CN106749551A - A kind of method that antibody is produced with multi-epitope peptide fragment combined antigen stimulating immune system - Google Patents
A kind of method that antibody is produced with multi-epitope peptide fragment combined antigen stimulating immune system Download PDFInfo
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- 229940124736 hepatitis-B vaccine Drugs 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000003116 impacting effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005897 peptide coupling reaction Methods 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 230000009385 viral infection Effects 0.000 description 1
- 230000009447 viral pathogenesis Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
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- C12N2770/00011—Details
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- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
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- C12N2770/00011—Details
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Abstract
The invention discloses a kind of method that antibody is produced with multi-epitope peptide fragment combined antigen stimulating immune system, belong to biotechnology, detection of specific antibody and vaccine preparation field.The mode of present invention application peptide fragment combination, is synthesized to substitute former pathogenic microorganism by several important small peptides, can be more pure, and corresponding side reaction is smaller, and immune specific aim will get well compared to complete pathogenic microorganism.
Description
Technical field
The present invention relates to a kind of method that antibody is produced with multi-epitope peptide fragment combined antigen stimulating immune system, belong to biological
Technology, detection of specific antibody and vaccine preparation field.
Background technology
Hepatitis B is caused by hepatitis b virus infected.HBV particles are by hepatitis B surface antigen (HBsAg) and from place
Main lipid composition, is the diameter about spherical structure of 42nm.HBsAg include 3 kinds by membrane glycoprotein, i.e. large protein (LHBs),
Middle albumen (MHBs) and little albumen (SHBs).Hepatitis B high molecular weight protein surface is divided into preceding S1 areas, preceding S2 areas and S areas again.Have
Research shows that PreS1 is existed only in the high molecular weight protein of surface antigen, is the index of early diagnosis of hepatitis B and virus replication,
Played an important role in the assembling and infection of virion；Two important epitopes of greatest concern at present：21-47 amino acid
, there is virus and liver in the epitope (abbreviation P94 epitopes) where the epitope (abbreviation P21 epitopes) and 94-117 amino acid at place
The binding site of cell membrane.
According to HBV whole genome sequence difference>HBV points is nine genotype of A-I by 8%, according to the difference of S genes>4%
HBV gene type can be divided into 20 hypotypes.At present, existed in the world different genotype canonical sequence and as according to
According to establishing Multi-genotype detection method, but these reference strains are generally single patient source, inherently there are various bases and replace
For there is larger difference between situation, and the homologous genes type or hypotype of different regions, corresponding being adapted to need to be set up for different regions
Canonical sequence, polypeptide sequence selection used when this is for specific research causes very big difficulty.
At present, polypeptide is mainly prepared by chemical synthesis and biosynthesis, and the chemical synthesis of wherein polypeptide has liquid phase
Synthesis divides with synthesis in solid state, and every kind of polypeptide synthesis method application mode is different and cuts both ways.For the conjunction of small fragment polypeptide
Solid phase progressively synthetic method is used into (within 30 amino acid, minority is within 50 amino acid), the method is accurate and has more
Specific aim, but with the increase of amino acid quantity, combined coefficient progressively declines, and non-purpose peptide content progressively increases, purpose peptide
Purity is accordingly reduced, and follow-up purifying and repeatability is further difficult.For large fragment polypeptide (containing 100 with upper amino acid) at present
Application liquid phase step synthetic method mostly, principle is according to polypeptide fragment selectivity in the solution or chemo-selective, spontaneous company
Peptide long, but complex steps are connected into, are not easily-synthesized, and precision and specific aim are left to be desired.
Vaccine is by pathogenic microorganism (such as bacterium, rickettsia, virus) and its metabolite, by manually subtracting
Poison, inactivation or using the methods such as genetic engineering be made for the active immunity preparation that keeps off infection.The preparation of current vaccine
Still imperfection, can produce different degrees of side reaction after different crowd vaccine inoculation, and effect is unstable.
The content of the invention
In order to the antigen peptide fragment for solving same gene type is complicated and changeable or antigenic mutation strain hyperplasia, large fragment Peptide systhesis
More difficult present situation, and the unstable problem of effect that vaccine is present at present, the present invention provide a kind of multi-epitope peptide fragment combination
Antigen, and the method that antibody is produced with combined antigen stimulating immune system.The mode of present invention application peptide fragment combination, by several
The synthesis of important small peptide substitutes former pathogenic microorganism, can be more pure, and corresponding side reaction is smaller, and immune specific aim is compared
To be got well in complete pathogenic microorganism.
