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CN109342722A - It is a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement - Google Patents

It is a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement Download PDF

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CN109342722A
CN109342722A CN201811158165.6A CN201811158165A CN109342722A CN 109342722 A CN109342722 A CN 109342722A CN 201811158165 A CN201811158165 A CN 201811158165A CN 109342722 A CN109342722 A CN 109342722A
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gly
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reagent
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侯志波
张伟
温建超
翁琪墁
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Shenzhen Hongmei Diagnostic Technology Co Ltd
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Shenzhen Hongmei Diagnostic Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

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Abstract

The invention discloses a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, include the steps that preparing R2 reagent: S1, extracting Heliobacter pylori antigen (urease) albumen, urease gene is cloned into pGEX-4T-1 plasmid, it is expressed by Escherichia coli fermentation, obtains recombinant helicobacterpylori antigen (urease) by affinitive layer purification；S2, anti-helicobacter pylori antigen (urease) polyclonal antibody is prepared；The latex particle of S3, preparation coating anti-helicobacter pylori antigen (urease) polyclonal antibody；S4, it is scattered in buffer solution and obtains R2 reagent.This method is Immunoturbidimetry, and reagent obtained is a kind of solution that helicobacter pylori can be directly detected by excrement, it is only necessary to which detection can be completed in fecal specimens, sampling is convenient, and to human body without security risk, detection efficiency is high, stability is good, high specificity is not easy to be disturbed.

Description

It is a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement
Technical field
The invention belongs to medical detection method technical field, it is anti-to be related to helicobacter pylori in a kind of quantitative determination excrement It is anti-to relate in particular to helicobacter pylori in a kind of latex immunoturbidimetry quantitative determination excrement for the method for former (urease) The preparation method of former reagent.
Background technique
A large number of studies show that helicobacter pylori (Helicobacter pylori, Hp) infection is chronic gastritis, digestibility The Important cause of disease of ulcer, and it is closely related with gastric cancer, mucosa-associated Lymphoid Tissue Lymphoma.Currently, clinically having more Whether kind method evaluation Hp infection, it is broadly divided into two major classes: invasive and Noninvasive detection method.
Wherein, invasive detection method is detected after obtaining gastric biopsy using endoscope, generally comprises urase Test method(s), pathology detection method and bacterial cultivation.Quick urease test method has abundant decomposable according to helicobacter pylori The urease of urea, urease can decompose urea and generate ammonia, measure helicobacter pylori by the variation of pH, this method is easy Rapidly, but there is a problem of that accuracy is insufficient, may because other bacteriums containing urease there are due to generate false positive or inspection Surveying to try out in the recent period reduces stomach amount of bacteria or directly the sample of the drug of urease activity is inhibited to can produce false negative.Pathology Detection method can directly display helicobacter pylori thallus and confirm, but difference is sick by being sliced chromoscopy to mucosa tissue The otherness of neo-confucian observation has certain difficulty for the diagnosis of lipogastry sample.Bacterial cultivation is by taking gastric mucosa Tissue does heiicobacter pylori cultivation, but there are problems that culture bacterium is less, operating process is complicated, somewhat expensive.In addition, invading There is also endoscopes to invade the problem of leading to person's pain to be detected in person's body to be detected or being also easy to produce infection for entering property detection method.
Noninvasive detection method refers to the method for not taking biopsy specimen diagnosing helicobacter pylori sample to infect by gastroscope.This Class method includes Serum Antibody Detection, 13C/14C breath test etc..Existing method includes enzyme-linked exempts to Serum Antibody Detection at present Epidemic disease method, Western blot, colloidal gold method etc., but due to still resisting containing corresponding in human serum after elimination helicobacter pylori Body, therefore helicobacter pylori infections are determined by serum antibody there are specificity issues really whether to reflect stomach There are also helicobacter pylori presence.13C/14C breath test is as a kind of method for detecting helicobacter pylori infections, clinically extensively General application, but there are problems that radioactivity, while needing special equipment, thus the application at family and the center She Kang just by Considerable restraint.
The present invention is tested using stool sample, acquires Heliobacter pylori antigen by dedicated quantitative fecal sampler (urease) is measured the content of helicobacter pylori by Immunoturbidimetry, has convenient and efficient and sanitation and hygiene The characteristics of, it can be widely applied in family and the center She Kang.Immunoturbidimetry is one kind by arriving antibody coupling On the latex particle of certain partial size, one is formed with Ag-Ab-latex particle compound is formed after antigen-reactive in sample Fixed turbidity, measure the variation of turbidity therefore, it is determined that in sample antigen concentration method.Currently, helicobacter pylori is by the world Health organization is determined as the carcinogenic cause of disease of I class, therefore how to prepare a kind of reagent that helicobacter pylori can be quickly detected without invasive As current urgent problem to be solved.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in pass through Immunoturbidimetry, rapid quantitative detection Heliobacter pylori antigen (urease) in excrement.
In order to solve the above technical problems, the technical solution of the present invention is as follows:
The present invention provides a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement comprising system The step of standby R2 reagent:
S1, Heliobacter pylori antigen albumen is extracted, urease gene is cloned into plasmid, Escherichia coli fermentation table is passed through It reaches, obtains recombinant helicobacterpylori antigen；
S2, anti-helicobacter pylori antigen polyclonal antibody is prepared；
The latex particle of S3, preparation coating anti-helicobacter pylori antigen polyclonal antibody；
S4, by be coated with anti-helicobacter pylori antigen polyclonal antibody latex particle with containing 0.1%-10% electrolyte, The MES buffering of 0.1%-10% stabilizer, 0.1%-10% surfactant, 0.1%-5% preservative and 1-1000mmol/L Liquid mixing to get.
Preferably, the step S1 is specifically, selection urease is as Heliobacter pylori antigen, the urease includes UreA and UreB protein protomer is expressed and affine by UreA or UreB gene cloning into plasmid by Escherichia coli fermentation For chromatographic purifying to get Heliobacter pylori antigen, the plasmid includes but is not limited to pGEX-4T-1 plasmid, and the plasmid includes egg White label gene, the protein tag include but is not limited to GST albumen.
Preferably, the step S2 includes:
S21, Heliobacter pylori antigen equivalent Freund's complete adjuvant is emulsified；
S22, the antigen after emulsification is subjected to animal subcutaneous injection, is immunoreacted；
S23, it takes after immune serum carries out affinity chromatography processing up to anti-helicobacter pylori antigen polyclonal antibody.
Preferably, the step S3 includes the following steps:
S31, latex particle and buffer solution are mixed and added into surfactant, obtain latex particle solution, the cream The mass percent that glue particle accounts for the latex particle solution is 0.01-10%；
S32, it the anti-helicobacter pylori antigen polyclonal antibody is dissolved in buffer solution obtains antibody-solutions, it is described anti- The mass percent that Heliobacter pylori antigen polyclonal antibody accounts for antibody-solutions is 0.01-10%；
S33, after mixing the latex particle solution with the antibody-solutions, 1- (3- dimethylamino-propyl) -3- is added Ethyl-carbodiimide hydrochloride, 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride account for the matter of mixed solution Amount percentage is 0.01%-5%, is reacted 2-4 hours at room temperature, and the cream of coating anti-helicobacter pylori antigen polyclonal antibody is obtained Glue particle.
Preferably, further include the steps that prepare R1 reagent, the R1 reagent include 0.1%-10% electrolyte, The stabilizer of 0.1%-10%, the surfactant of 0.1%-10%, 0.1%-5% preservative, surplus be concentration 1- The MES buffer of 1000mmol/L.
Preferably, further including the steps that preparing calibration object: using recombinant helicobacterpylori antigen as mother liquor, with cow's serum egg White solution gradient dilutes the recombinant helicobacterpylori antigen to get the calibration object of various concentration, the bovine serum albumen solution Concentration be 1ng/ml-100g/l.
Preferably, the electrolyte in the R1 reagent, R2 reagent includes but is not limited to sodium chloride, the stabilizer Including but not limited to mannitol, the surfactant include but is not limited to Tween 80, and preservative includes but is not limited to Sodium azide.
Preferably, the partial size of the latex particle is 50-500nm.
It is carried out 4 times altogether preferably, being injected into row immune response under Animal Skin described in the step S22；The step In S23, through affinity chromatography, treated that polyclonal antibody is placed in TBS buffer in bag filter, the dialysed overnight at 4 DEG C, The TBS buffer solution includes the Tween-20 of the NaCl of Tris, 500mM containing 20mM, 0.05%, pH 7.4.
Preferably, the buffer solution that the step S32 is used buffers for the MES buffer solution or PBS of 50mM, pH 6.0 Solution.
The above technical solution of the present invention has the following advantages over the prior art:
The preparation method of the present invention for being used to quantitative determine Heliobacter pylori antigen in excrement (urease) reagent, packet The step of including preparation R2 reagent: Heliobacter pylori antigen (urease) albumen S1, is extracted, urease gene is cloned into pGEX- It in 4T-1 plasmid, is expressed by Escherichia coli fermentation, obtains recombinant helicobacterpylori antigen (urease)；S2, anti-pylorus is prepared Pylori antigen (urease) polyclonal antibody；S3, preparation are coated with anti-helicobacter pylori antigen (urease) polyclonal antibody Latex particle；S4, by be coated with anti-helicobacter pylori antigen (urease) polyclonal antibody latex particle and contain 0.1%- 10% electrolyte, 0.1%-10% stabilizer, 0.1%-10% surfactant, 0.1%-5% preservative and 1- The MES buffer of 1000mmol/L mixes to arrive R2 reagent.Reagent made from this method is that one kind can be examined directly by excrement The solution of helicobacter pylori is surveyed, the present invention is tested using stool sample, measures pylorus by Immunoturbidimetry The content of helicobacter has the characteristics that convenient and efficient and sanitation and hygiene, can be widely applied in family and the center She Kang, it is only necessary to Detection can be completed in fecal specimens, and sampling is convenient, and to human body without security risk, detection efficiency is high, stability is good, high specificity, It is not easy to be disturbed.
