CN100558747C - With the diphtheria toxin muton CRM 197 immunogen and preparation method thereof and application of carrier - Google Patents

Abstract

The present invention relates to a kind of immunogen and preparation method thereof, be to be carrier proteins with the diphtheria toxin muton CRM 197, by special-shaped bi-functional cross-linking agent ε-dimaleoyl imino caproic acid N-hydroxy-succinamide ester (EMCS) and the mutually crosslinked G17CRM197 that gets of gastrin G17 molecule.Carrier proteins used in the present invention has rate of recovery height, is easy to purifying, is suitable for the advantage of large-scale industrial production, while cross-linking products crosslinking rate height, and preparation time is short, has reduced production cost.The present invention also relates to the immunogen of this method preparation effective constituent the application of medicine in oncotherapy of making as auxiliary material with suitable immunological adjuvant as therapeutic vaccine.

Description

With the diphtheria toxin muton CRM 197 immunogen and preparation method thereof and application of carrier

(1) technical field

The present invention relates to a kind of is the immunogen G17CRM197 and preparation method thereof of carrier with the diphtheria toxin muton CRM 197, and the application of this immunogen in the digestive tract tumor treatment, belongs to medical technical field.

(2) background technology

CRM197 (cross-reacting materials 197) is that diphtheria toxin has been lost toxic a kind of mutant, referring to people such as Wu Qida, diphtheria toxin separates with associated protein I. and describes diphtheria toxin serotype related mutants bacterial strain, biological chemistry, 1973.248:3838-3844. (Uchida, T., A.M.Pappenheimer, Jr.and R.Gregory.al., Diphtheria toxin and related proteins I.Isolation and properties of mutantproteins serologically related to diphtheria toxin.J.Biol.Chem.1973.248:3838-3844.), it is by sporting A by a bases G in the base sequence of wild-type diphtheria toxin, thereby caused the 52nd amino acids GLY to sport GLU, referring to people such as Jian Nini, the aminoacid sequence of two kinds of diphtheria toxin nontoxicity mutant CRM45 and CRM197, nucleic acids research, 1984.Vol.12No.10, P4063-4070 (G.Giannini, R.Rappuoli and G.Ratti al., The amino-acid sequence of two non-toxic mutants ofdiphtheria toixn:CRM45and CRM197..Nucleic Acids Research.1984.Vol.12 No.10, P4063-4070).On structure, CRM197 has complete diphtheria toxin functional structure, but the experiment show, the A segment of diphtheria toxin can combine with NAD and CRM197 can not, this show the NAD binding site variable effect the enzymic activity and the toxicity of diphtheria toxin.The difference that studies have shown that CRM197 and diphtheria toxin afterwards is that 52 GLY sports GLU, this GLY that has just proved 52 plays an important role at diphtheria toxin NAD binding site, this just causes the diphtheria toxin enzyme active sites---changes with NAD:EF2 ADP ribose transferring enzyme land, cause the CRM197 segment A can not the EF2 combination, can not play toxic action by pair cell, referring to: Mo Yina, Ke Lisi days are gloomy. adopt the comparison of gel-filtration and saturated ammonium sulphate precipitation method purifying diphtheria toxin and diphtheria toxoid method. and pathogenic micro-organism and immunotherapy annual meeting .1984,92:17-23 (K.Moyner, G.Christiansen, Comparison of gel filtration and ammonium sulphateprecipitation in the purification of diphtheria toxin and toxoid, Acta pathmicrobiol immunol scand sect C, 1984,92:17-23).CRM197 does not have enzymic activity and toxicity, but has the immunogenicity of diphtheria toxin, so CRM197 also is often used as crosslinked other haptens of a kind of immune protein carrier together as vaccine.Just utilize the immunogenicity of CRM197 as far back as U.S. scientist in 1985, the polysaccharide on bloodthirsty influenza bacterium surface is linked on the protein carrier of diphtheria toxoid and CRM197 and makes the vaccine control respiratory tract infection, on prevention effect, two kinds of crosslinked vaccines do not have significant difference on effect, but can both make child produce stronger immunological memory, referring to: Peter. the Anderson, rice cut and, Pi Qiqieluo, Rui Kade. adopt the polysaccharide on diphtheria toxoid or the crosslinked bloodthirsty influenza bacterium b hypotype of diphtheria toxin PROTEIN C RM197 surface to prepare influenza immunogen. clinical investigation magazine .1985:52-59 (Porter Anderson, Micheal E.Pichichero, and Richard A.Insel.Immunogens Consisting ofOligosaccharides from the Capsule of Haemophilus influenzae Type b Coupled toDiphtheria Toxiod or the Toxin protein CRM197.J.Clin.Invest.1985:52-59).U.S. scientist at children's before 2 years old for Pnu-Imune 23 (coccus surface polysaccharide, PnPs) can not produce immunological memory, therefore with coccus surface septivalency polysaccharide crosslinked in the multiple protein carrier so that before children's is 2 years old, can produce antibody to streptococcus pneumoniae, animal after process multiple protein carrier is crosslinked and clinical effectiveness are relatively, find that PnPs-CRM197 can produce immune effect preferably, and safety non-toxic negative interaction, referring to Bu Laike, roach moral in thanking is cut down and graceful, the Louis, auspicious, the Chinese is gloomy, Ai Erwen, the grace plug and, Haake and, Si Bo and, Man Liluosike, horse is taken by force, Chang Yi, section's white square, Wo Ke, Ao Siting, like moral Ward .2000, crosslinked septivalency streptococcus pneumoniae surface polysaccharide vaccine is in the intravital validity of children, security and immunogenicity. transmissible disease journal .9:187-195 (Black, S., H.Shinefield, B.Fireman, E.Lewis, P.Ray, J.R.Hansen, L.Elvin, K.M.Ensor, J.Hackell, G.Siber, F.Malinoski, D.Madore, I.Chang, R.Kohberger, W.Watson, R.Austrian, K.Edwards, et al.2000.Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine inchildren.Pediatr.Infect.Dis.J.9:187-195).This product also goes on the market by drugs approved by FDA at present, and commodity are called Prevnar[BIOPHARMA:Biopharmaceutical Products in the U.S.Market].Italy scientist Francesco has also adopted at epidemic meningitis and has utilized the crosslinked CRM197 of its germ surface polysaccharide to make vaccine, they have analyzed crosslinked feasibility from the structure of CRM197, wherein CRM197 has 39 Lysin amino-acid residues and 16 Arg residues, and the free amine group of more crosslinked usefulness can be provided.From experimental result, though the chemically crosslinked rate is higher, but all do not reach theoretical crosslinking rate, and its crosslinking rate of different sugar also there are differences, and this explanation crosslinking rate is also relevant with cross-linking agent, referring to: Francisco. the shellfish body, Borrow. restrain this altar and replace Mike. not Lay should, the. Lu Qila difficult to understand of Crow is grown. water-soluble, the deposition property of polysaccharide protein cross-link vaccine and kinetic character.(Francesco Berti，Paolo Costantino，Marco Fragai，y and Claudio Luchinatz。Water Accessibility，Aggregation，and Motional Features of Polysaccharide-Protein Conjugate Vaccines.Biophysical Journal Volume 86 January 2004 3-9)。

