CN103087173A - Synthetic peptide vaccine of B cell epitope based on TGF(transforming growth factor)-beta1 and application thereof - Google Patents
Synthetic peptide vaccine of B cell epitope based on TGF(transforming growth factor)-beta1 and application thereof Download PDFInfo
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Abstract
The invention discloses a synthetic peptide vaccine of B cell epitope based on TGF(transforming growth factor)-beta1 and application thereof, inhibits auxo-action of the cell factor TGF-B1-BB during the development process of liver fibrosis so as to inhibit the development of liver fibrosis to a some extent by constructing two epitope peptides of AY25 and LS30, coupling carrier proteins to synthesize an TGF-beta1 peptide vaccine, immunizing an ICR mice by intraperitoneal injection to induce the same to generate an anti-TGF-beta1 antibody, and neutralizing the cell factor TGF-B1-BB over expressed during CCL4 induced hepatic fibrosis process.
Description
Technical field
The invention belongs to the Prevention Technique field of hepatic fibrosis, relate to synthetic peptide vaccine and application thereof based on the B cell antigen epi-position of TGF-β 1.
Background technology
Hepatic fibrosis be multiple chronic hepatopathy to the liver cirrhosis development must through pathologic process, be the main factor that determines the chronic hepatopathy prognosis.At present, most of chronic hepatopathy all lacks effective etiological treatment, and studies confirm that the part hepatopathy removes heptic fibrosis in the cause of disease and still can continue progress, therefore thereby retardance or the genesis that delays hepatic fibrosis prevent from making progress and become one of main path of chronic hepatopathy treatment for liver cirrhosis.But there is no at present truly effectively anti-hepatic fibrosis medicines or measure, the anti-hepatic fibrosis measure safely and effectively of exploring is the important topic that present hepatopathy research field faces.
The cytokine vaccine is a focus in vaccinology research in recent years, be mainly will play a major role in a certain disease cytokine adopt the method active immunity body of vaccine, induce body to produce neutrality antibody, this cytokine that neutralizes to be stoping the performance of its corresponding biological function, thereby plays the purpose of control relative disease.Synthetic peptide vaccine is comparatively extensively and day by day one of ripe new generation vaccine of Recent study, and in stability, easily the property obtained, security, purifying be simple etc., and the aspect has obvious advantage, existing lot of documents is reported its feasibility and validity.This vaccine does not contain nucleic acid, is based on a class vaccine of the epitope development and Design of protein molecular, is ideal safe new generation vaccine.
Transforming growth factor-beta 1 (transforming growth factor beta, TGF-β 1) be the polypeptide that a class can be regulated Growth of Cells and differentiation, possess the various biological effect, possess activated hepatic stellate cells in hepatic fibrosis generation, evolution, promote collagen gene expression.Promoting extracellular matrix to synthesize with deposition and wait effect, is the present known the strongest cytokine of short fibrosis effect.The effect of TGF-β 1 in hepatic fibrosis is in particular in:
Promote the various kinds of cell epimatrix compositions such as hepatic stellate cell expression and secretion I, III, type Ⅳ collagen fiber, ln;
Suppress the activity of proteolytic enzyme in extracellular matrix, as the expression to matrix metalloproteinase and tissue plasminogen activator, restraining effect is arranged, suppress the degraded of extracellular matrix;
Strengthen the expression of some cytokine such as PDGF mRNA;
Inoblast, monocyte had chemotaxis;
Promote the expression of TGF-β 1 self by Autocrine.
The keying action of TGF β 1 in liver tissue during hepatic fibrogenesis obtained generally acknowledging of investigators, and the focus and the utmost point that also become anti-hepatic fibrosis research for the anti-hepatic fibrosis research of TGF β 1 have one of research of application prospect.The subject matter that faces at present is to explore to block safely and effectively TGF β 1 measure.The TGF β 1 that has been reported excessive generation when making hepatic fibrosis with the TGF beta II type acceptor of TGF beta 1 antibodies, soluble T GF β1receptor and adenovirus mediated brachymemma etc. by neutralizing effect reduces, shown significant anti hepatic fibrosis and reduced the hepatocellular apoptosis effect in experimentation on animals, and having found no obvious liver part or systemic side effects.There is the research of the organ fibrosis such as lung, kidney, the heart of similar genesis mechanism also to draw similar conclusion to hepatic fibrosis.But from the angle of clinical application, above method all has preparation method's complexity, needs repeated multiple times administration, uses the outstanding shortcomings such as inconvenient, and application prospect is not good enough.Seeking the more convenient means of effectively blocking TGF β 1 is one of present problem demanding prompt solutions.
Summary of the invention
The problem that the present invention solves is to provide synthetic peptide vaccine and the application thereof based on the B cell antigen epi-position of TGF-β 1, by inducing body to produce the antibody of anti-TGF-beta 1, with in and the promoter action of TGF-β 1 in liver fibrosis process, reach the purpose that suppresses the hepatic fibrosis process.
The present invention is achieved through the following technical solutions:
The B cell antigen epi-position of a kind of TGF-β 1 comprises:
The AY25 epi-position of amino acid residue sequence as shown in SEQ.ID.NO.1;
The LS30 epi-position of amino acid residue sequence as shown in SEQ.ID.NO.2.