Detailed description of the invention
Fig. 1 is the schematic diagram of cloning site in the embodiment of the present invention；
Fig. 2 is absorbance test chart in the embodiment of the present invention；
Fig. 3 is the canonical plotting of antigen standard in the embodiment of the present invention.
Specific embodiment
In order to make the content of the present invention more clearly understood, below according to specific embodiments of the present invention to this hair It is bright to be described in further detail.
Embodiment 1
This implementation provides a kind of for quantitative determining the preparation method of the reagent of Heliobacter pylori antigen in excrement, the examination Agent includes R1 reagent and R2 reagent, wherein by percentage to the quality, the R1 reagent includes electrolyte, the 0.1- of 0.1-10% 10% stabilizer, the surfactant of 0.1-10%, 0.1-5% preservative, the MES that surplus is 1-100mmol/L buffers Liquid.
1, R2 reagent is prepared
The R2 reagent includes the latex particle of coating anti-helicobacter pylori antigen (urease) polyclonal antibody, electrolysis Matter, stabilizer, surfactant, preservative and buffer, the R2 reagent are prepared by following technique:
S1, select any protein protomer of urease as Heliobacter pylori antigen, urease is by two albumen Asias Base composition, is UreA and UreB respectively, by UreA or UreB gene cloning into pGEX-4T-1 plasmid, cloning site is BamHI and EcoRI (as shown in Figure 1) in pGEX-4T-1 plasmid, the pGEX-4T-1 plasmid contain GST (Glutathione-S-Transferase) protein tag gene can carry out weight with any protein protomer of urease Group；The gene order of UreA, UreB, pGEX-4T-1 plasmid is shown in Table 1；By the UreA or UreB of GST label and urease Protein subunit is attached, and the pGEX-4T-1 plasmid of clone's urease UreA or UreB gene is transformed into coli strain, GST fusion urease (UreA albumen or UreB albumen) is given expression to by Escherichia coli fermentation, then uses Glutathione Sepharose 4B gel (purchasing from GE company) carries out affinitive layer purification, the use of eluent system is 5mM glutathione (GSH) buffer (Tris-HCl containing 25mM, 100mM NaCl, 1mM EDTA, 1mM dithiothreitol (DTT), pH 7.4), then Eluent is put into bag filter, then is dialysed (25mM Tris-HCl, pH 7.4,100mM NaCl, 1mM with dialyzate EDTA, 1mM dithiothreitol (DTT)), removing glutathione (GSH), (GST merges urea enzyme subunit egg to get Heliobacter pylori antigen It is white), abbreviation Heliobacter pylori antigen (urease), it is dense that last Heliobacter pylori antigen (urease) passes through BCA method measurement albumen Degree, mass ratio 1.0mg/ml-10.0mg/ml.Obtained Heliobacter pylori antigen (urease) can be used for next antibody Preparation, while also can be used as the mother liquor of standard items and quality-control product.
S2, Heliobacter pylori antigen (urease) polyclonal antibody is prepared:
S21,100 μ g Heliobacter pylori antigens (urease) are emulsified with equivalent Freund's complete adjuvant.
S22, by its dorsal sc multi-point injection in the New Zealand White Rabbit body of 2.2~2.5kg, thereafter every two weeks with not Family name's Freund's incomplete adjuvant emulsifies antigen, carries out booster immunization 3 times with method duplicate injection, amounts to 4 times immune.
S23, then from venous puncture 100ml rabbit blood, centrifugation obtains serum.10ml rabbit anteserum is taken to pass through affine in immunity Chromatographic column, with 1000ml TBS (Tris containing 20mM, 500mM NaCl, 0.05% Tween-20, pH 7.4), buffer is repeatedly Chromatographic column is rinsed, efflux is discarded, after flushing, is eluted with glycine/HCl (100mM, the pH 2.5) buffer of 10ml In conjunction with antibody, be collected in bag filter, and be placed in 1000ml TBS (Tris containing 20mM, 500mM NaCl, 0.05% Tween-20, pH 7.4) in buffer, 4 DEG C of dialysed overnights in refrigerator obtain more grams of anti-helicobacter pylori antigen (urease) Grand antibody.
S3, the latex that anti-helicobacter pylori antigen (urease) polyclonal antibody is coated with by the method preparation of chamical binding Particle specifically includes:
S31, it will be mixed in the latex particle of 100mg and 5ml MES buffer (50mM, pH 6.0) or PBS buffer Afterwards, surfactant sodium dodecyl base sodium sulfonate (ultimate density 0.01%) is added, obtains latex particle solution.
S32, anti-helicobacter pylori antigen (urease) polyclonal antibody is dissolved in (50mM, pH in 5ml MES buffer 6.0) or in PBS buffer solution, ultimate density reaches 1~10 μm of ol/ml, and it is polyclonal to obtain anti-helicobacter pylori antigen (urease) Antibody-solutions.
S33, latex particle solution and anti-helicobacter pylori antigen (urease) Anti-TNF-α liquid solution are sufficiently mixed, so 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) that 100mg is added afterwards is dissolved in mixed liquor, room temperature Lower reaction 2~4 hours, successfully obtains the latex particle of coating anti-helicobacter pylori antigen (urease) polyclonal antibody.
S4, by the latex particle of coating anti-helicobacter pylori antigen (urease) polyclonal antibody of 4mg/mL with contain 3% sodium chloride, 2.5% mannitol, 5% Tween 80, the MES buffer (pH 7.4) of 0.5% Sodium azide and 250mmol/L are mixed It closes to get R2 reagent.
Table 1
2, R1 reagent is prepared
Mannitol that sodium chloride that the R1 reagent is 3% by mass concentration, mass concentration are 2.5%, mass concentration are The MES buffer solution (pH 7.4) that the Sodium azide and molar concentration that 5% Tween 80, mass concentration are 0.5% are 250mmol/L Composition, mixes the above components evenly in proportion to obtain the final product.
After the preparation of R1, R2 reagent, it is respectively placed in R1 kit and R2 kit.
3, calibration object is prepared
With the Heliobacter pylori antigen (urease) by the purification of gene recombination technology described in step S1 for mother liquor, make With bovine serum albumin gradient dilution mother liquor, the calibration object of various concentration: S5:40 ng/mL, S4:20ng/mL, S3:10ng/ is prepared ML, S2:5ng/mL, S1:2.5ng/mL, wherein bovine serum albumin(BSA) concentration is 1ng/ml-100g/l.
4, quality-control product is prepared
With the Heliobacter pylori antigen (urease) by the purification of gene recombination technology described in step S1 for mother liquor, Mother liquor is diluted using bovine serum albumin gradient, prepares the quality-control product of two kinds of concentration: C1:4.0ng/mL, C2:1.0ng/mL, Middle bovine serum albumin(BSA) concentration 1ng/ml-100g/l.
5, the formulation of standard curve
Kit measurement detects dominant wavelength: 450nm, commplementary wave length: 800nm using automatic clinical chemistry analyzer.
Reagent dosage: 15 μ l of fecal sample；305 μ l of R1 reagent；25 μ l of R2 reagent, the fecal sample pass through containing as follows It is obtained after the lysate processing of ingredient: 20mM Tris-HCl (pH7.5), 1 mM DTT, 2mM EDTA, 2mM EGTA, 25mM NaF, 25mM β-glycerophosphate, 0.1mM Na3VO4,0.5mM phenylmethylsulfonyl fluoride (PMSF) and 0.3% Nonidet P-40。
Measuring method (Two point end assay): 305 μ l R1 reagents be added 15 μ l samples, in 37 DEG C react 5 minutes, then plus Enter 25 μ l R2 reagents and start read point to measure after absorbance A 1,10 minutes that read point measures absorbance A 2 again, calculates absorbance Difference DELTA A=A2-A1, as shown in Figure 2.
It makes standard curve: using Heliobacter pylori antigen obtained (urease) calibration object, concentration is respectively S5: 40ng/mL, S4:20ng/mL, S3:10ng/mL, S2:5ng/mL, S1:2.5 ng/mL.It is deep and remote that the present invention is measured according to above-mentioned steps The standard curve of Helicobacter pylori antigen (urease) standard items, as shown in Figure 3.Each point in Fig. 3 on curve represents one and contains The standard items of amount, wherein x axis indicates that the concentration of Heliobacter pylori antigen (urease), y-axis indicate the difference of absorbance.
6, the determination of the range of linearity
By close to 100 ng/mL of the Heliobacter pylori antigen of the range of linearity upper limit (urease) high concentration sample, physiology is used Salt water is configured to the solution of 7 various concentrations by 1/2,1/4,1/8,1/16,1/32,1/64 dilution altogether, separately to be free of pylorus The physiological saline of pylori antigen (urease) is as blank solution.Each concentration is detected with the method for production standard curve, will be surveyed Determine concentration value and theoretical concentration carries out linear regression analysis, calculates regression equation are as follows: y=1.0214x+2.2577, correlation coefficient r =0.986, show kit of the present invention good relationship in 0~100ng/mL range of linearity.
7, accuracy determination
Excrement measurement is carried out to 20 people using automatic clinical chemistry analyzer, sample gushes people from Shenzhen roc new district certain herbaceous plants with big flowers People hospital.Reference value: less than be equal to 0.5ng/mL for ' negative ' specimens, being greater than 1.0ng/mL is positive sample, between 0.5- 1.0ng/mL numerical value is uncertain sample, needs to retest, test result is as shown in table 2.
Table 2
The above results show that the correlation of obtained reagent and clinical disease is high.
8, sensitivity determination
Sensitivity definition is the variation of unit concentration absorbance, with Heliobacter pylori antigen (urease) calibration object to pylorus Pylori antigen (urease) reagent records calibration object (concentration 10ng/mL) and examination in automatic clinical chemistry analyzer upscaling The absorbance value of agent reaction is 0.08, i.e. sensitivity of the reagent when calibration concentration is 10ng/mL is 0.08, high sensitivity.
9, the measurement of withinrun precision
It measures by reagent of the present invention with a sample 10 times, calculates measurement mean value and withinrun precision, test knot Fruit is as shown in table 3.