At the deficiency of existing CRM197, Hainan Tianyuan Kangze Pharmaceutical Technology Co., Ltd improves the gene order of original CRM197, has made up to express the recombinant expression plasmid of CRM197 and be converted among the escherichia coli expression host with the inclusion body formal representation.Purpose product C RM197 expression amount height accounts for more than 24% in whole bacterial protein as a result, and the rate of recovery height of purpose product is easy to purifying, is suitable for large-scale industrial production.Referring to Chinese patent CN200610042194.7.

Gastrin (gastrin) is a kind of peptide hormone, can be divided into big gastrin (big gastrin G34) according to molecular structure, Shg17NS (little gastrin G17), little Shg17NS (minigastrin G14), big big gastrin (big big gastrin) and composition I (component I) are by being positioned at GI G emiocytosis.Its traditional role is to stimulate the secretion of hydrochloric acid in gastric juice and pepsinogen and to the trophism of digestive tube cell, referring to: Zou Jihong, Li Jinrui, the new development of Zhang Zhongping gastrin research. radioimmunology magazine the 8th the 4th phase of volume of nineteen ninety-five.Bigger variation has taken place as the notion of simple intestinal hormone in gastrin and cholecystokinin (CCK) in recent years, its regulating and controlling effect to gastroenteric tumor becomes the focus that people pay close attention to gradually, referring to: soften and falsely charge Gothic E, Wal stone J.H. gastrin, cholecystokinin, signal conduction and tumour.(Rozengurt E，Walsh JH.Gastrin，CCK，signaling，and cancer.Annu Rev Physiol，2001，63：49-76)。Most of digestive tract tumor cells can synthesize and secrete gastrin, and express corresponding acceptor, its excretory gastrin by approach performance behind the acceptor of self to the regulating effect of tumour, this is the Gastrin/CCK autocrine regulation loop (autocrine loop) of digestive tract tumor, this may be one of the most important biological characteristics of digestive tract tumor cell and autonomy regulation mechanism, referring to: Ba Er German GS, the effect in normal canceration gi tract growth of gastrin and cholecystokinin.(Baldwin GS.The role of gastrin andcholecystokinin in normaland neoplastic gastrointestinal growth.J Gastroenterol Hepatol，1995，10(2)：581-584)。Studies show that in recent years, except sophisticated amidation molecular form, the digestive tract tumor cell still can be secreted the gastrin of regulating effect precursor and glycine extension type intermediate product.Verifiedly in cancer of the stomach, large bowel cancer, carcinoma of the pancreas and cholangiocarcinoma, all there is a complete gastrin autocrine regulation loop, this loop all has regulating and controlling effect to generation, growth and the metastasis and invasion of digestive tract tumor, referring to: Zhang Fengshen, He Zhenping, the progress of Ma Kuansheng digestive tract tumor cell Gastrin/CCK autocrine regulation loop. gastroenterology and hepatopathy magazine, 2002,19:92-94).