A kind of synthetic peptide vaccine comprises the B cell antigen epi-position of TGF-β 1, and it is coupled on carrier proteins;
Shown epitope is the AY25 epi-position as shown in SEQ.ID.NO.1, or the LS30 epi-position as shown in SEQ.ID.NO.2.
Described carrier proteins is BSA or KLH.
The application of described synthetic peptide vaccine in the antibody of preparation anti-TGF-beta 1.
The application of described synthetic peptide vaccine in the medicine of preparation anti-hepatic fibrosis.
Described medicine is the medicine of inhibition or anti-TGF-beta 1B activity short of money.
Described medicine is for suppressing the medicine of hepatocellular apoptosis.
Compared with prior art, the present invention has following useful technique effect:
The B cell antigen epi-position of TGF-β 1 provided by the invention adopts synthetic its peptide sequence of chemical process, preparation coupling peptide vaccine, and by the method immune mouse of abdominal injection, the inducing mouse body produces corresponding antibody, and passes through CCl
4Build Liver Fibrosis Model, anti-TGF-beta 1 antibody that produces with observation post can in and TGF-β 1 to reach the effect of anti-hepatic fibrosis.
Result shows, the epi-position AY-25 that screens and LS-30 all have antigenicity, detect with ELISA and Western blot method, for control group and normal group, experimental mice internal antibody titre can reach 1:5120 after immune four times, sustainable 4-6 week of this antibody titers, then decay gradually, after 6 months, antibody titers decays to 1:640-1:1280.Can be with commercialization, the restructuring TGF-β 1B specific reaction of biologic activity is arranged; Can be combined with the TGF-of prokaryotic expression β 1 purifying protein; And the antibody that produces shows reactivity and biologic activity preferably.
TGF-β 1 synthetic peptide vaccine provided by the invention, immune mouse, inducing mouse produce anti-TGF-beta 1 antibody, in and at CCL
4The cytokine TGF-β 1 of overexpression in the liver fibrosis process of inducing suppresses its promoter action in the hepatic fibrosis evolution, thereby suppresses to a certain extent the development of hepatic fibrosis:
After immunity, at CCL
4In the liver fibrosis process of inducing, the hepatic tissue structure is more complete, and in liver, deposition of fibrous tissue is few, and the degree of hepatic fibrosis classification significantly reduces; The expression of α-SMA and a matter district PCNA obviously reduces; Between matter district PCNA index obviously reduce; Hepatic tissue Hyp content all significantly reduces.Confirm significantly to suppress CCl after TGF-β 1 synthetic peptide vaccine immune mouse
4The hepatic fibrosis in mice of inducing.Show that TGF-β 1 vaccine is expected to become a kind of simple, safe and effective hepatic fibrosis and prevents and treats novel method
Description of drawings
The immune serum antibody titers is respectively organized for ELISA detects in Fig. 1-1.
Fig. 1-2 is that Western blot detects the reactivity of respectively organizing mice serum antibody.
The biologic activity of respectively organizing mice serum antibody is detected for the experiment of MV1LU Growth of Cells in Fig. 1-3.
Fig. 2 is each group murine liver tissue Masson coloration result.
Fig. 3 is each group murine liver tissue hydroxyproline content result.
Fig. 4-1 is each group murine liver tissue α-SMA coloration result.
Fig. 4-2 are each group murine liver tissue α-SMA positive region statistic analysis result.
Fig. 4-3 respectively organize murine liver tissue pSmad2/3 for Western blot detects and Smad2/3 expresses.
Fig. 5-1 respectively organizes HSC-T6 cell pSmad2/3, Smad2/3 for Western blot detects and α-SMA expresses.
Express for the real-time fluorescence quantitative PCR method detects COL1A2mRNA Fig. 5-2.
Express for the real-time fluorescence quantitative PCR method detects PAI-1 Fig. 5-3.
Express for the real-time fluorescence quantitative PCR method detects TIMP-1 Fig. 5-4.
Fig. 6-1 is each group murine liver tissue TUNEL coloration result.
Fig. 6-2 are each group mouse liver cell apoptotic index statistic analysis result.
Fig. 7-1 is each group murine liver tissue PCNA coloration result.
Fig. 7-2 are each group mouse liver cell PCNA index statistic analysis result.
Fig. 8-1 is each group murine liver tissue DESMIN coloration result.
Fig. 8-2 are each group murine liver tissue DESMIN stained positive range statistics analytical results.
Embodiment
The invention provides synthetic peptide vaccine and application thereof based on the B cell antigen epi-position of TGF-β 1, screening has antigenic epitope peptide and synthetic TGF-β 1 peptide vaccine of coupling carrier albumen, adopt the method immune mouse of abdominal injection, repeat immunity and detect mice serum antibody titers, CCl
4The inducing mouse Liver Fibrosis Model.HE, Masson dyeing is observed level of fibrosis and is learned change.Immunohistochemical staining detects in hepatic tissue.Alkali hydrolysis method detects respectively organizes the hepatic tissue hydroxyproline content, observes the murine liver tissue fibroplasia situation of respectively organizing, relatively the difference of degree of hepatic fibrosis between vaccine immunity group and modeling group, KLH group.The present invention is described in further detail below in conjunction with specific embodiment, and the explanation of the invention is not limited.