Table 3
10, anti-Interference Analysis
Chaff interferent selection formula and test result are as shown in table 4.
Table 4
The above results show that reagent anti-interference ability made from method of the present invention is good.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or It changes still within the protection scope of the invention.
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Ala Gly Cys Gly Thr Ala Gly Gly Thr Cys Cys Thr Gly Cys Thr Ala
340 345 350
Cys Thr Gly Ala Ala Gly Cys Cys Thr Thr Ala Gly Cys Cys Gly Gly
355 360 365
Thr Gly Ala Ala Gly Gly Thr Thr Thr Gly Ala Thr Cys Gly Thr Ala
370 375 380
Ala Cys Thr Gly Cys Thr Gly Gly Thr Gly Gly Thr Ala Thr Thr Gly
385 390 395 400
Ala Cys Ala Cys Ala Cys Ala Cys Ala Thr Cys Cys Ala Cys Thr Thr
405 410 415
Cys Ala Thr Thr Thr Cys Ala Cys Cys Cys Cys Ala Ala Cys Ala Ala
420 425 430
Ala Thr Cys Cys Cys Thr Ala Cys Ala Gly Cys Thr Thr Thr Thr Gly
435 440 445
Cys Ala Ala Gly Cys Gly Gly Thr Gly Thr Ala Ala Cys Ala Ala Cys
450 455 460
Cys Ala Thr Gly Ala Thr Thr Gly Gly Thr Gly Gly Cys Gly Gly Ala
465 470 475 480
Ala Cys Thr Gly Gly Thr Cys Cys Thr Gly Cys Thr Gly Ala Thr Gly
485 490 495
Gly Cys Ala Cys Thr Ala Ala Thr Gly Cys Gly Ala Cys Thr Ala Cys
500 505 510
Thr Ala Thr Cys Ala Cys Thr Cys Cys Ala Gly Gly Cys Ala Gly Ala
515 520 525
Ala Gly Ala Ala Ala Thr Thr Thr Ala Ala Ala Ala Thr Gly Gly Ala
530 535 540
Thr Gly Cys Thr Cys Ala Gly Ala Gly Cys Gly Gly Cys Thr Gly Ala
545 550 555 560
Ala Gly Ala Ala Thr Ala Thr Thr Cys Thr Ala Thr Gly Ala Ala Cys
565 570 575
Thr Thr Ala Gly Gly Thr Thr Thr Cys Thr Thr Gly Gly Cys Thr Ala
580 585 590
Ala Ala Gly Gly Thr Ala Ala Cys Gly Cys Thr Thr Cys Thr Ala Ala
595 600 605
Cys Gly Ala Cys Gly Cys Gly Ala Gly Cys Thr Thr Ala Gly Cys Cys
610 615 620
Gly Ala Thr Cys Ala Ala Ala Thr Thr Gly Ala Ala Gly Cys Thr Gly
625 630 635 640
Gly Thr Gly Cys Gly Ala Thr Thr Gly Gly Cys Thr Thr Thr Ala Ala
645 650 655
Ala Ala Thr Cys Cys Ala Cys Gly Ala Ala Gly Ala Cys Thr Gly Gly
660 665 670
Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Cys Thr Thr Cys Thr Gly
675 680 685
Cys Ala Ala Thr Cys Ala Ala Thr Cys Ala Thr Gly Cys Gly Thr Thr
690 695 700
Ala Gly Ala Thr Gly Thr Thr Gly Cys Ala Gly Ala Cys Ala Ala Ala
705 710 715 720
Thr Ala Cys Gly Ala Thr Gly Thr Gly Cys Ala Ala Gly Thr Cys Gly
725 730 735
Cys Thr Ala Thr Cys Cys Ala Cys Ala Cys Ala Gly Ala Cys Ala Cys
740 745 750
Thr Thr Thr Gly Ala Ala Thr Gly Ala Ala Gly Cys Cys Gly Gly Thr
755 760 765
Thr Gly Cys Gly Thr Gly Gly Ala Ala Gly Ala Cys Ala Cys Thr Ala
770 775 780
Thr Gly Gly Cys Ala Gly Cys Thr Ala Thr Thr Gly Cys Cys Gly Gly
785 790 795 800
Ala Cys Gly Cys Ala Cys Thr Ala Thr Gly Cys Ala Cys Ala Cys Thr
805 810 815
Thr Thr Cys Cys Ala Cys Ala Cys Thr Gly Ala Ala Gly Gly Thr Gly
820 825 830
Cys Thr Gly Gly Cys Gly Gly Cys Gly Gly Ala Cys Ala Cys Gly Cys
835 840 845
Thr Cys Cys Thr Gly Ala Thr Ala Thr Thr Ala Thr Thr Ala Ala Ala
850 855 860
Gly Thr Ala Gly Cys Thr Gly Gly Thr Gly Ala Ala Cys Ala Cys Ala
865 870 875 880
Ala Cys Ala Thr Thr Cys Thr Thr Cys Cys Cys Gly Cys Thr Thr Cys
885 890 895
Cys Ala Cys Thr Ala Ala Cys Cys Cys Cys Ala Cys Thr Ala Thr Cys
900 905 910
Cys Cys Thr Thr Thr Cys Ala Cys Thr Gly Thr Gly Ala Ala Thr Ala
915 920 925
Cys Ala Gly Ala Ala Gly Cys Ala Gly Ala Ala Cys Ala Cys Ala Thr
930 935 940
Gly Gly Ala Cys Ala Thr Gly Cys Thr Thr Ala Thr Gly Gly Thr Gly
945 950 955 960
Thr Gly Cys Cys Ala Cys Cys Ala Cys Thr Thr Gly Gly Ala Thr Ala
965 970 975
Ala Ala Ala Gly Cys Ala Thr Thr Ala Ala Ala Gly Ala Ala Gly Ala
980 985 990
Thr Gly Thr Thr Cys Ala Gly Thr Thr Cys Gly Cys Thr Gly Ala Thr
995 1000 1005
Thr Cys Ala Ala Gly Gly Ala Thr Cys Cys Gly Cys Cys Cys Thr Cys
1010 1015 1020
Ala Ala Ala Cys Cys Ala Thr Thr Gly Cys Gly Gly Cys Thr Gly Ala
1025 1030 1035 1040
Ala Gly Ala Cys Ala Cys Thr Thr Thr Gly Cys Ala Thr Gly Ala Cys
1045 1050 1055
Ala Thr Gly Gly Gly Gly Ala Thr Thr Thr Thr Cys Thr Cys Ala Ala
1060 1065 1070
Thr Cys Ala Cys Cys Ala Gly Cys Thr Cys Thr Gly Ala Cys Thr Cys
1075 1080 1085
Thr Cys Ala Ala Gly Cys Thr Ala Thr Gly Gly Gly Thr Cys Gly Thr
1090 1095 1100
Gly Thr Gly Gly Gly Thr Gly Ala Ala Gly Thr Thr Ala Thr Cys Ala
1105 1110 1115 1120
Cys Thr Ala Gly Ala Ala Cys Thr Thr Gly Gly Cys Ala Ala Ala Cys
1125 1130 1135
Ala Gly Cys Thr Gly Ala Cys Ala Ala Ala Ala Ala Cys Ala Ala Ala
1140 1145 1150
Ala Ala Ala Gly Ala Ala Thr Thr Thr Gly Gly Cys Cys Gly Cys Thr
1155 1160 1165
Thr Gly Ala Ala Ala Gly Ala Ala Gly Ala Ala Ala Ala Ala Gly Gly
1170 1175 1180
Cys Gly Ala Thr Ala Ala Cys Gly Ala Cys Ala Ala Cys Thr Thr Cys
1185 1190 1195 1200
Ala Gly Gly Ala Thr Cys Ala Ala Ala Cys Gly Cys Thr Ala Cys Thr
1205 1210 1215
Thr Gly Thr Cys Thr Ala Ala Ala Thr Ala Cys Ala Cys Cys Ala Thr
1220 1225 1230
Thr Ala Ala Cys Cys Cys Ala Gly Cys Gly Ala Thr Cys Gly Cys Thr
1235 1240 1245
Cys Ala Thr Gly Gly Gly Ala Thr Thr Ala Gly Cys Gly Ala Gly Thr
1250 1255 1260
Ala Thr Gly Thr Ala Gly Gly Thr Thr Cys Thr Gly Thr Ala Gly Ala
1265 1270 1275 1280
Ala Gly Thr Gly Gly Gly Cys Ala Ala Ala Gly Thr Gly Gly Cys Thr
1285 1290 1295
Gly Ala Cys Thr Thr Gly Gly Thr Ala Thr Thr Gly Thr Gly Gly Ala
1300 1305 1310
Gly Thr Cys Cys Cys Gly Cys Ala Thr Thr Cys Thr Thr Thr Gly Gly
1315 1320 1325
Cys Gly Thr Ala Ala Ala Ala Cys Cys Cys Ala Ala Cys Ala Thr Gly
1330 1335 1340
Ala Thr Cys Ala Thr Cys Ala Ala Ala Gly Gly Cys Gly Gly Gly Thr
1345 1350 1355 1360
Thr Cys Ala Thr Thr Gly Cys Gly Thr Thr Gly Ala Gly Thr Cys Ala
1365 1370 1375
Ala Ala Thr Gly Gly Gly Thr Gly Ala Cys Gly Cys Gly Ala Ala Cys
1380 1385 1390
Gly Cys Thr Thr Cys Thr Ala Thr Cys Cys Cys Thr Ala Cys Cys Cys
1395 1400 1405
Cys Ala Cys Ala Ala Cys Cys Ala Gly Thr Thr Thr Ala Thr Thr Ala
1410 1415 1420
Cys Ala Gly Ala Gly Ala Ala Ala Thr Gly Thr Thr Cys Gly Cys Thr
1425 1430 1435 1440
Cys Ala Thr Cys Ala Thr Gly Gly Thr Ala Ala Ala Gly Cys Cys Ala
1445 1450 1455
Ala Ala Thr Ala Cys Gly Ala Thr Gly Cys Ala Ala Ala Cys Ala Thr
1460 1465 1470
Cys Ala Cys Thr Thr Thr Thr Gly Thr Gly Thr Cys Thr Cys Ala Ala
1475 1480 1485
Gly Cys Gly Gly Cys Thr Thr Ala Thr Gly Ala Cys Ala Ala Ala Gly
1490 1495 1500