(3) summary of the invention

The present invention is directed to the deficiencies in the prior art, providing a kind of is the immunogen and preparation method thereof of carrier with the diphtheria toxin muton CRM 197.

Another task of the present invention be by experimental evaluation in external and the body immunogen to the digestive tube antitumor activity against various tumors.

Immunogen of the present invention, be to be carrier proteins with the diphtheria toxin muton CRM 197, by special-shaped bi-functional cross-linking agent ε-dimaleoyl imino caproic acid N-hydroxy-succinamide ester (EMCS) and the mutually crosslinked G17CRM197 that gets of gastrin G17 molecule, wherein, the aminoacid sequence of described G17 molecule is as follows:

(a) sequence signature

Length: 16 amino acid

Type: amino acid

(b) molecule type: polypeptide

(c) sequence description

pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Ser-Ser-Pro-Pro-Pro-Pro-Cys

Described diphtheria toxin muton CRM 197 has following sequence:

(a) sequence signature

* length: 536 amino acid

* type: amino acid

(b) molecule type: protein

(c) sequence description

Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn Phe Ser Ser

1 5 10 15 20

Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys

21 25 30 35 40

Ser Gly Thr Gln Gly Asn Tyr Asp Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys

41 45 50 55 60

Tyr Asp Ala Ala Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly

61 65 70 75 80

Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val Asp Asn Ala

81 85 90 95 100

Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu Pro Leu Met Glu Gln Val Gly

101 105 110 115 120

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

121 125 130 135 140

Phe Ala Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu

141 145 150 155 160

Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp Ala Met Tyr

161 165 170 175 180

Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu

181 185 190 195 200

Ser Cys Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser

201 205 210 215 220

Leu Lys Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser

221 225 230 235 240

Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu His Pro Glu

241 245 250 255 260

Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala

261 265 270 275 280

Ala Trp Ala Val Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys

281 285 290 295 300

Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly

301 305 310 315 320

Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met

321 325 330 335 340

Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn

341 345 350 355 360

Phe Val Glu Ser Ile Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala

361 365 370 375 380

Tyr Ser Pro Gly His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn

381 385 390 395 400

Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His Asp Ile Lys

401 405 410 415 420

Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val Leu Leu Pro Thr Ile Pro Gly

421 425 430 435 440

Lys Leu Asp Val Asn Lys Ser Lys Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met

441 445 450 455 460

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

461 465 470 475 480

Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser Glu Lys Ile

481 485 490 495 500

His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp

501 505 510 515 520

His Thr Lys Val Asn Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys Ser

521 525 530 535

The preparation method of immunogen G17CRM197 of the present invention is as follows:

With the diphtheria toxin muton CRM 197 is carrier proteins, select special-shaped bi-functional cross-linking agent ε-dimaleoyl imino caproic acid N-hydroxy-succinamide ester (EMCS) for use, pass through crosslinking reaction, gastrin G17 molecule is mutually crosslinked with CRM197, measure crosslinking rate, crosslinked 15-20 G17 molecule on each CRM197 molecule.

The quantity of the free sulfhydryl group of above-mentioned G17 should be not less than 60%.Therefore need measure the quantity of free sulfhydryl group among the G17 in advance, be less than 60%, then need it is reduced as sulfhydryl content.Concrete method of reducing can use Ellman reagent (Ellman ' s Reagent) to carry out according to the operational guidance in the working instructions.

Free amino quantity should be not less than 5 moles/every mole CRM197 among the above-mentioned CRM197.Preferred free amine group quantitative range is to contain 10-30 mole free amine group among every mole of CRM197.Therefore need adopt the fluorescamine method to measure free amino quantity among the CRM197 in advance.Be lower than 5 moles as if the free amine group among every mole of CRM197 and then can not prepare immunogen with greater activity.

Carry out as for the method that crosslinking reaction particularly can adopt those skilled in the art to be familiar with, as G.L. Jones, H.M. the Kenneth Adelman moral is gloomy, L. this Bender, J. Gai Er, A. that continues waits and is utilizing dimaleoyl imino caproic acid succinimide base ester to prepare the (G.L.Jones of institute's reported method in the maleoyl peptide carrier immunogen, H.M.Edmundson, L.Spender.J.Gale, A.Saul.The use of maleimidocaproyloxysuccinimide to prepare malarial peptide carrier immunogens.Journal of immunological methods, 123 (1989) 211-216).The method that the measuring method of crosslinking rate can select for use those skilled in the art to be familiar with, as: SDS-PAGE method, chemical quantitative method or amino acid analysis method etc.

The invention provides following concrete operations step, but be not limited thereto:

(1) at first make G17 and linking agent EMCS reaction, the reaction times is 2 hours, obtains activatory G17;

(2) by the G10 desalting column activatory G17 is carried out purifying;

(3) G17 behind the activation purifying carries out crosslinking reaction with CRM197 again, and the reaction times is 2 hours;

(4) cross-linking products is carried out purifying.