First purpose of the invention there is provided a kind of combined antigen, and the combined antigen is by multiple epitope peptide fragment groups
Antigen obtained from conjunction.
In one embodiment of the invention, the combined antigen, is for detecting the antibody of hepatitis B virus Pres 1, second
Hepatovirus PreS2 antibody, hepatitis A virus VP1 antibody, hepatitis A virus VP3 antibody, hepatitis C virus E2 antibody etc..
In one embodiment of the invention, the combined antigen, is for detecting anti-hepatitis B virus antibody
Combined antigen.
In one embodiment of the invention, P21 epitopes, the P94 of the antibody of the epitope, including hepatitis B virus Pres 1
Epitope, the 120-145 amino acid epitopes of PreS2 antibody；The 11-25 amino acid epitopes of hepatitis A VP1 antibody, VP3 antibody
102-121 amino acid epitopes；The 384-410 amino acid epitopes of hepatitis C virus E2 antibody, 412-423 amino acid epitopes,
523-535 amino acid epitopes etc..
In one embodiment of the invention, the combined antigen is SEQ ID NO.1 and SEQ comprising amino acid sequence
The peptide fragment of ID NO.2.
In one embodiment of the invention, the amino acid sequence is P21 epitopes for the peptide fragment of SEQ ID NO.1
Peptide fragment；Amino acid sequence is the peptide fragment of P94 epitopes for the peptide fragment of SEQ ID NO.2.
In one embodiment of the invention, the combined antigen be by sequence be SEQ ID NO.1 and SEQ ID
After the peptide fragment of NO.2 is coupled large protein respectively, the compound after coupling is mixed to get.
In one embodiment of the invention, the large protein can be any one in KLH, OVA, BSA etc..
In one embodiment of the invention, amino acid sequence is SEQ ID NO.1, SEQ ID in the combined antigen
The mol ratio of the peptide fragment of NO.2 is 1:10~10:Between 1.
Second object of the present invention is to provide a kind of side with combined antigen stimulating immune system generation specific antibody
Method, methods described is to replace total length peptide fragment to produce specificity as antigenic stimulus immune system in the way of being combined using multi-epitope peptide fragment
Antibody.
In one embodiment of the invention, methods described, is, using the peptide fragment of two or more epitope, to be coupled respectively big
Albumen, then has more than two peptide fragments of large protein as antigen being coupled.
In one embodiment of the invention, the specific antibody refers to the antibody of hepatitis B virus Pres 1, hepatitis B
PreS2 antibody, hepatitis A virus VP1 antibody, hepatitis A virus VP3 antibody, hepatitis C virus E2 antibody etc..
In one embodiment of the invention, P21 epitopes, the P94 of the antibody of the epitope, including hepatitis B virus Pres 1
Epitope, the 120-145 amino acid epitopes of PreS2 antibody；The 11-25 amino acid epitopes of hepatitis A VP1 antibody, VP3 antibody
102-121 amino acid epitopes；The 384-410 amino acid epitopes of hepatitis C virus E2 antibody, 412-423 amino acid epitopes,
523-535 amino acid epitopes etc..
In one embodiment of the invention, the peptide fragment includes that amino acid sequence is SEQ ID NO.1 and SEQ ID
The peptide fragment of NO.2.
In one embodiment of the invention, the amino acid sequence is P21 epitopes for the peptide fragment of SEQ ID NO.1
Peptide fragment；Amino acid sequence is the peptide fragment of P94 epitopes for the peptide fragment of SEQ ID NO.2.
In one embodiment of the invention, described two peptide fragments above, each peptide fragment mol ratio each other exists
1:10~10:Between 1.
In one embodiment of the invention, the large protein can be any one in KLH, OVA, BSA etc..
Beneficial effects of the present invention：
The mode of present invention application peptide fragment combination, is synthesized to substitute former pathogenic microorganism, meeting by several important small peptides
More pure, corresponding side reaction is smaller, and immune specific aim will get well compared to complete pathogenic microorganism.