Gly Cys Ala Thr Thr Ala Ala Ala Gly Ala Ala Gly Ala Ala Thr Thr
1505 1510 1515 1520
Ala Gly Gly Gly Cys Thr Thr Gly Ala Ala Ala Gly Ala Cys Ala Ala
1525 1530 1535
Gly Thr Gly Thr Thr Gly Cys Cys Gly Gly Thr Ala Ala Ala Ala Ala
1540 1545 1550
Ala Thr Thr Gly Cys Ala Gly Ala Ala Ala Cys Ala Thr Cys Ala Cys
1555 1560 1565
Thr Ala Ala Ala Ala Ala Ala Gly Ala Cys Ala Thr Gly Cys Ala Ala
1570 1575 1580
Thr Thr Cys Ala Ala Cys Gly Ala Cys Ala Cys Thr Ala Cys Cys Gly
1585 1590 1595 1600
Cys Thr Cys Ala Cys Ala Thr Thr Gly Ala Ala Gly Thr Cys Ala Ala
1605 1610 1615
Thr Cys Cys Thr Gly Ala Ala Ala Cys Thr Thr Ala Cys Cys Ala Thr
1620 1625 1630
Gly Thr Gly Thr Thr Cys Gly Thr Gly Gly Ala Thr Gly Gly Cys Ala
1635 1640 1645
Ala Ala Gly Ala Ala Gly Thr Ala Ala Cys Thr Thr Cys Thr Ala Ala
1650 1655 1660
Ala Cys Cys Ala Gly Cys Cys Ala Ala Thr Ala Ala Ala Gly Thr Gly
1665 1670 1675 1680
Ala Gly Cys Thr Thr Gly Gly Cys Gly Cys Ala Ala Cys Thr Cys Thr
1685 1690 1695
Thr Thr Ala Gly Cys Ala Thr Thr Thr Thr Cys Thr Ala Gly
1700 1705 1710
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Ala Cys Gly Thr Thr Ala Thr Cys Gly Ala Cys Thr Gly Cys Ala Cys
1 5 10 15
Gly Gly Thr Gly Cys Ala Cys Cys Ala Ala Thr Gly Cys Thr Thr Cys
20 25 30
Thr Gly Gly Cys Gly Thr Cys Ala Gly Gly Cys Ala Gly Cys Cys Ala
35 40 45
Thr Cys Gly Gly Ala Ala Gly Cys Thr Gly Thr Gly Gly Thr Ala Thr
50 55 60
Gly Gly Cys Thr Gly Thr Gly Cys Ala Gly Gly Thr Cys Gly Thr Ala
65 70 75 80
Ala Ala Thr Cys Ala Cys Thr Gly Cys Ala Thr Ala Ala Thr Thr Cys
85 90 95
Gly Thr Gly Thr Cys Gly Cys Thr Cys Ala Ala Gly Gly Cys Gly Cys
100 105 110
Ala Cys Thr Cys Cys Cys Gly Thr Thr Cys Thr Gly Gly Ala Thr Ala
115 120 125
Ala Thr Gly Thr Thr Thr Thr Thr Thr Gly Cys Gly Cys Cys Gly Ala
130 135 140
Cys Ala Thr Cys Ala Thr Ala Ala Cys Gly Gly Thr Thr Cys Thr Gly
145 150 155 160
Gly Cys Ala Ala Ala Thr Ala Thr Thr Cys Thr Gly Ala Ala Ala Thr
165 170 175
Gly Ala Gly Cys Thr Gly Thr Thr Gly Ala Cys Ala Ala Thr Thr Ala
180 185 190
Ala Thr Cys Ala Thr Cys Gly Gly Cys Thr Cys Gly Thr Ala Thr Ala
195 200 205
Ala Thr Gly Thr Gly Thr Gly Gly Ala Ala Thr Thr Gly Thr Gly Ala
210 215 220
Gly Cys Gly Gly Ala Thr Ala Ala Cys Ala Ala Thr Thr Thr Cys Ala
225 230 235 240
Cys Ala Cys Ala Gly Gly Ala Ala Ala Cys Ala Gly Thr Ala Thr Thr
245 250 255
Cys Ala Thr Gly Thr Cys Cys Cys Cys Thr Ala Thr Ala Cys Thr Ala
260 265 270
Gly Gly Thr Thr Ala Thr Thr Gly Gly Ala Ala Ala Ala Thr Thr Ala
275 280 285
Ala Gly Gly Gly Cys Cys Thr Thr Gly Thr Gly Cys Ala Ala Cys Cys
290 295 300
Cys Ala Cys Thr Cys Gly Ala Cys Thr Thr Cys Thr Thr Thr Thr Gly
305 310 315 320
Gly Ala Ala Thr Ala Thr Cys Thr Thr Gly Ala Ala Gly Ala Ala Ala
325 330 335
Ala Ala Thr Ala Thr Gly Ala Ala Gly Ala Gly Cys Ala Thr Thr Thr
340 345 350
Gly Thr Ala Thr Gly Ala Gly Cys Gly Cys Gly Ala Thr Gly Ala Ala
355 360 365
Gly Gly Thr Gly Ala Thr Ala Ala Ala Thr Gly Gly Cys Gly Ala Ala
370 375 380
Ala Cys Ala Ala Ala Ala Ala Gly Thr Thr Thr Gly Ala Ala Thr Thr
385 390 395 400
Gly Gly Gly Thr Thr Thr Gly Gly Ala Gly Thr Thr Thr Cys Cys Cys
405 410 415
Ala Ala Thr Cys Thr Thr Cys Cys Thr Thr Ala Thr Thr Ala Thr Ala
420 425 430
Thr Thr Gly Ala Thr Gly Gly Thr Gly Ala Thr Gly Thr Thr Ala Ala
435 440 445
Ala Thr Thr Ala Ala Cys Ala Cys Ala Gly Thr Cys Thr Ala Thr Gly
450 455 460
Gly Cys Cys Ala Thr Cys Ala Thr Ala Cys Gly Thr Thr Ala Thr Ala
465 470 475 480
Thr Ala Gly Cys Thr Gly Ala Cys Ala Ala Gly Cys Ala Cys Ala Ala
485 490 495
Cys Ala Thr Gly Thr Thr Gly Gly Gly Thr Gly Gly Thr Thr Gly Thr
500 505 510
Cys Cys Ala Ala Ala Ala Gly Ala Gly Cys Gly Thr Gly Cys Ala Gly
515 520 525
Ala Gly Ala Thr Thr Thr Cys Ala Ala Thr Gly Cys Thr Thr Gly Ala
530 535 540
Ala Gly Gly Ala Gly Cys Gly Gly Thr Thr Thr Thr Gly Gly Ala Thr
545 550 555 560
Ala Thr Thr Ala Gly Ala Thr Ala Cys Gly Gly Thr Gly Thr Thr Thr
565 570 575
Cys Gly Ala Gly Ala Ala Thr Thr Gly Cys Ala Thr Ala Thr Ala Gly
580 585 590
Thr Ala Ala Ala Gly Ala Cys Thr Thr Thr Gly Ala Ala Ala Cys Thr
595 600 605
Cys Thr Cys Ala Ala Ala Gly Thr Thr Gly Ala Thr Thr Thr Thr Cys
610 615 620
Thr Thr Ala Gly Cys Ala Ala Gly Cys Thr Ala Cys Cys Thr Gly Ala
625 630 635 640
Ala Ala Thr Gly Cys Thr Gly Ala Ala Ala Ala Thr Gly Thr Thr Cys
645 650 655
Gly Ala Ala Gly Ala Thr Cys Gly Thr Thr Thr Ala Thr Gly Thr Cys
660 665 670
Ala Thr Ala Ala Ala Ala Cys Ala Thr Ala Thr Thr Thr Ala Ala Ala
675 680 685
Thr Gly Gly Thr Gly Ala Thr Cys Ala Thr Gly Thr Ala Ala Cys Cys
690 695 700
Cys Ala Thr Cys Cys Thr Gly Ala Cys Thr Thr Cys Ala Thr Gly Thr
705 710 715 720
Thr Gly Thr Ala Thr Gly Ala Cys Gly Cys Thr Cys Thr Thr Gly Ala
725 730 735
Thr Gly Thr Thr Gly Thr Thr Thr Thr Ala Thr Ala Cys Ala Thr Gly
740 745 750
Gly Ala Cys Cys Cys Ala Ala Thr Gly Thr Gly Cys Cys Thr Gly Gly
755 760 765
Ala Thr Gly Cys Gly Thr Thr Cys Cys Cys Ala Ala Ala Ala Thr Thr
770 775 780
Ala Gly Thr Thr Thr Gly Thr Thr Thr Thr Ala Ala Ala Ala Ala Ala
785 790 795 800
Cys Gly Thr Ala Thr Thr Gly Ala Ala Gly Cys Thr Ala Thr Cys Cys
805 810 815
Cys Ala Cys Ala Ala Ala Thr Thr Gly Ala Thr Ala Ala Gly Thr Ala
820 825 830
Cys Thr Thr Gly Ala Ala Ala Thr Cys Cys Ala Gly Cys Ala Ala Gly
835 840 845
Thr Ala Thr Ala Thr Ala Gly Cys Ala Thr Gly Gly Cys Cys Thr Thr
850 855 860
Thr Gly Cys Ala Gly Gly Gly Cys Thr Gly Gly Cys Ala Ala Gly Cys
865 870 875 880
Cys Ala Cys Gly Thr Thr Thr Gly Gly Thr Gly Gly Thr Gly Gly Cys
885 890 895
Gly Ala Cys Cys Ala Thr Cys Cys Thr Cys Cys Ala Ala Ala Ala Thr
900 905 910
Cys Gly Gly Ala Thr Cys Thr Gly Gly Thr Thr Cys Cys Gly Cys Gly
915 920 925
Thr Gly Gly Ala Thr Cys Cys Cys Cys Gly Gly Ala Ala Thr Thr Cys
930 935 940
Cys Cys Gly Gly Gly Thr Cys Gly Ala Cys Thr Cys Gly Ala Gly Cys
945 950 955 960
Gly Gly Cys Cys Gly Cys Ala Thr Cys Gly Thr Gly Ala Cys Thr Gly
965 970 975
Ala Cys Thr Gly Ala Cys Gly Ala Thr Cys Thr Gly Cys Cys Thr Cys
980 985 990
Gly Cys Gly Cys Gly Thr Thr Thr Cys Gly Gly Thr Gly Ala Thr Gly
995 1000 1005
Ala Cys Gly Gly Thr Gly Ala Ala Ala Ala Cys Cys Thr Cys Thr Gly
1010 1015 1020
Ala Cys Ala Cys Ala Thr Gly Cys Ala Gly Cys Thr Cys Cys Cys Gly
1025 1030 1035 1040
Gly Ala Gly Ala Cys Gly Gly Thr Cys Ala Cys Ala Gly Cys Thr Thr