The described purifying of step (4) can adopt the method for dialysis method (the dialysis tubing interception is 8000-10000 dalton), ultrafiltration process and gel column desalination to carry out purifying.Concrete preferred G25 desalting column carries out purifying.

Measure the immunogenicity of prepared cross-linking products.Adopt the method for immune animal to measure the situation that prepared cross-linking products stimulates animal generation antibody, carry out immunity, adopt enzyme-linked immunosorbent assay (ELISA) to detect the antibody titer that produces according to the method for routine.

With prepared G17CRM197 immunogen immune animal, two weeks of back of immunity for the third time are with sacrifice of animal and gather serum.The serum of being gathered is carried out purifying obtain anti-G17CRM197 antibody, and resulting antibody is carried out evaluating drug effect in external, the body.The tumour cell that experiment in vitro adopted is stomach cancer cell MKN45, BGC823 and colorectal cancer cells LOVO, SW480; Experiment is carried out with nude mice in the body.Experimental results show that by pressing down knurl in tumor cell in vitro cultivation and the nude mouse immunogenic antibodies of the present invention has the obvious treatment effect to cancer of the stomach and large bowel cancer.Particular content will further describe in an embodiment.

With the diphtheria toxin muton CRM 197 is the immunogenic pharmaceutical applications of carrier, is used to prepare the medicine for the treatment of tumour.With the effective constituent of immunogen of the present invention as therapeutic vaccine, the pharmaceutical preparation of making as auxiliary material with suitable immunological adjuvant can be directly used in the human injection.This immunogen can produce corresponding anti-gastrin antibody by stimulating immune system in human body, this antibody can in and the Infusion in Patients with Digestive body in too much gastrin, thereby reach the treatment tumour purpose.

Compared with prior art, excellent results of the present invention mainly shows the following aspects:

1. protein carrier used in the present invention is a diphtheria toxin muton CRM 197, has rate of recovery height, is easy to purifying, is suitable for the advantage of large-scale industrial production;

2. the diphtheria toxin muton CRM 197 material of Shi Yonging is single, preparation technology simple, quality is easy to control;

3. preparation method's whole process of preparation of the present invention only needs 6 hours, shortens dramatically than prior art, greatly reduces production cost;

4. prepared cross-linking products crosslinking rate is much higher than prior art, that is to say each CRM197 molecule the G17 molecule on crosslinked obviously more than prior art; Crosslinking rate of the present invention is between 15-20 G17 molecule/CRM197 molecule, and prior art can only reach 7-15 G17 molecule/CRM197 molecule.

5. experimentation on animals result shows that compared with prior art, the immunogenicity of G17CRM197 is stronger, and the antibody titers that is produced can reach 1: 500000, and the antibody titers that can reach according to the bibliographical information prior art is 1: 64000.

6. press down the knurl experiment in the external and nude mouse and show that this cross-linking products has the good antitumor effect.

(1) description of drawings

Fig. 1 is the SDS-PAGE figure of immunogenic composition of the present invention, and wherein 1 is the molecular weight of albumen standard, and 2 is CRM197, and 3 are the CRM197 after the activation, and 4 is cross-linking products.

Fig. 2 is respectively at 14,35 and 56 days measured antibody titerss behind the prepared immunogenic composition immune animal of usefulness the present invention.Wherein X-coordinate represents to test fate, and ordinate zou is represented antibody titers.

Fig. 3 adopts the ELISA method to detect antiserum(antisera) antibody titers photo.Wherein 1 for using the resulting sero-fast detected result of prior art G17DT immune animal, 2 for using the prepared resulting sero-fast detected result of immunogen immune animal of the present invention, 3 positive contrasts, 4 negative contrasts, laterally numeral is sero-fast extension rate.

(2) embodiment

Describe the preparation method of immunogenic composition G17CRM197 of the present invention in detail below in conjunction with embodiment.

Embodiment 1:

1. material therefor

1.1 main agents

Single hydration cysteine hydrochloride (Cysteine Hydrochloride Monohydrate), sulfydryl are measured reagent E llman reagent, linking agent EMCS: Pierre Si (Pierce) company product; Fluorescamine: western GAMA (Sigma) company product; Other is a domestic reagent.

1.2 key instrument equipment

TGL-16 type high speed tabletop centrifuge: Shanghai medicine equipment six factory's products.

Micropipet: this doffer Eppendof company product is liked by Germany.