Brief description of the drawings
Fig. 1：Different coating mode antibody test absorbances and Saliency maps；
Fig. 2：The antibody test absorbance figure of coating P21＆P94-KLH and PreS1 total lengths；
Fig. 3：It is coated with the antibody test absorbance figure of P21-KLH, P94-KLH and PreS1 total length
Fig. 4：The antibody test absorbance Saliency maps of coating P21＆P94-KLH and PreS1 total lengths；
Fig. 5：The antibody test absorbance Saliency maps of coating P21-KLH and PreS1 total lengths；
Fig. 6：The antibody test absorbance Saliency maps of coating P94-KLH and PreS1 total lengths.
Specific embodiment
Below in conjunction with the accompanying drawings, by better embodiment of the present invention, i.e., by by taking 1 B gene type as an example two epitope peptide fragment groups
Close instead of total length detection hepatitis B virus front S 1 antibody to illustrate the inventive method, but do not limit the present invention.
Embodiment 1：Anti-hepatitis B virus former S 1 immune body is detected by way of two epitope peptide fragments are combined instead of total length
AC in indirect ELISA method detection HBV patients serums, the selection of wherein envelope antigen is most important.Often
Rule ELISA method need in advance determine the genotype of HBV patient, according to the suitable coating polypeptide sequence of its selection.But have two
Individual problem：First, at present, HBV points is nine genotype of A-I；Even for the reference strain of syngeneic type, due to regional disparity
And own bases are situations such as replace, there are various different amino acid sequences, select which kind of polypeptide sequence is caused during for research
Difficulty；Second, at present for small fragment polypeptide (being less than 30 amino acid) more using the method for synthesis in solid state, it is easy to synthesize,
But with the increase of amino acid quantity, combined coefficient progressively declines, follow-up purifying and repeatability is further difficult；
Large fragment polypeptide (being more than 100 amino acid) uses liquid phase step synthetic method, but complex steps more, is not easily-synthesized,
And precision and specific aim are left to be desired.
According to the research method of the application, with reference to actual conditions, the method for detection antibody is：Determine several on total length peptide fragment
Important epitope peptide fragment, its immunogenicity is improved by being coupled large protein, by two in total length peptide fragment important epitope peptide fragment groups
Close to carry out detection of specific antibody as antigen instead of total length peptide fragment.
Specific implementation step design is as follows：
Step one：Extract the viral DNA in Chronic Hepatitis B serum
Chronic Hepatitis B blood sample 213 is obtained, blood sample supplier's age range is larger (- 73 years old 7 years old), and male accounts for 61%
(130/213), with Research Significance.Use DNA extraction kit (the MiniBEST Viral RNA/DNA of TAKARA companies
Extraction Kit Ver.5.0, Catalog No.9766), viral gene extraction is carried out to 213 samples, by what is extracted
Hepatitis B virus DNA enters performing PCR and expands as template strand.
Step 2：S1 regions and it is sequenced before PCR amplicon virus
213 DNA samples are entered respectively by PCR (Polymerase Chain Reaction, PCR)
The amplification of row DNA profiling chain, while setting carries out reference substance (being not added with the blank group of DNA profiling), reaction system is 50 μ L,
Each reagent dosage refers to table 1.
To improve product purity, expanded using nest-type PRC, the primer sequence for using is shown in Table 2.
First round PCR response procedures are set to 95 DEG C of predegenerations 3min, 95 DEG C of denaturation 40sec, 55 DEG C of anneal 30sec, 72
DEG C chain elongation 40sec, 72 DEG C of stabilization 3min, carry out 36 circulations altogether.Second wheel PCR response procedures are set to 95 DEG C of predegenerations
3min, 95 DEG C of denaturation 40sec, 50 DEG C of annealing 30sec, 72 DEG C of chain elongations 40sec, 72 DEG C of stabilization 3min, carries out 36 circulations altogether.
Amplified production is identified using 1.2% agarose gel electrophoresis.
213 DNA are extracted in sample, and once expanding unsuccessful sample will carry out second amplification, to exclude PCR experiment
Error.If expanding failed (relevant position is not presented band) twice, then it is assumed that the DNA extraction steps of the sample not into
Work(.
PCR amplifications are：202 DNA extract sample amplification success；11 sample amplification failures.
The PCR reaction system reagent dosage tables of table 1
The primer table that the PCR of table 2 amplifications are used
Step 3：P21 and P94 peptide section sequences in S1 regions before comparing, determine patient's infection P21 and P94 genotype and
Corresponding amino acid sequence
202 pcr amplification products are carried out DNA sequencing by student on commission's work bioengineering (Shanghai) limited company, sequencing
Primer sequence is GGTTGGTCTTCCAAACCTCG (SEQ ID NO:7) it is, unidirectional to survey logical, sequencing fragment length about 600bp.Will be each
After the sequencing result of sample is translated as amino acid sequence, the canonical sequence with P21 and P94 compares respectively, it is determined that sense
Contaminate 14, patients serum's sample of 1 B gene type.