1045 1050 1055
Gly Thr Cys Thr Gly Thr Ala Ala Gly Cys Gly Gly Ala Thr Gly Cys
1060 1065 1070
Cys Gly Gly Gly Ala Gly Cys Ala Gly Ala Cys Ala Ala Gly Cys Cys
1075 1080 1085
Cys Gly Thr Cys Ala Gly Gly Gly Cys Gly Cys Gly Thr Cys Ala Gly
1090 1095 1100
Cys Gly Gly Gly Thr Gly Thr Thr Gly Gly Cys Gly Gly Gly Thr Gly
1105 1110 1115 1120
Thr Cys Gly Gly Gly Gly Cys Gly Cys Ala Gly Cys Cys Ala Thr Gly
1125 1130 1135
Ala Cys Cys Cys Ala Gly Thr Cys Ala Cys Gly Thr Ala Gly Cys Gly
1140 1145 1150
Ala Thr Ala Gly Cys Gly Gly Ala Gly Thr Gly Thr Ala Thr Ala Ala
1155 1160 1165
Thr Thr Cys Thr Thr Gly Ala Ala Gly Ala Cys Gly Ala Ala Ala Gly
1170 1175 1180
Gly Gly Cys Cys Thr Cys Gly Thr Gly Ala Thr Ala Cys Gly Cys Cys
1185 1190 1195 1200
Thr Ala Thr Thr Thr Thr Thr Ala Thr Ala Gly Gly Thr Thr Ala Ala
1205 1210 1215
Thr Gly Thr Cys Ala Thr Gly Ala Thr Ala Ala Thr Ala Ala Thr Gly
1220 1225 1230
Gly Thr Thr Thr Cys Thr Thr Ala Gly Ala Cys Gly Thr Cys Ala Gly
1235 1240 1245
Gly Thr Gly Gly Cys Ala Cys Thr Thr Thr Thr Cys Gly Gly Gly Gly
1250 1255 1260
Ala Ala Ala Thr Gly Thr Gly Cys Gly Cys Gly Gly Ala Ala Cys Cys
1265 1270 1275 1280
Cys Cys Thr Ala Thr Thr Thr Gly Thr Thr Thr Ala Thr Thr Thr Thr
1285 1290 1295
Thr Cys Thr Ala Ala Ala Thr Ala Cys Ala Thr Thr Cys Ala Ala Ala
1300 1305 1310
Thr Ala Thr Gly Thr Ala Thr Cys Cys Gly Cys Thr Cys Ala Thr Gly
1315 1320 1325
Ala Gly Ala Cys Ala Ala Thr Ala Ala Cys Cys Cys Thr Gly Ala Thr
1330 1335 1340
Ala Ala Ala Thr Gly Cys Thr Thr Cys Ala Ala Thr Ala Ala Thr Ala
1345 1350 1355 1360
Thr Thr Gly Ala Ala Ala Ala Ala Gly Gly Ala Ala Gly Ala Gly Thr
1365 1370 1375
Ala Thr Gly Ala Gly Thr Ala Thr Thr Cys Ala Ala Cys Ala Thr Thr
1380 1385 1390
Thr Cys Cys Gly Thr Gly Thr Cys Gly Cys Cys Cys Thr Thr Ala Thr
1395 1400 1405
Thr Cys Cys Cys Thr Thr Thr Thr Thr Thr Gly Cys Gly Gly Cys Ala
1410 1415 1420
Thr Thr Thr Thr Gly Cys Cys Thr Thr Cys Cys Thr Gly Thr Thr Thr
1425 1430 1435 1440
Thr Thr Gly Cys Thr Cys Ala Cys Cys Cys Ala Gly Ala Ala Ala Cys
1445 1450 1455
Gly Cys Thr Gly Gly Thr Gly Ala Ala Ala Gly Thr Ala Ala Ala Ala
1460 1465 1470
Gly Ala Thr Gly Cys Thr Gly Ala Ala Gly Ala Thr Cys Ala Gly Thr
1475 1480 1485
Thr Gly Gly Gly Thr Gly Cys Ala Cys Gly Ala Gly Thr Gly Gly Gly
1490 1495 1500
Thr Thr Ala Cys Ala Thr Cys Gly Ala Ala Cys Thr Gly Gly Ala Thr
1505 1510 1515 1520
Cys Thr Cys Ala Ala Cys Ala Gly Cys Gly Gly Thr Ala Ala Gly Ala
1525 1530 1535
Thr Cys Cys Thr Thr Gly Ala Gly Ala Gly Thr Thr Thr Thr Cys Gly
1540 1545 1550
Cys Cys Cys Cys Gly Ala Ala Gly Ala Ala Cys Gly Thr Thr Thr Thr
1555 1560 1565
Cys Cys Ala Ala Thr Gly Ala Thr Gly Ala Gly Cys Ala Cys Thr Thr
1570 1575 1580
Thr Thr Ala Ala Ala Gly Thr Thr Cys Thr Gly Cys Thr Ala Thr Gly
1585 1590 1595 1600
Thr Gly Gly Cys Gly Cys Gly Gly Thr Ala Thr Thr Ala Thr Cys Cys
1605 1610 1615
Cys Gly Thr Gly Thr Thr Gly Ala Cys Gly Cys Cys Gly Gly Gly Cys
1620 1625 1630
Ala Ala Gly Ala Gly Cys Ala Ala Cys Thr Cys Gly Gly Thr Cys Gly
1635 1640 1645
Cys Cys Gly Cys Ala Thr Ala Cys Ala Cys Thr Ala Thr Thr Cys Thr
1650 1655 1660
Cys Ala Gly Ala Ala Thr Gly Ala Cys Thr Thr Gly Gly Thr Thr Gly
1665 1670 1675 1680
Ala Gly Thr Ala Cys Thr Cys Ala Cys Cys Ala Gly Thr Cys Ala Cys
1685 1690 1695
Ala Gly Ala Ala Ala Ala Gly Cys Ala Thr Cys Thr Thr Ala Cys Gly
1700 1705 1710
Gly Ala Thr Gly Gly Cys Ala Thr Gly Ala Cys Ala Gly Thr Ala Ala
1715 1720 1725
Gly Ala Gly Ala Ala Thr Thr Ala Thr Gly Cys Ala Gly Thr Gly Cys
1730 1735 1740
Thr Gly Cys Cys Ala Thr Ala Ala Cys Cys Ala Thr Gly Ala Gly Thr
1745 1750 1755 1760
Gly Ala Thr Ala Ala Cys Ala Cys Thr Gly Cys Gly Gly Cys Cys Ala
1765 1770 1775
Ala Cys Thr Thr Ala Cys Thr Thr Cys Thr Gly Ala Cys Ala Ala Cys
1780 1785 1790
Gly Ala Thr Cys Gly Gly Ala Gly Gly Ala Cys Cys Gly Ala Ala Gly
1795 1800 1805
Gly Ala Gly Cys Thr Ala Ala Cys Cys Gly Cys Thr Thr Thr Thr Thr
1810 1815 1820
Thr Gly Cys Ala Cys Ala Ala Cys Ala Thr Gly Gly Gly Gly Gly Ala
1825 1830 1835 1840
Thr Cys Ala Thr Gly Thr Ala Ala Cys Thr Cys Gly Cys Cys Thr Thr
1845 1850 1855
Gly Ala Thr Cys Gly Thr Thr Gly Gly Gly Ala Ala Cys Cys Gly Gly
1860 1865 1870
Ala Gly Cys Thr Gly Ala Ala Thr Gly Ala Ala Gly Cys Cys Ala Thr
1875 1880 1885
Ala Cys Cys Ala Ala Ala Cys Gly Ala Cys Gly Ala Gly Cys Gly Thr
1890 1895 1900
Gly Ala Cys Ala Cys Cys Ala Cys Gly Ala Thr Gly Cys Cys Thr Gly
1905 1910 1915 1920
Cys Ala Gly Cys Ala Ala Thr Gly Gly Cys Ala Ala Cys Ala Ala Cys
1925 1930 1935
Gly Thr Thr Gly Cys Gly Cys Ala Ala Ala Cys Thr Ala Thr Thr Ala
1940 1945 1950
Ala Cys Thr Gly Gly Cys Gly Ala Ala Cys Thr Ala Cys Thr Thr Ala
1955 1960 1965
Cys Thr Cys Thr Ala Gly Cys Thr Thr Cys Cys Cys Gly Gly Cys Ala
1970 1975 1980
Ala Cys Ala Ala Thr Thr Ala Ala Thr Ala Gly Ala Cys Thr Gly Gly
1985 1990 1995 2000
Ala Thr Gly Gly Ala Gly Gly Cys Gly Gly Ala Thr Ala Ala Ala Gly
2005 2010 2015
Thr Thr Gly Cys Ala Gly Gly Ala Cys Cys Ala Cys Thr Thr Cys Thr
2020 2025 2030
Gly Cys Gly Cys Thr Cys Gly Gly Cys Cys Cys Thr Thr Cys Cys Gly
2035 2040 2045
Gly Cys Thr Gly Gly Cys Thr Gly Gly Thr Thr Thr Ala Thr Thr Gly
2050 2055 2060
Cys Thr Gly Ala Thr Ala Ala Ala Thr Cys Thr Gly Gly Ala Gly Cys
2065 2070 2075 2080
Cys Gly Gly Thr Gly Ala Gly Cys Gly Thr Gly Gly Gly Thr Cys Thr
2085 2090 2095
Cys Gly Cys Gly Gly Thr Ala Thr Cys Ala Thr Thr Gly Cys Ala Gly
2100 2105 2110
Cys Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Ala Thr Gly Gly
2115 2120 2125
Thr Ala Ala Gly Cys Cys Cys Thr Cys Cys Cys Gly Thr Ala Thr Cys
2130 2135 2140
Gly Thr Ala Gly Thr Thr Ala Thr Cys Thr Ala Cys Ala Cys Gly Ala
2145 2150 2155 2160
Cys Gly Gly Gly Gly Ala Gly Thr Cys Ala Gly Gly Cys Ala Ala Cys
2165 2170 2175
Thr Ala Thr Gly Gly Ala Thr Gly Ala Ala Cys Gly Ala Ala Ala Thr
2180 2185 2190
Ala Gly Ala Cys Ala Gly Ala Thr Cys Gly Cys Thr Gly Ala Gly Ala
2195 2200 2205
Thr Ala Gly Gly Thr Gly Cys Cys Thr Cys Ala Cys Thr Gly Ala Thr
2210 2215 2220
Thr Ala Ala Gly Cys Ala Thr Thr Gly Gly Thr Ala Ala Cys Thr Gly
2225 2230 2235 2240
Thr Cys Ala Gly Ala Cys Cys Ala Ala Gly Thr Thr Thr Ala Cys Thr
2245 2250 2255
Cys Ala Thr Ala Thr Ala Thr Ala Cys Thr Thr Thr Ala Gly Ala Thr
2260 2265 2270
Thr Gly Ala Thr Thr Thr Ala Ala Ala Ala Cys Thr Thr Cys Ala Thr