HD95-1 Protein Detection instrument and 3057 type registering instruments: (going up Haikang China biochemical instrument manufactory).Peristaltic pump: Lange pump, YZ1515, Baoding LanGe constant flow pump Co., Ltd.BHW-IV electric heating constant temperature water temperature case: Beijing Medical Equipment Plant.J2-MC high speed low temperature centrifugal machine: Beckman company product.Ice-making machine (GRANT), 1-15 whizzer (Sigma), Spectrumlad54 ultraviolet-visible pectrophotometer (Prism Optical Technology Co), FR-200 gel imaging system (Shanhai Furi Science and Technology Co., Ltd.), DK-8D type electric heating constant temperature tank (the gloomy reliable Instr Ltd. that tests in Shanghai), DYY-12 type computer three is permanent many with electrophoresis apparatus (Beijing Liuyi Instrument Factory), the mini vibrator of MS2 (IKA, Guangzhou company), the accurate PH meter of PHS-3C type (Shanghai Precision Scientific Apparatus Co., Ltd), MILLEXTMGP 0.22um millipore filter (Millipore), Bechtop, electronic balance.

1.3 antibody and two anti-, adjuvants

Horseradish peroxidase mark goat-anti rabbit two anti-(Beijing Huamei Bio-Engrg Co.), diphtheria toxoid antibody (Sigma company), freund's adjuvant (BBI company).

2. the preparation method of immunogenic composition

2.1 sulfydryl and amino mensuration

(Cysteine Hydrochloride Monohydrate) is contrast with single hydration cysteine hydrochloride, adopts the method for measuring of Ellman reagent to make the mark curve.Accurately the G17 solution of preparation 0.1mM adopts Ellman reagent to measure equally, calculates the quantity of free sulfhydryl group by typical curve.Among the present invention employed G17 after measured, wherein sulfhydryl content is 97%, need not to handle.If sulfhydryl content is lower, then need G17 is reduced, can adopt dithiothreitol (DTT) to handle.

Adopt fluorescamine reagent to measure free amino quantity among the CRM197.With the glycine is that typical curve is made in contrast.Accurately the CRM197 solution of configuration 0.1M is measured with fluorescamine equally, by contrasting the quantity that calculates free amine group with typical curve.The amino quantity of dissociating among the employed CRM197 among the present invention is 30mol amino/molCRM197.

2.2 the preparation of cross-linking agent

Method one:

Take by weighing 20mgG17, be dissolved in the saturated sodium phosphate buffer of 1ml nitrogen (pH6.5-7.0); Take by weighing 6mgEMCS and be dissolved in the 100ul dimethyl formamide, treat that it is slow in 15 minutes after fully dissolving, gradation is added in the G17 solution oscillatory reaction 2 hours.This step reaction requires EMCS definitely to be higher than the molecular amounts of G17 in molecular amounts.

Above-mentioned reaction product is carried out purifying to remove unreacted EMCS.Purification process has multiple, can adopt G10 desalting column method, also can adopt the method for dialysis.

Take by weighing 10mgCRM197, slowly be added among the G17 that above-mentioned purifying crosses, oscillatory reaction is spent the night.Promptly obtain cross-linking products after removing unreacted G17.Can adopt dialysis or G25 desalting column to remove unreacted G17 equally.

Method two:

Taking by weighing 20mgCRM197 is dissolved in the sodium phosphate buffer; Take by weighing 6mgEMCS and be dissolved in the 100ul dimethyl formamide, treat that it is slow in 15 minutes after fully dissolving, gradation is added in the CRM197 solution oscillatory reaction 2 hours.

Above-mentioned reaction product is carried out purifying to remove unreacted EMCS.Purification process has multiple, can adopt G10 desalting column method, also can adopt the method for dialysis.

Take by weighing 10mgG17, slowly be added among the CRM197 that above-mentioned purifying crosses, oscillatory reaction is spent the night, and adopts nitrogen protection simultaneously.Promptly obtain cross-linking products after removing unreacted G17.Can adopt dialysis or G25 desalting column to remove unreacted G17 equally.

2.3 the mensuration of crosslinking rate

The crosslinking rate of indication of the present invention is meant the number of the bonded G17 of institute molecule on the CRM197 of each molecule, and its measuring method is various.Can pass through the peptide content signature analysis, peptide content can increase such as weight by many methods that those skilled in the art suppress and amino acid analysis waits and measures.Also can be undertaken, calculate crosslinking rate by the variation of CRM197 on molecular weight before and after the contrast reaction by the electrophoretic method of SDS-PAGE.Also can come the indirect calculation crosslinking rate in addition by the variation of calculating the last bonded EMCS quantity of CRM197.Exactly concrete: as to make with EMCS reaction back and purified CRM197 and cultivate with cysteine hydrochloride, then with the Ellman reagent react of 10mM, with the sulfydryl of Ellman reagent in the molar extinction coefficient 13600 calculating free halfcystines at 412nm place.Calculate the quantity of EMCS by same method again after reaction finishes, the difference of twice mensuration numerical value is the quantity of crosslinked G17 molecule.Wherein protein concentration adopts Lowry method or Bradford method to measure.

3. immunogenic mensuration

3.1 the immunity of animal

Prepared cross-linking agent G17CRM197 is dissolved in physiological saline, and mixes, make that the ultimate density of cross-linking agent is 1mg/ml with the equivalent freund's adjuvant.The immunity time is 0,21,42 day; Immunization method adopts the intramuscular injection of rabbit back leg, injects right leg for the first time, injects left leg for the second time, injects right leg for the third time, and injection point is a little more than the injection point first time.Injection for the first time adopts Freund's complete adjuvant to handle sample, and the back adopts for twice Freund's incomplete adjuvant to handle, and immunizing dose is 1mg for the first time, and the immunizing dose that the back is twice is 0.5mg.