1, the screening of the B cell antigen epi-position of TGF-β 1 and synthetic
At first adopt the bioinformatics method analysis, specifically by the B cell antigen epi-position of online webserver TGF-β 1 albumen and the physicochemical property of TGF-β 1 albumen, screening has immunogenic epi-position, has specifically screened two epi-position AY25 and LS30:
Shown in TGF-β 125:41ANFCLGPCPYIWSLDTQYSKVLALY65(SEQ.ID.NO.1);
Shown in TGF-β 1-30:83LEPLPIVYYVGRKPKVEQLSNMIVRSCKCS112(SEQ.ID.NO.2);
And the epi-position of screening be also T and mouse TGF-β 1 molecule reply fragment height homology, and be the position that plays a crucial role in TGF-β 1 its receptor corresponding with it.
Then adopt automatic Peptide synthesizer to synthesize corresponding peptide sequence, synthetic polypeptide is analyzed through high performance liquid chromatography (high performance liquid chromatography, HPLP), and purity all can reach 99.00%.
2, the preparation of coupling peptide
Epitope peptide is coupled on carrier proteins, because carrier proteins has immunogenicity, is conducive to the body generation for the antibody of epitope peptide.
The operation steps of the coupling of KLH/BSA-AY25 and KLH/BSA-LS30:
(1) take: AY255mg, LS305mg, EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) 20mg, KLH(Yao Kong Qi hemocyanin) 4mg, the BSA(ovalbumin) 4mg(please provides respectively Chinese), be loaded on respectively in corresponding bottle and mark.
(2) dissolving: add respectively 400ul0.1M MES damping fluid (laboratory preparation) in KLH and BSA, visible dissolving rapidly.
(3) dissolving: add respectively approximately 1.2ml0.1M MES damping fluid in AY25 and LS30, fully vibration impels its dissolving, as seen its incomplete dissolving; Add wherein 100ul dinethylformamide (organic solvent) short molten, as seen its rapid dissolving; Add wherein more a small amount of hydrochloric acid to regulate its pH value, it is fully dissolved.
(4) mix: AY25 and LS30 are divided into respectively two equal portions (approximately 600ul/ part), add respectively in KLH solution and BSA solution, be labeled as KLH-AY25, BSA-AY25, KLH-LS30 and BSA-LS30.
(5) coupling: add the 2ml deionized water dissolving in 20mg EDC, respectively get immediately 100ul and add respectively in KLH-AY25, BSA-AY25, KLH-LS30 and BSA-LS30, after mixing, room temperature is standing 2 hours.
(6) dialysis: liquid in each bottle is put in the PBS damping fluid of use 0.01M in dialysis tubing in 4 ℃ of dialysis 24 hours, changed liquid 1 time in every 8 hours, totally 3 times, visible dialysis tubing is bulging gradually.
(7) packing: dialyzate is pressed the packing of 0.5ml/ pipe, each 8 pipe.Frozen standby in-20 ℃ of refrigerators.
3, immune mouse
Male ICR mouse is divided into normal group, CCl at random
4Group, KLH group, KLH-AY25 group and KLH-LS30 group, wherein normal group is 6, CCl
4Each 8 of group and KLH groups, each 10 of KLH-AY25 group and KLH-LS30 groups.
The immunity flow process:
1) the first week: OVA-VF16 and KLH-QK16 are respectively got 2 (1ml), mix with isopyknic Freund's complete adjuvant respectively, give the mouse peritoneal injection after fully emulsified, 0.2ml/ only.
2) second week: OVA-VF16 and KLH-QK16 are respectively got 1 (0.5ml), with mixing with isopyknic Freund's incomplete adjuvant respectively after equivalent PBS dilution, give the mouse peritoneal injection, 0.2ml/ after fully emulsified.
3) the 3rd week: same second week.
4) 4th week: OVA-VF16 and KLH-QK16 are respectively got 1 (0.5ml), and with giving the mouse peritoneal injection after equivalent PBS dilution mixing, 0.1ml/ only.
5) the 5th week: same 4th week.
6) the 8th week: same second week.
7) the tenth week: same second week.
4, indirect ELISA detects
Blank when collecting mouse mouse tail serum for the ELISA detection before immune mouse, frozen standby in-20 ℃ of refrigerators.Carry out ELISA detection serum antibody titer respectively at adopting mouse tail blood in the 4th week, the 6th week, the 8th week after immunity, put to death mouse after experiment finishes, collection serum carries out indirect ELISA and detects.
Operation steps:
1) antigen coated: the carbonate buffer solution of 0.05M PH9.6 dilution coupling peptide OVA-AY25, not coupling of OVA-LS30(OVA) (50ng/ hole) and commercial TGF-β 1 recombinant cytokine (20ng/ hole) that biologic activity the arranged different hole of coated polystyrene board respectively.Add 0.1ml in the reacting hole of each polystyrene board, 4 ℃ are spent the night.Discard solution in the hole next day, adds wherein 300ul/ hole 10% bovine serum/PBS sealing, and 4 ℃ are spent the night.The 3rd day, discard solution in the hole, pat dry, frozen standby in-20 ℃ of refrigerators.(washing 1 time with washing lotion with front need).