2275 2280 2285
Thr Thr Thr Thr Ala Ala Thr Thr Thr Ala Ala Ala Ala Gly Gly Ala
2290 2295 2300
Thr Cys Thr Ala Gly Gly Thr Gly Ala Ala Gly Ala Thr Cys Cys Thr
2305 2310 2315 2320
Thr Thr Thr Thr Gly Ala Thr Ala Ala Thr Cys Thr Cys Ala Thr Gly
2325 2330 2335
Ala Cys Cys Ala Ala Ala Ala Thr Cys Cys Cys Thr Thr Ala Ala Cys
2340 2345 2350
Gly Thr Gly Ala Gly Thr Thr Thr Thr Cys Gly Thr Thr Cys Cys Ala
2355 2360 2365
Cys Thr Gly Ala Gly Cys Gly Thr Cys Ala Gly Ala Cys Cys Cys Cys
2370 2375 2380
Gly Thr Ala Gly Ala Ala Ala Ala Gly Ala Thr Cys Ala Ala Ala Gly
2385 2390 2395 2400
Gly Ala Thr Cys Thr Thr Cys Thr Thr Gly Ala Gly Ala Thr Cys Cys
2405 2410 2415
Thr Thr Thr Thr Thr Thr Thr Cys Thr Gly Cys Gly Cys Gly Thr Ala
2420 2425 2430
Ala Thr Cys Thr Gly Cys Thr Gly Cys Thr Thr Gly Cys Ala Ala Ala
2435 2440 2445
Cys Ala Ala Ala Ala Ala Ala Ala Cys Cys Ala Cys Cys Gly Cys Thr
2450 2455 2460
Ala Cys Cys Ala Gly Cys Gly Gly Thr Gly Gly Thr Thr Thr Gly Thr
2465 2470 2475 2480
Thr Thr Gly Cys Cys Gly Gly Ala Thr Cys Ala Ala Gly Ala Gly Cys
2485 2490 2495
Thr Ala Cys Cys Ala Ala Cys Thr Cys Thr Thr Thr Thr Thr Cys Cys
2500 2505 2510
Gly Ala Ala Gly Gly Thr Ala Ala Cys Thr Gly Gly Cys Thr Thr Cys
2515 2520 2525
Ala Gly Cys Ala Gly Ala Gly Cys Gly Cys Ala Gly Ala Thr Ala Cys
2530 2535 2540
Cys Ala Ala Ala Thr Ala Cys Thr Gly Thr Cys Cys Thr Thr Cys Thr
2545 2550 2555 2560
Ala Gly Thr Gly Thr Ala Gly Cys Cys Gly Thr Ala Gly Thr Thr Ala
2565 2570 2575
Gly Gly Cys Cys Ala Cys Cys Ala Cys Thr Thr Cys Ala Ala Gly Ala
2580 2585 2590
Ala Cys Thr Cys Thr Gly Thr Ala Gly Cys Ala Cys Cys Gly Cys Cys
2595 2600 2605
Thr Ala Cys Ala Thr Ala Cys Cys Thr Cys Gly Cys Thr Cys Thr Gly
2610 2615 2620
Cys Thr Ala Ala Thr Cys Cys Thr Gly Thr Thr Ala Cys Cys Ala Gly
2625 2630 2635 2640
Thr Gly Gly Cys Thr Gly Cys Thr Gly Cys Cys Ala Gly Thr Gly Gly
2645 2650 2655
Cys Gly Ala Thr Ala Ala Gly Thr Cys Gly Thr Gly Thr Cys Thr Thr
2660 2665 2670
Ala Cys Cys Gly Gly Gly Thr Thr Gly Gly Ala Cys Thr Cys Ala Ala
2675 2680 2685
Gly Ala Cys Gly Ala Thr Ala Gly Thr Thr Ala Cys Cys Gly Gly Ala
2690 2695 2700
Thr Ala Ala Gly Gly Cys Gly Cys Ala Gly Cys Gly Gly Thr Cys Gly
2705 2710 2715 2720
Gly Gly Cys Thr Gly Ala Ala Cys Gly Gly Gly Gly Gly Gly Thr Thr
2725 2730 2735
Cys Gly Thr Gly Cys Ala Cys Ala Cys Ala Gly Cys Cys Cys Ala Gly
2740 2745 2750
Cys Thr Thr Gly Gly Ala Gly Cys Gly Ala Ala Cys Gly Ala Cys Cys
2755 2760 2765
Thr Ala Cys Ala Cys Cys Gly Ala Ala Cys Thr Gly Ala Gly Ala Thr
2770 2775 2780
Ala Cys Cys Thr Ala Cys Ala Gly Cys Gly Thr Gly Ala Gly Cys Thr
2785 2790 2795 2800
Ala Thr Gly Ala Gly Ala Ala Ala Gly Cys Gly Cys Cys Ala Cys Gly
2805 2810 2815
Cys Thr Thr Cys Cys Cys Gly Ala Ala Gly Gly Gly Ala Gly Ala Ala
2820 2825 2830
Ala Gly Gly Cys Gly Gly Ala Cys Ala Gly Gly Thr Ala Thr Cys Cys
2835 2840 2845
Gly Gly Thr Ala Ala Gly Cys Gly Gly Cys Ala Gly Gly Gly Thr Cys
2850 2855 2860
Gly Gly Ala Ala Cys Ala Gly Gly Ala Gly Ala Gly Cys Gly Cys Ala
2865 2870 2875 2880
Cys Gly Ala Gly Gly Gly Ala Gly Cys Thr Thr Cys Cys Ala Gly Gly
2885 2890 2895
Gly Gly Gly Ala Ala Ala Cys Gly Cys Cys Thr Gly Gly Thr Ala Thr
2900 2905 2910
Cys Thr Thr Thr Ala Thr Ala Gly Thr Cys Cys Thr Gly Thr Cys Gly
2915 2920 2925
Gly Gly Thr Thr Thr Cys Gly Cys Cys Ala Cys Cys Thr Cys Thr Gly
2930 2935 2940
Ala Cys Thr Thr Gly Ala Gly Cys Gly Thr Cys Gly Ala Thr Thr Thr
2945 2950 2955 2960
Thr Thr Gly Thr Gly Ala Thr Gly Cys Thr Cys Gly Thr Cys Ala Gly
2965 2970 2975
Gly Gly Gly Gly Gly Cys Gly Gly Ala Gly Cys Cys Thr Ala Thr Gly
2980 2985 2990
Gly Ala Ala Ala Ala Ala Cys Gly Cys Cys Ala Gly Cys Ala Ala Cys
2995 3000 3005
Gly Cys Gly Gly Cys Cys Thr Thr Thr Thr Thr Ala Cys Gly Gly Thr
3010 3015 3020
Thr Cys Cys Thr Gly Gly Cys Cys Thr Thr Thr Thr Gly Cys Thr Gly
3025 3030 3035 3040
Gly Cys Cys Thr Thr Thr Thr Gly Cys Thr Cys Ala Cys Ala Thr Gly
3045 3050 3055
Thr Thr Cys Thr Thr Thr Cys Cys Thr Gly Cys Gly Thr Thr Ala Thr
3060 3065 3070
Cys Cys Cys Cys Thr Gly Ala Thr Thr Cys Thr Gly Thr Gly Gly Ala
3075 3080 3085
Thr Ala Ala Cys Cys Gly Thr Ala Thr Thr Ala Cys Cys Gly Cys Cys
3090 3095 3100
Thr Thr Thr Gly Ala Gly Thr Gly Ala Gly Cys Thr Gly Ala Thr Ala
3105 3110 3115 3120
Cys Cys Gly Cys Thr Cys Gly Cys Cys Gly Cys Ala Gly Cys Cys Gly
3125 3130 3135
Ala Ala Cys Gly Ala Cys Cys Gly Ala Gly Cys Gly Cys Ala Gly Cys
3140 3145 3150
Gly Ala Gly Thr Cys Ala Gly Thr Gly Ala Gly Cys Gly Ala Gly Gly
3155 3160 3165
Ala Ala Gly Cys Gly Gly Ala Ala Gly Ala Gly Cys Gly Cys Cys Thr
3170 3175 3180
Gly Ala Thr Gly Cys Gly Gly Thr Ala Thr Thr Thr Thr Cys Thr Cys
3185 3190 3195 3200
Cys Thr Thr Ala Cys Gly Cys Ala Thr Cys Thr Gly Thr Gly Cys Gly
3205 3210 3215
Gly Thr Ala Thr Thr Thr Cys Ala Cys Ala Cys Cys Gly Cys Ala Thr
3220 3225 3230
Ala Ala Ala Thr Thr Cys Cys Gly Ala Cys Ala Cys Cys Ala Thr Cys
3235 3240 3245
Gly Ala Ala Thr Gly Gly Thr Gly Cys Ala Ala Ala Ala Cys Cys Thr
3250 3255 3260
Thr Thr Cys Gly Cys Gly Gly Thr Ala Thr Gly Gly Cys Ala Thr Gly
3265 3270 3275 3280
Ala Thr Ala Gly Cys Gly Cys Cys Cys Gly Gly Ala Ala Gly Ala Gly
3285 3290 3295
Ala Gly Thr Cys Ala Ala Thr Thr Cys Ala Gly Gly Gly Thr Gly Gly
3300 3305 3310
Thr Gly Ala Ala Thr Gly Thr Gly Ala Ala Ala Cys Cys Ala Gly Thr
3315 3320 3325
Ala Ala Cys Gly Thr Thr Ala Thr Ala Cys Gly Ala Thr Gly Thr Cys
3330 3335 3340
Gly Cys Ala Gly Ala Gly Thr Ala Thr Gly Cys Cys Gly Gly Thr Gly
3345 3350 3355 3360
Thr Cys Thr Cys Thr Thr Ala Thr Cys Ala Gly Ala Cys Cys Gly Thr
3365 3370 3375
Thr Thr Cys Cys Cys Gly Cys Gly Thr Gly Gly Thr Gly Ala Ala Cys
3380 3385 3390
Cys Ala Gly Gly Cys Cys Ala Gly Cys Cys Ala Cys Gly Thr Thr Thr
3395 3400 3405
Cys Thr Gly Cys Gly Ala Ala Ala Ala Cys Gly Cys Gly Gly Gly Ala
3410 3415 3420
Ala Ala Ala Ala Gly Thr Gly Gly Ala Ala Gly Cys Gly Gly Cys Gly
3425 3430 3435 3440
Ala Thr Gly Gly Cys Gly Gly Ala Gly Cys Thr Gly Ala Ala Thr Thr
3445 3450 3455
Ala Cys Ala Thr Thr Cys Cys Cys Ala Ala Cys Cys Gly Cys Gly Thr
3460 3465 3470
Gly Gly Cys Ala Cys Ala Ala Cys Ala Ala Cys Thr Gly Gly Cys Gly
3475 3480 3485
Gly Gly Cys Ala Ala Ala Cys Ala Gly Thr Cys Gly Thr Thr Gly Cys
3490 3495 3500