Gather rabbit blood respectively at the 14th, 35,56 day and carry out the antibody titer detection.Wherein preceding twice is the rabbit ear edge vein exploitating blood, for the carotid artery blood sampling, places 1 hour under the serum room temperature of collection for the third time, solidifies rearmounted 4 ℃ and spends the night, and separates out serum, and 2000rpm is centrifugal, separation of serum.Serum is stored in-4 ℃, and is standby in 2 weeks.

3.2 the detection of antibody titers

It is enzyme-linked immunosorbent assay (ELISA) that antiserum titre detects method the most commonly used.Detection method used in the present invention is the ELISA method.Be specially: with immunogen G17BSA coating buffer (Na ₂CO ₃-NaHCO ₃, pH9.6) be diluted to 0.1ug/ml, wrap by polystyrene 96 orifice plates, the 50ul/ hole, 4 ℃ of cultivations are spent the night.Wash behind the plate three times with confining liquid (2%BSA-PBS) sealing, 50ul/ hole, 37 ℃, 2 hours.Add antiserum(antisera), the antiserum(antisera) of gathering is done following multiple dilution: 2000,8000,16000,32000,64000,128000,256000,512000, every hole 50ul cultivated 2 hours for 37 ℃.With the negative contrast of the rabbit anteserum of injecting normal saline,, compare with the antiserum(antisera) that produces with the rabbit of injecting G17DT simultaneously with the positive contrast of commodity diphtheria toxin antibody.Wash after three times and to add the two anti-of horseradish peroxidase-labeled, dilution in 1: 4000 was hatched 1 hour for 37 ℃.Add the substrate colour developing after washing plate 10 times, hatch termination reaction after 40 minutes for 37 ℃, measure A ₄₉₁Value.

The result as shown in Figure 3, as can be seen from the figure with the anti-gastrin antibody that has produced high titre behind the prepared immunogen immune animal of the present invention three times, the highest tiring reaches 1: 512000, and is 1: 64000 with the antibody titers that produces behind the prior art G17DT immune animal three times.Experimental result proves that fully the prepared immunogenic composition of the present invention can stimulate body to produce the anti-gastrin antibody of high titre, and its immunogenicity will be apparently higher than G17DT of the prior art.

Embodiment 2:

1. the external knurl that presses down is tested

1.1 the detection of the gastrin that is produced in the Cultural Course of Tumor Cells

The present invention adopts gastrin detection kit (ELISA method) to measure the production of gastrin in the Cultural Course of Tumor Cells.

Get cultured tumor cell line, observation of cell is individual layer and is paved with the whole growth face under the inverted microscope, shows well-grown.Digestion and make single cell suspension in sterilisable chamber.Obtained cell suspension is counted on blood counting chamber, adjusts the amount of RPMI 1640 liquid, and making the final cell number is 1 * 10 ⁹Individual/L, get the 1ml cell suspension inoculation in the T25 culturing bottle with transfer pipet, other adds the about 4ml of RPMI1640 liquid.Change liquid every day 1 time, the RPMI 1640 liquid 5ml that change are collected, give over to ELISA and measure.Get 2 bottles when changing liquid at random, make single cell suspension by above-mentioned steps, obtained cell suspension is counted on blood counting chamber, gets its mean, represents the cell count of this moment in every bottle.All this bottle cells of having counted no longer are included within the experiment.Continuous 3 times, cultivated altogether 72 hours.

The result is added up, see the following form.

The production of gastrin in the table 1a stomach cancer cell MKN45 culturing process

Time (hour)	Cell count (10 9/L)	Gastrin (ng/L)
			0	1.0	0
24	Increase to 3.2 by 1.0	15.2
			48	Increase to 5.1 by 3.2	16.1
72	Increase to 5.9 by 5.1	16.9

The production of gastrin in the table 1b stomach cancer cell BCG823 culturing process

Time (hour)	Cell count (10 9/L)	Gastrin (ng/L)
			0	1.0	0
24	Increase to 3.0 by 1.0	13.1
			48	Increase to 4.8 by 3.0	14.2
72	Increase to 5.3 by 4.8	14.9

The production of gastrin in the table 1c colorectal cancer cells LOVO culturing process

Time (hour)	Cell count (10 9/L)	Gastrin (ng/L)
			0	1.0	0
24	Increase to 4.1 by 1.0	17.2
			48	Increase to 5.5 by 4.1	18.1
72	Increase to 5.9 by 5.5	18.6

The production of gastrin in the table 1d colorectal cancer cells SW480 culturing process

Time (hour)	Cell count (10 9/L)	Gastrin (ng/L)
			0	1.0	0
24	Increase to 3.6 by 1.0	16.1
			48	Increase to 4.8 by 3.6	16.9
72	Increase to 5.5 by 4.8	17.4

Above result shows that stomach cancer cell MKN45, BCG823 and colorectal cancer cells LOVO, SW480 can secrete gastrin in culturing process, and along with cell is constantly bred, its excretory gastrin quantity also increases, and both are proportionate.