2) add primary antibodie: add with 10% bovine serum/PBS liquid and (do simultaneously blank hole and negative control hole) by each experimental mice serum 0.1ml of gradient dilution proportion in above-mentioned coated reacting hole, put 37 ℃ and hatched 1 hour.Pat dry after washing 4 times.
3) add ELIAS secondary antibody: add the goat anti-mouse IgG 0.1ml (pressing 1:5000 dilution with 10% bovine serum/PBS liquid) of the HRP mark of fresh dilution in each reacting hole, hatched 1 hour for 37 ℃, pat dry after washing 4 times.
4) add substrate solution and nitrite ion: add each 1 of substrate solution and nitrite ion in each reacting hole, 37 ℃ of 10min.
5) termination reaction: add 1 of stop buffer in each reacting hole.
6) result is judged: a, direct observations on white background with the naked eye: reacting hole in, color is darker, and positive degree is stronger, and negative reaction is colourless or extremely shallow, according to the depth of be color, number represents with "+", "-".B, microplate reader are measured: measure its OD value in the 450nm wavelength, and positive to treat gaging hole OD value 〉=2.1 times of negative control hole OD values.
Through the equal test positive in corresponding antigens coated hole of the mice serum after OVA-AY25, OVA-LS30 epitope peptide vaccine immunity, and negative in KLH, PBS coated hole, the specific antibody that has produced after epi-position peptide vaccine immune mouse for corresponding epitope peptide is described.
ELISA detect respectively organize the immune serum antibody titers detected result as Figure 1-1, experimental mice internal antibody titre can reach 1:5120 after immune four times, sustainable 4-6 week of this antibody titers, then decay gradually, after 6 months, antibody titers decays to 1:640-1:1280.And can be with commercialization, have the restructuring TGF-β 1 of biologic activity that specific reaction occurs.
5, Western blot detects
1) preparation 12%Tris-glycine SDS-polyacrylamide gel:
(1) preparation separation gel 5m1:H
2O1.6ml, 30% acrylamide 2.0ml, 1.5M Tris (PH8.8) 1.3ml, 10%SDS50ul, 10% Ammonium Persulfate 98.5 50ul, TEMED2ul adds in order successively, ordinary method encapsulating after mixing, after 30min, gelling is solid.
(2) preparation spacer gel 2ml:H2O1.4ml, 30% acrylamide 330ul, 1.0M Tris (PH6.8) 20ul, 10%SDS20ul, 10% Ammonium Persulfate 98.5 20ul, TEMED2ul adds in order successively, ordinary method encapsulating after mixing, insert comb, 30min after coagulation, but loading.
2) sample preparation: 6his-TGF-β 1 chain gene Prokaryotic expression, purification protein sample is mixed with isopyknic sample-loading buffer (6 * loading buffer:DTT is 9:1), boil 5min in metal bath, ice bath 5min littlely namely can be used for loading after centrifugal.
3) loading: electrophoresis apparatus is installed, is poured wherein into 1 * SDS-glycine electrophoretic buffer, press 3ul/ hole sample size loading, 2 * loading buffer loading of blank well use equivalent.
4) protein electrophoresis: connect electrophoresis apparatus, voltage 80V, approximately stop electrophoresis after 120min, take off gel, carries out transferring film.
5) transferring film
(1) take out gel, first gel is put into transferring film damping fluid balance 30min, carry out according to a conventional method transferring film.
(2) be followed successively by 3MM Whatman filter paper (3 layers), gel (connecing negative pole), nylon membrane (NC film) (connecing positive pole), 3MM Whatman filter paper (3 layers) from bottom to upper strata.
(3) constant current transferring film, 1mA/cm2,30min.
(4) take out the NC film, sealed 1 hour in room temperature with the TBST liquid that contains 5% skim-milk.
6) dye film
(1) after washing once with TBST liquid the NC film, add with the mice serum (primary antibodie) of the TBST liquid that contains 5% skim-milk by the 1:1000 dilution, spend the night in 4 ℃ after incubated at room 1h.
(2) wash film 4 times with TBST liquid, each 5-10min adds with the goat anti-mouse IgG of the TBST liquid that contains 5% skim-milk by the HRP mark of 1:2000 dilution, incubated at room 1h.
(3) wash film 4 times with TBST liquid, each 5-10min adds moulding sheet exposure after ECL colour developing lmin, observations and scan image in the darkroom.
Western Blot detected result is as shown in Fig. 1-2, from left to right be respectively No. KLH-AY25-1, No. KLH-AY25-2, No. KLH-AY25-3, No. KLH-LS30-1, No. KLH-LS30-2, No. KLH-LS30-3, KLH-1 mice serum antibody and PBS, antibody dilution is 1:800.Result shows: the KLH that compares group, the TGF-beta 1 antibodies in the experimental mice immune serum can be combined with the TGF-of prokaryotic expression β 1 purifying protein, shows reactivity and specificity preferably.
6, detect the TGF-beta 1 antibodies to the impact of MV1LU cell proliferation
(this is the antibody neutralization test, because TGF-β 1 can promote cell proliferation, add after the TGF-beta 1 antibodies can anti-TGF-beta 1 short of money proliferation, for detection of antibodies specific, reactivity and antibody biologic activity)
Taking the logarithm goes down to posterity vegetative period cultivates the MV1LU cell, and after tryptic digestion, counting cells also is adjusted into 1 * 10 with the DMEM serum-free medium with cell concn
3/ ml adds 96 aseptic porocyte culture plates with every hole 200 μ l volumes with cell suspension, is placed in 37 ℃, 5%CO
2Cell culture incubator in cultivated 48 hours.
