepatocarcinoma invasive ability, improves the five year survival rate of liver cancer patient.What RACO-1 monoclonal antibody adopted is people source RACO-1 monoclonal antibody, and it has the plurality of advantages such as specificity is good, affinity is high, immunogenicity is little, side effect is low.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Attached voluntary hospital of <110> Medical College of Zhejiang Univ.
The medicine of <120> Hepatoma therapy and the application of RACO-1 monoclonal antibody
<160> 4
<170> PatentIn version 3.3
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<213> engineer
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Claims (1)
1. a medicine for Hepatoma therapy, is characterized in that, comprises the RACO-1 monoclonal antibody of medicine effective quantity, described RACO-1 monoclonal antibody behaviour source RACO-1 monoclonal antibody; Described people source RACO-1 monoclonal antibody is prepared by following steps:
(1) by reverse transcription PCR method clone RACO-1 DNA;
(2) described RACO-1 DNA is adopted to carry out phage antibody library structure;
(3) screen described phage antibody library and obtain object antibacterial;
(4) extract the plasmid DNA in described object antibacterial, excise bacteriophage coat protein III gene in described plasmid DNA, the described plasmid DNA of remainder is carried out express, purification obtains described people source RACO-1 monoclonal antibody;
The sequence of the forward primer that described clone RACO-1 DNA is used is the sequence shown in SEQ ID NO:1, and the sequence of downstream primer is the sequence shown in SEQ ID NO:2; Or the sequence of the forward primer that described clone RACO-1 DNA is used is the sequence shown in SEQ ID NO:3, and the sequence of downstream primer is the sequence shown in SEQ ID NO:4.
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Non-Patent Citations (3)
| Title |
|---|
| Is there a general autoantibody signature for cancer?;Ludwig n et al;《European Journal of Cancer》;20121031;第48卷(第15期);2451-2461 * |
| Transcriptional Profiling of Mouse Uterus at Pre-Implantation Stage under VEGF Repression;ji y et al;《PloS one》;20130228;第8卷(第2期);e57287 * |
| Tumor necrosis factor α stimulates endothelin-1 synthesis in rat hepatic stellate cells in hepatic wound healing through a novel IKK/JNK pathway;zhan sx et al;《Experimental Cell Research》;20110415;第317卷(第7期);1040-1048 * |
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