Thr Gly Ala Thr Thr Gly Gly Cys Gly Thr Thr Gly Cys Cys Ala Cys
3505 3510 3515 3520
Cys Thr Cys Cys Ala Gly Thr Cys Thr Gly Gly Cys Cys Cys Thr Gly
3525 3530 3535
Cys Ala Cys Gly Cys Gly Cys Cys Gly Thr Cys Gly Cys Ala Ala Ala
3540 3545 3550
Thr Thr Gly Thr Cys Gly Cys Gly Gly Cys Gly Ala Thr Thr Ala Ala
3555 3560 3565
Ala Thr Cys Thr Cys Gly Cys Gly Cys Cys Gly Ala Thr Cys Ala Ala
3570 3575 3580
Cys Thr Gly Gly Gly Thr Gly Cys Cys Ala Gly Cys Gly Thr Gly Gly
3585 3590 3595 3600
Thr Gly Gly Thr Gly Thr Cys Gly Ala Thr Gly Gly Thr Ala Gly Ala
3605 3610 3615
Ala Cys Gly Ala Ala Gly Cys Gly Gly Cys Gly Thr Cys Gly Ala Ala
3620 3625 3630
Gly Cys Cys Thr Gly Thr Ala Ala Ala Gly Cys Gly Gly Cys Gly Gly
3635 3640 3645
Thr Gly Cys Ala Cys Ala Ala Thr Cys Thr Thr Cys Thr Cys Gly Cys
3650 3655 3660
Gly Cys Ala Ala Cys Gly Cys Gly Thr Cys Ala Gly Thr Gly Gly Gly
3665 3670 3675 3680
Cys Thr Gly Ala Thr Cys Ala Thr Thr Ala Ala Cys Thr Ala Thr Cys
3685 3690 3695
Cys Gly Cys Thr Gly Gly Ala Thr Gly Ala Cys Cys Ala Gly Gly Ala
3700 3705 3710
Thr Gly Cys Cys Ala Thr Thr Gly Cys Thr Gly Thr Gly Gly Ala Ala
3715 3720 3725
Gly Cys Thr Gly Cys Cys Thr Gly Cys Ala Cys Thr Ala Ala Thr Gly
3730 3735 3740
Thr Thr Cys Cys Gly Gly Cys Gly Thr Thr Ala Thr Thr Thr Cys Thr
3745 3750 3755 3760
Thr Gly Ala Thr Gly Thr Cys Thr Cys Thr Gly Ala Cys Cys Ala Gly
3765 3770 3775
Ala Cys Ala Cys Cys Cys Ala Thr Cys Ala Ala Cys Ala Gly Thr Ala
3780 3785 3790
Thr Thr Ala Thr Thr Thr Thr Cys Thr Cys Cys Cys Ala Thr Gly Ala
3795 3800 3805
Ala Gly Ala Cys Gly Gly Thr Ala Cys Gly Cys Gly Ala Cys Thr Gly
3810 3815 3820
Gly Gly Cys Gly Thr Gly Gly Ala Gly Cys Ala Thr Cys Thr Gly Gly
3825 3830 3835 3840
Thr Cys Gly Cys Ala Thr Thr Gly Gly Gly Thr Cys Ala Cys Cys Ala
3845 3850 3855
Gly Cys Ala Ala Ala Thr Cys Gly Cys Gly Cys Thr Gly Thr Thr Ala
3860 3865 3870
Gly Cys Gly Gly Gly Cys Cys Cys Ala Thr Thr Ala Ala Gly Thr Thr
3875 3880 3885
Cys Thr Gly Thr Cys Thr Cys Gly Gly Cys Gly Cys Gly Thr Cys Thr
3890 3895 3900
Gly Cys Gly Thr Cys Thr Gly Gly Cys Thr Gly Gly Cys Thr Gly Gly
3905 3910 3915 3920
Cys Ala Thr Ala Ala Ala Thr Ala Thr Cys Thr Cys Ala Cys Thr Cys
3925 3930 3935
Gly Cys Ala Ala Thr Cys Ala Ala Ala Thr Thr Cys Ala Gly Cys Cys
3940 3945 3950
Gly Ala Thr Ala Gly Cys Gly Gly Ala Ala Cys Gly Gly Gly Ala Ala
3955 3960 3965
Gly Gly Cys Gly Ala Cys Thr Gly Gly Ala Gly Thr Gly Cys Cys Ala
3970 3975 3980
Thr Gly Thr Cys Cys Gly Gly Thr Thr Thr Thr Cys Ala Ala Cys Ala
3985 3990 3995 4000
Ala Ala Cys Cys Ala Thr Gly Cys Ala Ala Ala Thr Gly Cys Thr Gly
4005 4010 4015
Ala Ala Thr Gly Ala Gly Gly Gly Cys Ala Thr Cys Gly Thr Thr Cys
4020 4025 4030
Cys Cys Ala Cys Thr Gly Cys Gly Ala Thr Gly Cys Thr Gly Gly Thr
4035 4040 4045
Thr Gly Cys Cys Ala Ala Cys Gly Ala Thr Cys Ala Gly Ala Thr Gly
4050 4055 4060
Gly Cys Gly Cys Thr Gly Gly Gly Cys Gly Cys Ala Ala Thr Gly Cys
4065 4070 4075 4080
Gly Cys Gly Cys Cys Ala Thr Thr Ala Cys Cys Gly Ala Gly Thr Cys
4085 4090 4095
Cys Gly Gly Gly Cys Thr Gly Cys Gly Cys Gly Thr Thr Gly Gly Thr
4100 4105 4110
Gly Cys Gly Gly Ala Thr Ala Thr Cys Thr Cys Gly Gly Thr Ala Gly
4115 4120 4125
Thr Gly Gly Gly Ala Thr Ala Cys Gly Ala Cys Gly Ala Thr Ala Cys
4130 4135 4140
Cys Gly Ala Ala Gly Ala Cys Ala Gly Cys Thr Cys Ala Thr Gly Thr
4145 4150 4155 4160
Thr Ala Thr Ala Thr Cys Cys Cys Gly Cys Cys Gly Thr Thr Ala Ala
4165 4170 4175
Cys Cys Ala Cys Cys Ala Thr Cys Ala Ala Ala Cys Ala Gly Gly Ala
4180 4185 4190
Thr Thr Thr Thr Cys Gly Cys Cys Thr Gly Cys Thr Gly Gly Gly Gly
4195 4200 4205
Cys Ala Ala Ala Cys Cys Ala Gly Cys Gly Thr Gly Gly Ala Cys Cys
4210 4215 4220
Gly Cys Thr Thr Gly Cys Thr Gly Cys Ala Ala Cys Thr Cys Thr Cys
4225 4230 4235 4240
Thr Cys Ala Gly Gly Gly Cys Cys Ala Gly Gly Cys Gly Gly Thr Gly
4245 4250 4255
Ala Ala Gly Gly Gly Cys Ala Ala Thr Cys Ala Gly Cys Thr Gly Thr
4260 4265 4270
Thr Gly Cys Cys Cys Gly Thr Cys Thr Cys Ala Cys Thr Gly Gly Thr
4275 4280 4285
Gly Ala Ala Ala Ala Gly Ala Ala Ala Ala Ala Cys Cys Ala Cys Cys
4290 4295 4300
Cys Thr Gly Gly Cys Gly Cys Cys Cys Ala Ala Thr Ala Cys Gly Cys
4305 4310 4315 4320
Ala Ala Ala Cys Cys Gly Cys Cys Thr Cys Thr Cys Cys Cys Cys Gly
4325 4330 4335
Cys Gly Cys Gly Thr Thr Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala
4340 4345 4350
Thr Thr Ala Ala Thr Gly Cys Ala Gly Cys Thr Gly Gly Cys Ala Cys
4355 4360 4365
Gly Ala Cys Ala Gly Gly Thr Thr Thr Cys Cys Cys Gly Ala Cys Thr
4370 4375 4380
Gly Gly Ala Ala Ala Gly Cys Gly Gly Gly Cys Ala Gly Thr Gly Ala
4385 4390 4395 4400
Gly Cys Gly Cys Ala Ala Cys Gly Cys Ala Ala Thr Thr Ala Ala Thr
4405 4410 4415
Gly Thr Gly Ala Gly Thr Thr Ala Gly Cys Thr Cys Ala Cys Thr Cys
4420 4425 4430
Ala Thr Thr Ala Gly Gly Cys Ala Cys Cys Cys Cys Ala Gly Gly Cys
4435 4440 4445
Thr Thr Thr Ala Cys Ala Cys Thr Thr Thr Ala Thr Gly Cys Thr Thr
4450 4455 4460
Cys Cys Gly Gly Cys Thr Cys Gly Thr Ala Thr Gly Thr Thr Gly Thr
4465 4470 4475 4480
Gly Thr Gly Gly Ala Ala Thr Thr Gly Thr Gly Ala Gly Cys Gly Gly
4485 4490 4495
Ala Thr Ala Ala Cys Ala Ala Thr Thr Thr Cys Ala Cys Ala Cys Ala
4500 4505 4510
Gly Gly Ala Ala Ala Cys Ala Gly Cys Thr Ala Thr Gly Ala Cys Cys
4515 4520 4525
Ala Thr Gly Ala Thr Thr Ala Cys Gly Gly Ala Thr Thr Cys Ala Cys
4530 4535 4540
Thr Gly Gly Cys Cys Gly Thr Cys Gly Thr Thr Thr Thr Ala Cys Ala
4545 4550 4555 4560
Ala Cys Gly Thr Cys Gly Thr Gly Ala Cys Thr Gly Gly Gly Ala Ala
4565 4570 4575
Ala Ala Cys Cys Cys Thr Gly Gly Cys Gly Thr Thr Ala Cys Cys Cys
4580 4585 4590
Ala Ala Cys Thr Thr Ala Ala Thr Cys Gly Cys Cys Thr Thr Gly Cys
4595 4600 4605
Ala Gly Cys Ala Cys Ala Thr Cys Cys Cys Cys Cys Thr Thr Thr Cys
4610 4615 4620
Gly Cys Cys Ala Gly Cys Thr Gly Gly Cys Gly Thr Ala Ala Thr Ala
4625 4630 4635 4640
Gly Cys Gly Ala Ala Gly Ala Gly Gly Cys Cys Cys Gly Cys Ala Cys
4645 4650 4655
Cys Gly Ala Thr Cys Gly Cys Cys Cys Thr Thr Cys Cys Cys Ala Ala
4660 4665 4670
Cys Ala Gly Thr Thr Gly Cys Gly Cys Ala Gly Cys Cys Thr Gly Ala
4675 4680 4685
Ala Thr Gly Gly Cys Gly Ala Ala Thr Gly Gly Cys Gly Cys Thr Thr
4690 4695 4700
Thr Gly Cys Cys Thr Gly Gly Thr Thr Thr Cys Cys Gly Gly Cys Ala
4705 4710 4715 4720
Cys Cys Ala Gly Ala Ala Gly Cys Gly Gly Thr Gly Cys Cys Gly Gly