Discard nutrient solution, cultivated 30 minutes with containing the sero-fast DMEM nutrient solution of different Dilution ratios.
The TGF-Β 1-BB and the BrdU that add 3ng/ml in each hole continue to cultivate 48 hours.
Discard nutrient solution, wash with PBS, add with anti-BrdU Ad and ELISA detection Growth of Cells situation in each hole.Above experiment repeats 2 times.
Experiment grouping situation is: blank group, KLH group, KLH-AY25 group and KLH-LS30 group.Establish 3 multiple holes for every group.Take mice serum to repeat above-mentioned experimentation before the beginning immune animal and when 2w, 4w, 6w, 8w after immunity, 10w, 12w, 14w, 16w, 18w, 20w, 22w, 24w respectively.
Each is organized MV1LU Growth of Cells experimental result and sees Fig. 1-3.With KLH group and CCL
4Group is compared, and KLH-AY25 group and KLH-LS30 group all can obviously suppress Mv1Lu cell proliferation, significant difference (P＜0.05); And the extent of dilution of 1:250 can suppress 50% Mv1Lu cell proliferation.
Above-mentioned detected result shows, two epitope peptide AY25 that synthesized and LS30, after vaccine is made in coupling, the antibody that afterwards all can be in the middle of body all can inducing mouse produces high titre in immunity, and the antibody that produces shows reactivity and specificity preferably, and all can suppress the growth of MV1LU cell, show the activity of anti-TGF-beta 1.
7, CCl
4The foundation of inducing mouse Liver Fibrosis Model
In detecting KLH-AY25 group and KLH-LS30 group mice serum antibody titers the 8th week of immunity to reaching 1:5120, begin to use CCl
4The inducing mouse Liver Fibrosis Model.CCl
4Be mixed with by volume 20% mixed solution with peanut oil, every modeling group mouse is pressed 5ml/kg abdominal injection CCl
4/ peanut oil mixed solution, 1 week 2 times, continuous 6 weeks.
8, laboratory animal is got blood and sample
1) after experiment finished, eyeball was got blood system and is not put into the good corresponding clean EP pipe of mark, placed approximately 30min under room temperature, observed blood coagulation in the EP pipe, when as seen having serum to separate out, and centrifugal 5min, 3000rpm/min.
2) supernatant after centrifugal is drawn in the EP pipe of respective markers, recentrifuge 5min,, 5000rpm/min.
3) supernatant after centrifugal is drawn in corresponding EP pipe ,-20 ℃ save backup.
4) get and respectively organize mouse liver, right lobe of liver is placed in 10% formalin fixing; Lobe of the liver corresponding to residue liver collected respectively, puts into-70 ℃ and saves backup.
Visual inspection: normal group mouse liver smooth surface, ruddy, matter is soft.OVA-VF16 group and KLH-QK16 group mouse liver color depth, the surface is more smooth without adhesion, and volume has no obviously and dwindles.KLH group and CCl
4Group mouse liver gross specimen shows that liver loses normal ruddy gloss, is brown color, and there are many white particles on the surface, and quality is more tough, and is not easily broken.
9, paraffin section and HE dyeing
Carry out paraffin section and HE dyeing according to conventional treatment method, can entrust laboratory or company to carry out, specifically make section and dyeing by Xi'an Communications University's Pathological Staff Room.
Each organizes mouse HE coloration result: in the section of normal group murine liver tissue, the liver lobule form shows no obvious abnormalities, without the infiltration of hepatic necrosis and inflammatory cell.OVA-VF16 group and the most of form of KLH-QK16 group liver cell are normal, small part hepatocellular degeneration, necrosis, cell infiltration.Observe the liver lobule normal configuration under KLH group and CCl4 arrangement of mirrors and disappear, pseudolobuli forms, and visible significantly liver cell cavity sample sex change is even downright bad, cell infiltration.
10, Masson dyeing
1) be that the hepatic tissue paraffin section of 6um is placed in 37 ℃ of incubators and toasts 1h with thickness.
2) dewaxing: order is: dimethylbenzene 2min → dimethylbenzene 2min → dimethylbenzene 2min → dehydrated alcohol 2min → dehydrated alcohol 2min → 95% alcohol 2min → 85% alcohol 2min → tap water is washed till few 3min → distillation washing 1min.
3) section is put in the masson dye liquor approximately 5min, the distillation washing is 5s approximately.
4) section is put in 1% phospho-molybdic acid approximately 3min, the distillation washing is 5s approximately.
5) section is put in 2% BG approximately 6min, the distillation washing is 5s approximately.
6) section is put in 0.2% glacial acetic acid approximately 5s, the distillation washing is 5s approximately.
7) dehydration: order is: 85% alcohol 5s → 95% alcohol 5s → 95% alcohol 5s → dehydrated alcohol 5s → dehydrated alcohol 5s → propyl carbinol 5s → dimethylbenzene 5s → dimethylbenzene 5s.
8) neutral gum is fixed, conventional mounting.