1.2 the growth-inhibiting of tumour cell is tested with the anti-G17CRM197 antibody that the prepared immunogen of the present invention is prepared

In well-grown tumour cell, add prepared antibody among the embodiment 1, measure the cell viability of different treatment group (add 17 groups of gastrins, add the antibody group, blank group) by mtt assay, to determine the restraining effect of antibody to tumour cell.Selected tumour cell is stomach cancer cell MKN45 and colorectal cancer cells SW480.

The tumor cell line cell in vitro is cultivated, and adds aseptic gastrin 17 and gastrin antibody respectively in the 10% calf serum RPMI1640 nutrient solution, is divided into three groups of blank groups, gastrin 17 groups of (25ug/ml) antibody group (30ug/ml).

The mensuration of cell viability: use thiophene nitrogen azoles indigo plant (MTT) colorimetric analysis and measure cell viability, (cell concn is 10 to get the celliferous nutrient solution of 100ul ⁵/ ml), being inoculated in 96 well culture plates, nutrient solution was removed in suction after cell pasted an ancient piece of jade, round, flat and with a hole in its centre; Add nutrient solution, gastrin 17 and antibody respectively by above-mentioned grouping, each group is all established 8 multiple holes, cultivate that every hole adds MTT (5mg/ml) 20ul after 66 hours, continue to cultivate 6 hours, the centrifugal supernatant of abandoning, every hole adds 20%SDS100ul, shakes 5 minutes, puts room temperature is measured 570nm wavelength place on microplate reader after half an hour absorbancy.

Each organizes A ₅₇₀Value is reflection viable cell quantity indirectly, is stomach cancer cell MKN45 or colorectal cancer cells SW480 no matter the result shows, gastrin group viable count is apparently higher than control group (P＜0.01), antibody group cell count obviously descend (P＜0.01).

The anti-G17CRM197 antibody of table 2 is to the growth-inhibiting situation of tumour cell

Grouping	MKN45 cell A 570	SW480 cell A 570
			17 groups of antibody groups of blank group gastrin	0.561±0.056 0.767±0.065 0.421±0.045	0.472±0.046 0.656±0.057 0.389±0.041

2. press down the knurl experiment in the nude mouse

The foundation of animal model for tumour: get the nutrient solution 70ul (cell count 10 that contains MKN45 cell and SW480 cell ⁷), being inoculated in one-tenth knurl in the right armpit back of the body of the nude mice intersection subcutaneous layer, the knurl piece is inoculated 2 times repeatedly, forms animal model for tumour, gets male nude mouse and is divided into three groups at random, six every group; The tumour that goes down to posterity is cut into cubic knurl piece of the same size, about 8mm under magnifying glass ³Size, it is subcutaneous to be inoculated in the right front armpit of nude mice back of the body intersection respectively with trochar.

Each group is all from beginning in the 1st day medication of knurl piece inoculation back.Control group: injection carboxymethyl cellulose suspending agent 200ul in every nude mice abdominal cavity, every day 2 times, treated 28 days altogether; The antibody group: every nude mice intravenous injection antibody 500mg/kg, every day 2 times, treated 28 days altogether.

Observation of curative effect: tumour begins to form in the time of the 5th day, with the major diameter and the vertical diameter of every nude mice lotus of vernier caliper measurement knurl, measures every other day 1 time; Nude mice all survives in the time of the 28th day, and the taking-up tumour is also weighed.Calculate tumour inhibiting rate.Each is organized difference and carries out statistical procedures with the t check.

The result: existing tumor growth when inoculating the 5th day, to measure and respectively organize the tumour area, size is close, there was no significant difference (P＞0.05), the homogeneity that shows tumor growth is good, obviously dwindles than control group since the 16th day antibody group tumour area, and difference has significance (P＜0.05); Antibody group tumour area is littler than control group in the time of the 28th day.

Calculate according to the changes in weight of tumour, reach 28% and 26% respectively for the tumour inhibiting rate of two kinds of tumours with the tumour inhibiting rate of the prepared antibody of the present invention.The tumor weight changing conditions is as shown in the table.