4725 4730 4735
Ala Ala Ala Gly Cys Thr Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly
4740 4745 4750
Cys Gly Ala Thr Cys Thr Thr Cys Cys Thr Gly Ala Gly Gly Cys Cys
4755 4760 4765
Gly Ala Thr Ala Cys Thr Gly Thr Cys Gly Thr Cys Gly Thr Cys Cys
4770 4775 4780
Cys Cys Thr Cys Ala Ala Ala Cys Thr Gly Gly Cys Ala Gly Ala Thr
4785 4790 4795 4800
Gly Cys Ala Cys Gly Gly Thr Thr Ala Cys Gly Ala Thr Gly Cys Gly
4805 4810 4815
Cys Cys Cys Ala Thr Cys Thr Ala Cys Ala Cys Cys Ala Ala Cys Gly
4820 4825 4830
Thr Ala Ala Cys Cys Thr Ala Thr Cys Cys Cys Ala Thr Thr Ala Cys
4835 4840 4845
Gly Gly Thr Cys Ala Ala Thr Cys Cys Gly Cys Cys Gly Thr Thr Thr
4850 4855 4860
Gly Thr Thr Cys Cys Cys Ala Cys Gly Gly Ala Gly Ala Ala Thr Cys
4865 4870 4875 4880
Cys Gly Ala Cys Gly Gly Gly Thr Thr Gly Thr Thr Ala Cys Thr Cys
4885 4890 4895
Gly Cys Thr Cys Ala Cys Ala Thr Thr Thr Ala Ala Thr Gly Thr Thr
4900 4905 4910
Gly Ala Thr Gly Ala Ala Ala Gly Cys Thr Gly Gly Cys Thr Ala Cys
4915 4920 4925
Ala Gly Gly Ala Ala Gly Gly Cys Cys Ala Gly Ala Cys Gly Cys Gly
4930 4935 4940
Ala Ala Thr Thr Ala Thr Thr Thr Thr Thr Gly Ala Thr Gly Gly Cys
4945 4950 4955 4960
Gly Thr Thr Gly Gly Ala Ala Thr Thr
4965

Claims (10)

1. a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, which is characterized in that including preparation The step of R2 reagent:
S1, Heliobacter pylori antigen albumen is extracted, urease gene is cloned into plasmid, is expressed by Escherichia coli fermentation, Obtain recombinant helicobacterpylori antigen；
S2, anti-helicobacter pylori antigen polyclonal antibody is prepared；
The latex particle of S3, preparation coating anti-helicobacter pylori antigen polyclonal antibody；
S4, by be coated with anti-helicobacter pylori antigen polyclonal antibody latex particle with containing 0.1%-10% electrolyte, The MES buffering of 0.1%-10% stabilizer, 0.1%-10% surfactant, 0.1%-5% preservative and 1-1000mmol/L Liquid mixing to get.
2. it is according to claim 1 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is that for the step S1 specifically, selection urease is as Heliobacter pylori antigen, the urease includes UreA and UreB Protein protomer, by UreA or UreB gene cloning into plasmid, by Escherichia coli fermentation expression and affinitive layer purification, i.e., Heliobacter pylori antigen is obtained, the plasmid includes but is not limited to pGEX-4T-1 plasmid, and the plasmid includes protein tag gene, The protein tag includes but is not limited to GST albumen.
3. it is according to claim 2 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is that the step S2 includes:
S21, Heliobacter pylori antigen equivalent Freund's complete adjuvant is emulsified；
S22, the antigen after emulsification is subjected to animal subcutaneous injection, is immunoreacted；
S23, it takes after immune serum carries out affinity chromatography processing up to anti-helicobacter pylori antigen polyclonal antibody.
4. it is according to claim 3 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is that the step S3 includes the following steps:
S31, latex particle and buffer solution are mixed and added into surfactant, obtain latex particle solution, the latex The mass percent that grain accounts for the latex particle solution is 0.01-10%；
S32, it the anti-helicobacter pylori antigen polyclonal antibody is dissolved in buffer solution obtains antibody-solutions, the anti-pylorus The mass percent that pylori antigen polyclonal antibody accounts for antibody-solutions is 0.01-10%；
S33, after mixing the latex particle solution with the antibody-solutions, 1- (3- dimethylamino-propyl) -3- ethyl is added Carbodiimide hydrochloride, 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride account for the quality hundred of mixed solution Divide than being 0.01%-5%, reacts 2-4 hours at room temperature, obtain the latex of coating anti-helicobacter pylori antigen polyclonal antibody Grain.
5. it is according to claim 4 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is, further includes the steps that preparing R1 reagent, the R1 reagent include the electrolyte of 0.1%-10%, 0.1%-10% it is steady Determine agent, the surfactant of 0.1%-10%, 0.1%-5% preservative, surplus be concentration 1-1000mmol/L MES buffer Liquid.
6. it is according to claim 5 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is, further includes the steps that preparing calibration object: using recombinant helicobacterpylori antigen as mother liquor, with bovine serum albumen solution gradient The recombinant helicobacterpylori antigen is diluted to get the calibration object of various concentration, and the concentration of the bovine serum albumen solution is 1ng/ml-100g/l。
7. it is according to claim 6 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is that the electrolyte in the R1 reagent, R2 reagent includes but is not limited to sodium chloride, and the stabilizer includes but unlimited In mannitol, the surfactant includes but is not limited to Tween 80, and preservative includes but is not limited to Sodium azide.
8. it is according to claim 7 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is that the partial size of the latex particle is 50-500nm.
9. it is according to claim 8 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is, row immune response is injected under Animal Skin described in the step S22 and is carried out 4 times altogether；In the step S23, through parent It is placed in bag filter in TBS buffer with the polyclonal antibody after Image processing, the dialysed overnight at 4 DEG C, the TBS buffering Solution includes the Tween-20 of the NaCl of Tris, 500mM containing 20mM, 0.05%, pH 7.4.
10. it is according to claim 9 for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement, it is special Sign is that the buffer solution that the step S32 is used is the MES buffer solution of 50mM, pH 6.0 or PBS buffer solution.
CN201811158165.6A 2018-09-30 2018-09-30 It is a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement Pending CN109342722A (en)

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