9) microscopically is observed coloration result: under opticmicroscope, visible endochylema, myofiber are red, and collegen filament, mucus are green.The liver cirrhosis pathology grade scale is as follows:
(1) 0 grade is that hepatic tissue is normal, collagen-free fibroplasia;
(2) the I level is that collegen filament slightly stretch out from the portal area or around central vein;
(3) the II level is that collegen filament extend obviously, but not yet connects, and holds whole liver lobule;
(4) the III level is that collegen filament extend obviously, and is interconnection, holds whole liver lobule;
(5) the IV level is that collegen filament hold and cut apart liver lobule, and the normal hepatocytes leaflet structure is destroyed, and pseudolobuli forms, but take the large square pseudolobuli as main;
(6) the V level is that the liver lobule structure is destroyed fully, and pseudolobuli forms the large square pseudolobuli and the small circular pseudolobuli respectively accounts for 50%;
(7) the VI level is to be covered with the small circular pseudolobuli in liver, and the collegen filament of thick hyperplasia are arranged in pseudolobuli.
Each organizes mouse Masson coloration result as shown in Figure 2, no abnormality seen fibroplasia after the dyeing of normal group murine liver tissue, and the portal area is without obvious proliferation of fibrous tissue.KLH-AY25 group and KLH-LS30 organize the portal area and see a small amount of proliferation of fibrous tissue, and liver lobule is cut apart not exclusively by the fibrous septum.KLH group and CCl
4The visible a large amount of proliferations of fibrous tissue of group, liver lobule are cut apart by the fibrous tissue of hyperplasia and are held into the liver cell group of differing in size, and form pseudolobuli, pseudolobuli endite central vein lack as, off normal or more than two.
Each is organized murine liver tissue and carries out pathological grading, with ANOVA one-way analysis of variance method, classification results is carried out statistical procedures, and result shows: 1. normal group mouse liver and other 4 experimental mice liver case classification othernesses remarkable (P＜0.05); 2. KLH-AY25 group, KLH-LS30 organize and KLH group, CCl
4Group is compared, and significant difference (P＜0.05) 3. KLH-AY25 group is compared with the KLH-LS30 group, KLH group and CCl
4Group is compared, there are no significant for difference (P〉0.05).
11, hydroxyproline determination
1) get 40 clean glass test tubees and mark.
2) accurately weigh hepatic tissue weight in wet base 60 ㎎ and put into the good glass test tube of mark, add the approximately hydrolyzed solution of 1ml, fully mixing.
3) test tube is put into 95 ℃ of constant water bath box water-bath 20min, every the 10min mixing once, made the more thorough of tissue hydrolysis.
4) after test tube is cooling, add 1 of indicator in each pipe, fully mixing.
5) add adjusting pH value first liquid 1ml in each pipe, abundant mixing, at this moment visible test tube, solution is red.
6) dropwise add the second liquid of transferring pH value in each test tube, need at once fully mixing liquid after often being added dropwise to, until in each test tube, liquid becomes yellow-green colour, every Guan Zhongyue adds 500 μ l to transfer the second liquid of pH values, and in this moment each pipe, the pH value of liquid is about the 6.0-6.8 left and right.
7) adding distilled water to final volume in each pipe is 10ml, fully mixing.
8) get 40 5ml centrifuge tubes that mark is good, get the diluent of 3ml and put into respectively centrifuge tube, weigh 25 ㎎ gacs and pour in each centrifuge tube, fully mixing, centrifugal, 3500rpm/min, 10min.
9) get supernatant liquor 1ml and do testing sample.
10) according to the form below operation:
Table 3-1 oxyproline operation table
11) with liquid blending in above-mentioned each pipe, water-bath 15min in 60 ℃ of constant water bath box, cooling rear centrifugal, 3500rpm/min, 10min.
12) open spectrophotometer, get in each centrifuge tube supernatant liquor at the 550nm place, the 1cm optical path after the distilled water zeroing, adds liquid in each pipe in cuvette successively, measures and respectively manages light absorption value, and light absorption value respectively managed in record.
13) calculate as follows every mouse liver hydroxyproline content:
Each organizes in murine liver tissue hydroxyproline content as shown in Figure 3, KLH group and CCl
4Organize Hyp content apparently higher than normal group, significant difference (P＜0.05); But between these two groups, relatively, difference is not remarkable; KLH-AY25 group and KLH-LS30 group hydroxyproline content and KLH group and CCl
4Group is compared, significant difference (P＜0.05); Between KLH-AY25 group and KLH-LS30 group, relatively, difference is not remarkable.The KLH-AY25 group is compared with normal group with KLH-LS30 group hydroxyproline content, and difference is not remarkable.
12, immunohistochemical staining detects the expression of α-SMA in liver
1) the hepatic tissue paraffin section is placed in 37 ℃ of incubators and toasts 1h.
2) dimethylbenzene is put in section and soaked 60min, change dimethylbenzene and soak 10min, again change dimethylbenzene and soak 5min.
3) dehydrated alcohol is put in section and soaked 5min, change soaked in absolute ethyl alcohol 5min.
4) section is put into 95% alcohol-pickled 5min.
5) section is put into 75% alcohol and soak 5min.
6) section is put into approximately 30min of 3% hydrogen peroxide incubated at room, distilled water flushing.
7) sealing: with 10% bovine serum/PBS sealing 1 hour, discard this confining liquid; Again sealed 1 hour with lowlenthal serum with the sealing in the SP detection kit again, discard this serum.