Table 3 in-vivo tumour changes in weight table

Grouping	Cancer of the stomach group nude mice tumor weight	Large bowel cancer group nude mice tumor weight
			Control group antibody group	1177.7±183.5mg 847.9±69.6mg	1089.3±156.2mg 802.1±52.3mg

Sequence table

＜110〉Hainan Tianyuan Kangze Pharmaceutical Technology Co., Ltd

＜120〉with the diphtheria toxin muton CRM 197 be the immunogen and preparation method thereof and application of carrier

<140>

<141>

<160>2

<170>Patent In3.1

<210>1

<211>16

＜212〉polypeptide

<400>1

pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Ser-Ser-Pro-Pro-Pro-Pro-Cys

5 10 15

<210>2

<211>536

＜212〉protein

＜213〉diphtheria corynebacterium (corynebacterium diphtheriae)

<400>2

Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn Phe Ser Ser

1 5 10 15 20

Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys

21 25 30 35 40

Ser Gly Thr Gln Gly Asn Tyr Asp Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys

41 45 50 55 60

Tyr Asp Ala Ala Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly

61 65 70 75 80

Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val Asp Asn Ala

81 85 90 95 100

Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu Pro Leu Met Glu Gln Val Gly

101 105 110 115 120

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

121 125 130 135 140

Phe Ala Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu

141 145 150 155 160

Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp Ala Met Tyr

161 165 170 175 180

Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu

181 185 190 195 200

Ser Cys Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser

201 205 210 215 220

Leu Lys Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser

221 225 230 235 240

Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu His Pro Glu

241 245 250 255 260

Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala

261 265 270 275 280

Ala Trp Ala Val Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys

281 285 290 295 300

Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly

301 305 310 315 320

Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met

321 325 330 335 340

Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn

341 345 350 355 360

Phe Val Glu Ser Ile Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala

361 365 370 375 380

Tyr Ser Pro Gly His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn

381 385 390 395 400

Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His Asp Ile Lys

401 405 410 415 420

Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val Leu Leu Pro Thr Ile Pro Gly

421 425 430 435 440

Lys Leu Asp Val Asn Lys Ser Lys Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met

441 445 450 455 460

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

461 465 470 475 480

Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser Glu Lys Ile

481 485 490 495 500

His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp

501 505 510 515 520

His Thr Lys Val Asn Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys Ser

521 525 530 535

Claims (3)

1, a kind of immunogen G17CRM197, it is characterized in that with the diphtheria toxin muton CRM 197 being carrier proteins, by special-shaped bi-functional cross-linking agent ε-dimaleoyl imino caproic acid N-hydroxy-succinamide ester and the mutually crosslinked G17CRM197 that gets of gastrin G17 molecule, crosslinked 15-20 G17 molecule on each CRM197 molecule; Wherein, the aminoacid sequence of described G17 molecule is as follows:

pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Ser-Ser-Pro-Pro-Pro-Pro-Cys

The aminoacid sequence of described diphtheria toxin muton CRM 197 is as follows:

Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn Phe Ser Ser

1 5 10 15 20

Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys

21 25 30 35 40

Ser Gly Thr Gln Gly Asn Tyr Asp Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys

41 45 50 55 60

Tyr Asp Ala Ala Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly

61 65 70 75 80

Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val Asp Asn Ala

81 85 90 95 100

Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu Pro Leu Met Glu Gln Val Gly

101 105 110 115 120

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

121 125 130 135 140

Phe Ala Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu

141 145 150 155 160

Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp Ala Met Tyr

161 165 170 175 180

Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu

181 185 190 195 200

Ser Cys Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser

201 205 210 215 220

Leu Lys Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser

221 225 230 235 240

Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu His Pro Glu

241 245 250 255 260

Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala

261 265 270 275 280

Ala Trp Ala Val Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys

281 285 290 295 300

Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly

301 305 310 315 320

Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met

321 325 330 335 340

Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn

341 345 350 355 360

Phe Val Glu Ser Ile Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala

361 365 370 375 380

Tyr Ser Pro Gly His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn

381 385 390 395 400

Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His Asp Ile Lys

401 405 410 415 420

Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val Leu Leu Pro Thr Ile Pro Gly

421 425 430 435 440

Lys Leu Asp Val Asn Lys Ser Lys Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met

441 445 450 455 460

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

461 465 470 475 480

Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser Glu Lys Ile

481 485 490 495 500

His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp

501 505 510 515 520

His Thr Lys Val Asn Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys Ser

521 525 530 535。

2, the preparation method of the immunogen G17CRM197 of claim 1, with the diphtheria toxin muton CRM 197 is carrier proteins, select special-shaped bi-functional cross-linking agent ε-dimaleoyl imino caproic acid N-hydroxy-succinamide ester for use, pass through crosslinking reaction, gastrin G17 molecule is mutually crosslinked with CRM197, crosslinked 15-20 G17 molecule on each CRM197 molecule, concrete steps are:

(1) at first make G17 and linking agent ε-dimaleoyl imino caproic acid N-hydroxy-succinamide ester reaction, the reaction times is 2 hours, obtains activatory G17; The content of free sulfhydryl group is no less than 60% among the described G17, and free amino quantity is 10-30 mole amino/mole CRM197 among the described CRM197;

(2) by the G10 desalting column activatory G17 is carried out purifying;

(3) G17 behind the activation purifying carries out crosslinking reaction with CRM197 again, and the reaction times is 2 hours;

(4) cross-linking products is carried out purifying.

3, the pharmaceutical applications of the described immunogen G17CRM197 of claim 1 is used to prepare the medicine for the treatment of digestive tract tumor.

Images