Step 4：By P21B genotype and P94B genotype peptide fragment coupling large protein, to improve its immunogenicity.
P21 the and P94 peptide fragments of 1 B gene type are ordered respectively, and are coupled KLH large proteins；Order the PreS1 of 1 B gene type
Total length peptide fragment (peace is than strange bio tech ltd).
P21 (1 B gene type) peptide section sequence is (SEQ ID NO:1)：PLGFFPDHQLDPAFKANSENPDWDLNP
P94 (1 B gene type) peptide section sequence is (SEQ ID NO:2)：PASTNRQSGRQPTPLSPPLRDTHP
PreS1 total lengths (1 B gene type) peptide section sequence is (SEQ ID NO:8)：
MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDNWPDANKVGVGAFGPGFTP
PHGGLLGWSPQAQGLLTTVPAAPPPASTNRQSGRQPTPLSPPLRDTHPQA
Step 5：Combination coating P21-KLH and P94-KLH peptide fragments, the blood sample of patient 14 of ELISA detection infection 1 B gene types
It is individual.
By 1 B gene type P21-KLH peptide fragments and 1 B gene type P94-KLH peptide fragments in 0.05mol/L, the carbonate buffer of pH9.6
After being diluted with the μ g/ml of concentration 10 in liquid, it is coated with overnight on 96 orifice plates (NEST).With the PBS solution containing 1%BSA by blood to be measured
Clear 125 times of Sample Dilution, 37 DEG C of incubation 1h.ELIAS secondary antibody (the people's IGg antibody isolated from goat body, catalog number (Cat.No.) A-8667,
Sigma Aldrich) dilution 20000 times after, 37 DEG C incubation 1h.Using the TMB of horseradish peroxidase-labeled as colour developing bottom
Thing, after incubation at room temperature 20min, 2M H2SO4Terminating reaction.OD is determined on ELIASA in 10min after terminating reaction450Value.
Each sample does 3 multiple holes.
Step 6：Single coating PreS1 total length peptide fragments, the blood sample of patient 14 of ELISA detection infection 1 B gene types.
After PreS1 total lengths peptide fragment is diluted in 0.05mol/L, the carbonate buffer solution of pH9.6 with the μ g/ml of concentration 10,
It is coated with overnight on 96 orifice plates (NEST).With the PBS solution containing 1%BSA by 125 times of test serum Sample Dilution, 37 DEG C of incubations
1h.ELIAS secondary antibody (the people's IGg antibody isolated from goat body, catalog number (Cat.No.) A-8667, Sigma Aldrich) dilution 20000
After times, 37 DEG C of incubation 1h.Using the TMB of horseradish peroxidase-labeled as chromogenic substrate, after incubation at room temperature 20min, 2M
H2SO4Terminating reaction.OD is determined on ELIASA in 10min after terminating reaction450Value.
Each sample does 3 multiple holes.
Step 7：Single coating P21-KLH peptide fragments, the blood sample of patient 14 of ELISA detection infection 1 B gene types.
After P21-KLH peptide fragments are diluted in 0.05mol/L, the carbonate buffer solution of pH9.6 with the μ g/ml of concentration 10,
It is coated with overnight on 96 orifice plates (NEST).With the PBS solution containing 1%BSA by 125 times of test serum Sample Dilution, 37 DEG C of incubation 1h.
ELIAS secondary antibody (the people's IGg antibody isolated from goat body, catalog number (Cat.No.) A-8667, Sigma Aldrich) dilutes 20000 times
Afterwards, 37 DEG C of incubation 1h.Using the TMB of horseradish peroxidase-labeled as chromogenic substrate, after incubation at room temperature 20min, 2M H2SO4
Terminating reaction.OD is determined on ELIASA in 10min after terminating reaction450Value.
Each sample does 3 multiple holes.
Step 8：Single coating P94-KLH peptide fragments, the blood sample of patient 14 of ELISA detection infection 1 B gene types.