8) add primary antibodie: dilute primary antibodie with 10% bovine serum/PBS, wherein anti-α-SMA Dilution ratio is 1:800, and anti-PCNA Dilution ratio is 1:400, and anti-Desmin Dilution ratio is 1:400, and wet box is put in section, and room temperature is placed after 1 hour and spent the night in 4 ℃.
9) take out section and at room temperature hatch 45min, rinse 5min/ time, totally 3 times with PBST.
10) adding two resists: drip biotin labeled two anti-working fluids in section, hatched 1 hour in 37 ℃ of constant water bath box.
11) rinse section with PBST, each 5min washes 3 times altogether.
12) drip the chain enzyme avidin working fluid of horseradish peroxidase-labeled in section, hatched 1 hour in 37 ℃ of constant water bath box.
13) DAB dyeing: get the 1ml deionized water in 1.5ml EP pipe, add successively wherein A liquid, B liquid, each 1 of C liquid (often adding 1 abundant mixing afterwards), be used for section statining after mixing.Dyeing 5-10min, microscopically is controlled dye levels.
14) tap water fully rinses section.
15) the phenodin dye liquor is redyed 2-10min, the hydrochloride alcohol differentiation.
16) tap water fully rinses section.
17) will cut into slices and put into successively 75% alcohol, 95% alcohol, dehydrated alcohol and dewater, each 5min.
18) taking out section, to put into dimethylbenzene transparent.
19) neutral gum is fixed, conventional mounting.
20) optical microphotograph Microscopic observation section.
Each organizes murine liver tissue α-SMA coloration result as shown in Fig. 4-1, has no obvious α-SMA positive cell in the normal group mouse liver; KLH group and CCl
4The group murine liver tissue has obvious brown yellow granule spaced apart along fibrosis around visible portal area, and painted darker; In KLH-AY25 group and KLH-LS30 group murine liver tissue, visible positive cell distributes on every side in the portal area, but painted more shallow, and positive cell number is starkly lower than CCl
4Group.
Each is organized murine liver tissue α-SMA stained positive zone employing area of computer aided semi-quantitative analysis system statistical analysis and the results are shown in Figure 4-2.With KLH group and CCl
4Group is compared, and α-SMA stained positive region area is less for KLH-AY25 group and KLH-LS30 group murine liver tissue, and its difference has significance (P＜0.001).
Western-blot detects and respectively organizes murine liver tissue pSmad2/3 and Smad2/3 expression.
The phosphorylation of Smad2/3 is one of sign of activating of TGF-signal β Signal Transduction Pathways, and each organizes murine liver tissue pSmad2/3 and Smad2/3 expression as shown in Fig. 4-3.Repeat to give CCL
4Abdominal injection can be induced the phosphorylation of Smad2/3, activates TGF-signal β Signal Transduction Pathways, and gives the phosphorylation that KLH-AY25 and KLH-LS30 can suppress Smad2/3 in Fibrotic murine liver tissue.
13, cell in vitro is learned the biologic activity that experiment detects the specific antibody of TGF-β 1 synthetic peptide vaccine inducing mouse generation.
As shown in Fig. 5-1,5-2,5-3,5-4, the HSC-T6 cell Smad2/3 phosphorylation of TGF-β 1 being induced by Western-blot detection specificity antibody and the impact of α-SMA, the impact that HSC-T6 cell COL1A2, the PAI-1 that TGF-β 1 is induced by real-time fluorescence quantitative PCR method detection specificity antibody and TIMP-1mRNA express.
Result as seen, 6ng/ml TGF-β 1 can significantly induce HSC-T6 cell Smad2/3 phosphorylation, promote the expression of α-SMA, COL1A2, PAI-1 and TIMP-1, give TGF-β 1 synthetic peptide specific antibody (1:200 dilution) and can obviously suppress the Smad2/3 phosphorylation of HSC-T6 cell and the expression of α-SMA, COL1A2, PAI-1 and TIMP-1.
The above results shows, by in and TGF-β 1 active and then the activation that suppresses hepatic stellate cell to CCL
4The hepatic fibrosis of inducing has obvious restraining effect.
14, TGF-β 1 synthetic peptide vaccine is to CCL
4The impact of the hepatic fibrosis mouse liver cell apoptosis of inducing.
Each organizes mouse TUNEL coloration result as shown in Fig. 6-1 and 6-2.Apoptotic index represents with the per-cent that the TUNEL positive cell accounts for the total cell number of counting.As seen repeat to give CCL
4Abdominal injection can obviously increase the hepatocellular quantity of mouse apoptosis, gives TGF-β 1 synthetic peptide vaccine and can significantly reduce CCL
4The hepatocellular ratio of apoptosis in the mouse liver fibrosis of inducing.The above results shows that cross expressing of TGF-β 1 can increase hepatocellular apoptosis in Fibrotic mouse liver, and in and the activity of TGF-β 1 can suppress its apoptosis.
15, TGF-β 1 synthetic peptide vaccine is to CCL
4The impact of the hepatic fibrosis mouse liver cell propagation of inducing.
PCNA and cell DNA compositive relation are close, play an important role in the startup of cell proliferation, are the good index of reflection proliferative activity; Positive painted mainly being distributed in karyon of PCNA, it is painted that endochylema has no, and the positive cell signal of brown occurs with karyon.