After P94-KLH peptide fragments are diluted in 0.05mol/L, the carbonate buffer solution of pH9.6 with the μ g/ml of concentration 10,
It is coated with overnight on 96 orifice plates (NEST).With the PBS solution containing 1%BSA by 125 times of test serum Sample Dilution, 37 DEG C of incubation 1h.
ELIAS secondary antibody (the people's IGg antibody isolated from goat body, catalog number (Cat.No.) A-8667, Sigma Aldrich) dilutes 20000 times
Afterwards, 37 DEG C of incubation 1h.Using the TMB of horseradish peroxidase-labeled as chromogenic substrate, after incubation at room temperature 20min, 2M H2SO4
Terminating reaction.OD is determined on ELIASA in 10min after terminating reaction450Value.
Each sample does 3 multiple holes.
Step 9：Can combine two peptide fragment combine detections using SPSS software analysis replace total length to detect specific antibody.
For can checking two peptide fragments of combination detection replace total length to detect specific antibody, this patent is soft using SPSS 19.0
Part carries out significance analysis to the detection absorbance of PreS1 antibody in HBV patients serums, and using t methods of inspection, confidence level is
95%.
(1) mass ratio：Combination P21＆P94-KLH peptide fragments, single PreS1 total lengths peptide fragment, single P21-KLH, single P94-
The coating concentration of KLH is 10 μ g/ml, i.e., target envelope antigen is identical in quality.
(2) mole ratio：P21 contains 27 amino acid, and P94 contains 24 amino acid, and PreS1 total lengths contain 119 ammonia
Base acid, KLH is about made up of 3400 amino acid.
Therefore the molal quantity of combination P21＆P94-KLH is only the 3.5% of PreS1 total lengths, it was demonstrated that P21＆P94-KLH peptide fragments
Antibody capture ability is stronger.
(3) significance analysis：(wherein conspicuousness is the single side test of t inspections)
The Analysis of test results of P21＆P94-KLH and PreS1 total lengths is as shown in table 3.
The P21＆P94-KLH of table 3 and PreS1 total lengths coating detection absorbance significance analysis result
AC (remove No. 11) of the AC of coating P21＆P94-KLH detections more than coating PreS1 total length detections
(Fig. 1, Fig. 2), the conspicuousness between two kinds of coating modes is respectively less than 0.05 (*) (Fig. 3).Prove that the combination of two epitope peptide fragments can be carried
The sensitivity of antibody test high, and two epitopes of P21 and P94 can preferably cover the antigen-antibody bound site of PreS1 antibody
The combination of the epitope peptide fragment of point, i.e. P21 and P94 two can effectively replace total length peptide fragment for PreS1 antibody tests.
The Analysis of test results of P21-KLH and PreS1 total lengths is as shown in table 4.
The P21-KLH of table 4 and PreS1 total lengths coating detection absorbance significance analysis result
The P94-KLH of table 5 and PreS1 total lengths coating detection absorbance significance analysis result
The Analysis of test results of P21-KLH and PreS1 total lengths is as shown in table 4.Wherein 12 sample coating P21-KLH detections
AC of the AC more than coating PreS1 total lengths detection, (No. 11 and No. 14) coating P21-KLH of 2 samples detect
AC be slightly less than the AC (Fig. 1, Fig. 3) of coating PreS1 total lengths detection；Wherein 13 the two of sample kinds of coating sides
Conspicuousness between formula is respectively less than 0.05 (*), and 1 conspicuousness of sample (No. 12) is more than 0.05 (Fig. 5).P94-KLH and PreS1
The Analysis of test results of total length is as shown in table 5.(No. 1) AC of coating P94-KLH detections of wherein 1 sample is more than coating
The AC of PreS1 total lengths detection, the AC of remaining 13 sample coating P94-KLH detection is complete less than coating PreS1
The AC (Fig. 1, Fig. 3) of detection long；Conspicuousness between wherein 11 the two of sample kinds of coating modes is respectively less than 0.05
(*), 3 conspicuousnesses of sample (No. 5, No. 7, No. 8) are more than 0.05 (Fig. 6).Detected during single coating P21-KLH or P94-KLH
Antibody is not unique with the notable sexual intercourse of coating total length detection antibody, it was demonstrated that the antigen antibody binding sites of PreS1 antibody are main
In two epitopes of P21 and P94, and single P21 epitopes or P94 epitopes can not be effective over the complete of PreS1 antibody
Portion's antigen antibody binding sites, i.e., single P21 epitopes or P94 epitopes can not effectively replace total length peptide fragment anti-for PreS1
Physical examination is surveyed.
In sum, detect that specificity is anti-by way of two in total length peptide fragment important epitope peptide fragments are combined as antigen
Body, can effectively can be used for effective over whole antigen antibody binding sites of antigen-antibody instead of total length peptide fragment
PreS1 antibody tests, can also improve the detection sensitivity of antibody.
Embodiment 2：Monoclonal antibody is prepared as antigenic stimulus mouse using the combination of two epitope peptide fragments
Step one：Peptide systhesis and coupling
Synthesis P21 and P94 epitope peptide fragments, each peptide fragment is coupled KLH and BSA respectively.The peptide fragment for being coupled KLH will be respectively used to exempt from
Two groups of animals of epidemic disease, and BSA coupled complexes will be used to screen, and be reflected with the antibody excluded for KLH.
Step 2：Animal immune
1) it is immune for the first time：With P21-KLH and P94-KLH according to 1:1 combination, animal is immunized as combined antigen respectively,
Mixing and emulsifying, subcutaneous initial immunity 6 are carried out in equal volume with Freund's complete adjuvant (MP Products, Cat No.642852)
Balb/C mouse, consumption is 60 μ g albumen/200 μ l/ mouse.
2) it is immunized for second：Subcutaneous inoculation, with incomplete Freund's adjuvant (MP Products, Cat No.642862) and group
The isometric mixing and emulsifying of antigen is closed, the amount of being immunized is 30 μ g albumen/200 μ l/ mouse.
3) blood is taken for the first time：Eye socket takes the μ l of blood 100, and after room temperature places half an hour, 2000g is centrifuged 5 minutes, collects serum,
For determining serum titer, measure is finished, and puts -20 DEG C of Cord bloods.
4) third time is immune:With step 2) it is second immune.
5) blood is taken for the second time：Blood is taken with the first time of step 3).
6) it is immunized for the 4th time：With step 2) it is second immune.
7) blood is taken for the third time：Blood is taken with the first time of step 3).
Step 3：Serum titer is determined
Step 4：Screening is compared with clinical sample and confirm
Step 5：Impact is immune
According to mice serum and clinical sample Evaluation result, choosing mouse carries out impacting immune.The immune step of impact
For：Polypeptide coupling KLH albumen is taken out into 50ng, is 0.3ml with normal saline dilution, mouse peritoneal is expelled to 1ml syringes,
This process operation needs slow, it is to avoid puncture mouse intestinal.
Step 6：Cell fusion
Step 7：Positive colony is screened
Step 8：The screening and confirmation of hybridoma cell strain
Step 9：The subclone of hybridoma cell strain
Result shows, combined antigen as antigenic stimulus mouse immune system can be produced corresponding anti-in Mice Body
Body, specific 1 B gene type monoclonal antibody can be obtained by screening and cloning.
To sum up, combination P21-KLH and the epitope peptide fragments of P94-KLH two replace total length peptide fragment as anti-as a combined antigen
Primary stimuli body, the immune system of body can produce corresponding PreS1 antibody, be conducive to body fight virus infection, play epidemic disease
The effect of seedling.Therefore in order to prevent the infection of hepatitis B, can replace with the combined antigen tradition hepatitis B vaccine (inactivation of viruses,
DNA recombinant expression antigen etc.) new generation vaccine is prepared, there are 3 advantages：(1) combined antigen is that multi-epitope small peptide coupling KLH etc. is big
Albumen, significantly enhances the immunogenicity of antigen, compared with original total length peptide fragment, can preferably stimulate body to produce corresponding
Antibody；(2) combined antigen vaccine is more pure, stimulates the side reaction produced after body smaller：The combined antigen is only that solid phase is closed
Into amino acid fragment, compared to inactivation of viruses, viral pathogenesis factor can be greatly reduced；(3) combined antigen vaccine is easier to pure
Change and be obtained：Compared to inactivation of viruses and DNA recombinant expression antigen, clear and definite amino may be selected when prepared by peptide fragment for combined antigen
Acid sequence, therefore the process of later-period purification can be greatly simplified.Therefore, preparing new generation vaccine using combined antigen can solve traditional epidemic disease
The problem that side reaction is larger in seedling preparation and later-period purification process is complicated, and combined antigen can replace the complete micro- life of cause of disease
Thing can strengthen the immune specific aim of vaccine.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes with modification, therefore protection model of the invention
Enclose being defined of being defined by claims.