Each is organized mouse PCNA coloration result and sees Fig. 7-1: the section of normal group murine liver tissue has no obvious PCNA positive cell; Repeat to give CCL
4Abdominal injection can obviously increase the PCNA positive cell number, and KLH-AY25 group and KLH-LS30 group are than KLH group and CCl
4Group is compared, and the increase of PCNA positive cell number is more remarkable, but prompting is by giving in TGF-β 1 synthetic peptide vaccine and the propagation of too much TGF-β 1 accelerating fibers mouse liver cell.Every PCNA stained random 5 visuals field of solid area and visual field, 5, district of matter selected under 40 times of object lens, count respectively PCNA positive cell and total cell under each visual field, through one-way analysis of variance, PCNA index and KLH group and the CCl of KLH-AY25 group and KLH-LS30 group mouse liver cell
4Group PCNA index differential has significance (P＜0.001), as shown in Fig. 7-2.
16, respectively organize murine liver tissue Desmin dyeing.
Each is organized murine liver tissue Desmin coloration result and sees Fig. 8-1.Have no obvious Desmin positive cell in the normal group mouse liver; KLH group and CCl
4Organize the interior visible positive cell of murine liver tissue more and painted darker, KLH-AY25 organizes and the interior positive cell of KLH-LS30 group mouse liver is less and dyed particles is painted more shallow.Each is organized the system statistical analysis of murine liver tissue Desmin stained positive zone employing area of computer aided semi-quantitative analysis and the results are shown in Figure 8-2.With KLH group and CCl
4Group is compared, and KLH-AY25 group and KLH-LS30 group murine liver tissue Desmin stained positive region area are less, and difference has significance (P＜0.001).
This shows, in murine liver tissue, hydroxyproline content is consistent with HE dyeing, Masson dyeing and immunohistochemical staining result.Produce on the basis of antibody at AY25 and LS30 epitope peptide vaccine, experimentation on animals confirms can significantly suppress the hepatic fibrosis in mice that CCl4 induces after TGF-β 1 synthetic peptide vaccine immune mouse.
To sum up result shows, screen and have antigenic peptide sequence AY25 and LS30 in TGF-β 1 molecule, after adopting chemical process synthetic with the coupling of carrier proteins phase after immune mouse, show that with ELISA and Western blot detection method the equal energy of 2 the 16 peptides inducing mouse that screens produces the antibody of high titre, and the antibody that produces shows reactivity and specificity preferably.Further, can significantly suppress CCl after QK16 and VF16 synthetic peptide vaccine immune mouse
4The hepatic fibrosis in mice of inducing.
Confirmation can produce specific antibody by inducing mouse through the TGF-of abdominal injection AY25 and LS30 β 1 synthetic peptide vaccine, can significantly suppress CCl
4The mouse experiment hepatic fibrosis of inducing.Show that TGF-β 1 vaccine is expected to become a kind of simple, safe and effective hepatic fibrosis and prevents and treats novel method.
Claims (7)
1. the B cell antigen epi-position of a TGF-β 1, is characterized in that, comprising:
The AY25 epi-position of amino acid residue sequence as shown in SEQ.ID.NO.1;
The LS30 epi-position of amino acid residue sequence as shown in SEQ.ID.NO.2.
2. a synthetic peptide vaccine, is characterized in that, comprises the B cell antigen epi-position of TGF-β 1, and it is coupled on carrier proteins;
Shown epitope is the AY25 epi-position as shown in SEQ.ID.NO.1, or the LS30 epi-position as shown in SEQ.ID.NO.2.
3. synthetic peptide vaccine as claimed in claim 2, is characterized in that, described carrier proteins is BSA or KLH.
4. the application of the described synthetic peptide vaccine of claim 2 or 3 in the antibody of preparation anti-TGF-beta 1.
5. the application of the described synthetic peptide vaccine of claim 2 or 3 in the medicine of preparation anti-hepatic fibrosis.
6. application as claimed in claim 5, is characterized in that, described medicine is the medicine of inhibition or anti-TGF-beta 1B activity short of money.
7. application as claimed in claim 5, is characterized in that, described medicine is for suppressing the medicine of hepatocellular apoptosis.
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| WO2020245264A1 (en) * | 2019-06-05 | 2020-12-10 | Io Biotech Aps | Tgf-beta vaccine |
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| CN108404125A (en) * | 2018-03-15 | 2018-08-17 | 西安交通大学医学院第附属医院 | Interleukin-1 beta recombinant vaccine for treating pulmonary fibrosis and its application |
| CN108404125B (en) * | 2018-03-15 | 2019-11-12 | 西安交通大学医学院第一附属医院 | For treating interleukin-1 beta recombinant vaccine and its application of pulmonary fibrosis |
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| CN111303271A (en) * | 2020-02-19 | 2020-06-19 | 西安交通大学医学院第一附属医院 | Platelet-derived growth factor recombinant vaccine for treating pulmonary fibrosis and application thereof |
| CN111303271B (en) * | 2020-02-19 | 2022-02-01 | 西安交通大学医学院第一附属医院 | Platelet-derived growth factor recombinant vaccine for treating pulmonary fibrosis and application thereof |
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