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Claims (10)
1. a kind of combined antigen, it is characterised in that the combined antigen is antigen obtained from multiple epitope peptide fragments are combined.
2. combined antigen according to claim 1, it is characterised in that the combined antigen is for detecting hepatitis B
PreS1 antibody, the antibody of hepatitis B virus Pres 2, hepatitis A (HAV) virus VP 1 antibody, hepatitis A virus VP3 antibody or hepatitis (HCV)
The antigen of viral E2 antibody.
3. combined antigen according to claim 1, it is characterised in that the epitope, including the antibody of hepatitis B virus Pres 1
P21 epitopes, P94 epitopes, the 120-145 amino acid epitopes of PreS2 antibody；The 11-25 amino acid tables of hepatitis A VP1 antibody
Position, the 102-121 amino acid epitopes of VP3 antibody；The 384-410 amino acid epitopes of hepatitis C virus E2 antibody, No. 412-423
Amino acid epitope, 523-535 amino acid epitopes.
4. combined antigen according to claim 1, it is characterised in that the combined antigen is SEQ comprising amino acid sequence
The peptide fragment of ID NO.1 and SEQ ID NO.2.
5. combined antigen according to claim 1, it is characterised in that it by sequence is SEQ ID that the combined antigen is
After the peptide fragment of NO.1 and SEQ ID NO.2 is coupled large protein respectively, the compound after coupling is mixed to get.
6. combined antigen according to claim 1, it is characterised in that amino acid sequence is SEQ ID in the combined antigen
The mol ratio of the peptide fragment of NO.1, SEQ ID NO.2 is 1:10~10:Between 1.
7. it is a kind of with combined antigen stimulating immune system produce specific antibody method, it is characterised in that methods described be with
The mode of multi-epitope peptide fragment combination replaces total length peptide fragment to produce specific antibody as antigenic stimulus immune system.
8. method according to claim 7, it is characterised in that methods described be using the peptide fragment of two or more epitope, point
Large protein is not coupled, then there are more than two peptide fragments of large protein as antigen being coupled.
9. method according to claim 7, it is characterised in that the specific antibody refer to the antibody of hepatitis B virus Pres 1,
The antibody of hepatitis B virus Pres 2, hepatitis A virus VP1 antibody, hepatitis A virus VP3 antibody, hepatitis C virus E2 antibody.
10. method according to claim 7, it is characterised in that the epitope includes the P21 of the antibody of hepatitis B virus Pres 1
Epitope, P94 epitopes, the 120-145 amino acid epitopes of PreS2 antibody；The 11-25 amino acid epitopes of hepatitis A VP1 antibody,
The 102-121 amino acid epitopes of VP3 antibody；The 384-410 amino acid epitopes of hepatitis C virus E2 antibody, 412-423 ammonia
Base acid epitope, 523-535 amino acid epitopes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363728A (en) * | 2020-01-20 | 2020-07-03 | 武汉大学 | Recombinant influenza A virus carrying hepatitis B virus gene, host cell, preparation method and application thereof |
| CN114907472A (en) * | 2022-04-07 | 2022-08-16 | 武汉爱博泰克生物科技有限公司 | Method for accurately copying antibody and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104781274A (en) * | 2012-11-12 | 2015-07-15 | 海德堡吕布莱希特-卡尔斯大学 | Lipopetides for use in treating liver diseases and cardiovascular diseases |
| CN104961806A (en) * | 2015-06-08 | 2015-10-07 | 吉林大学 | Method for detecting specific antibody through combination of peptide segments of conservation region |
-
2016
- 2016-12-30 CN CN201611253131.6A patent/CN106749551A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104781274A (en) * | 2012-11-12 | 2015-07-15 | 海德堡吕布莱希特-卡尔斯大学 | Lipopetides for use in treating liver diseases and cardiovascular diseases |
| CN104961806A (en) * | 2015-06-08 | 2015-10-07 | 吉林大学 | Method for detecting specific antibody through combination of peptide segments of conservation region |
Non-Patent Citations (5)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363728A (en) * | 2020-01-20 | 2020-07-03 | 武汉大学 | Recombinant influenza A virus carrying hepatitis B virus gene, host cell, preparation method and application thereof |
| CN114907472A (en) * | 2022-04-07 | 2022-08-16 | 武汉爱博泰克生物科技有限公司 | Method for accurately copying antibody and application |
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