CN106928327A - CSFV part epitope polypeptide SE242 and its application - Google Patents

CSFV part epitope polypeptide SE242 and its application Download PDF

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CN106928327A
CN106928327A CN201710213707.4A CN201710213707A CN106928327A CN 106928327 A CN106928327 A CN 106928327A CN 201710213707 A CN201710213707 A CN 201710213707A CN 106928327 A CN106928327 A CN 106928327A
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CN106928327B (en
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银梅
宁红梅
岳峰
刘海文
李鹏
王选年
徐东方
唐海蓉
孔令芸
朱艳萍
张秋雨
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Henan Institute of Science and Technology
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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • G—PHYSICS
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • G01—MEASURING; TESTING
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    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus

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Abstract

The invention discloses CSFV part epitope polypeptide SE242 and its application, the amino acid sequence of described polypeptide SE242 is：SAGPVRKTSCTFNYAKTGKNKYYEPRDSYF.Small peptide of the present invention design synthesis on SE24 polypeptides, has obtained polypeptide SE242.The combination of the cell of polypeptide SE242 and PK 15 and blocking test show, the average fluorescent strength of the cells of polypeptide SE242 combinations PK 15 is significantly higher than FITC positive controls, the average fluorescent strength of the cells of CSFV blocking SE242 combinations PK 15 is significantly lower than binding tests, confirm that polypeptide SE242 can effectively combine the cells of PK 15, and the combination of SE242 and the cells of PK 15 can be blocked by CSFV, the part epitope that SE242 is CSFV combination target cells is determined.Meanwhile, the cell average fluorescent strengths of SE242 combinations PK 15 of the present invention are higher than SE24, and SE24 is significantly higher than by CSFV barrier effects, show SE242 specificity more preferably.

Description

CSFV part epitope polypeptide SE242 and its application
Technical field
The invention belongs to molecular pathology and immunological technique field, and in particular to CSFV part epitope polypeptide SE242 and its application.
Background technology
Swine fever (CSF) is a kind of acute, the hot, highly contagious disease caused by CSFV (CSFV) infection. It is one of infectious disease of pig most serious, huge economic loss is caused to pig industry, is classified as strong by OIE Infectious disease, is also a class infectious disease of the Ministry of Agriculture of China regulation.CSFV belongs to one of flaviviridae, pestivirus member, its base Because group is wire single-stranded positive RNA, 12 kinds of ripe virus proteins can be encoded, wherein C, Erns, E1 and E2 is structural proteins, its Remaining is non-structural protein.Structural proteins Erns, E1 and E2 are in virus to having on the identification of host cell, absorption and virus antigenicity Play an important role.Therefore, the research to CSFV structural proteins is concentrated mainly on this three kinds of albumen.
Viral ligand typically refers to the virus surface molecule specifically bound with virus receptor, i.e., viral absorption egg In vain.Virus receptor refers to that the molecule for positioned at host cell surface being capable of identify that viral ligand and specifically binding therewith is combined Thing.The specific binding of viral ligand and target cells virus receptor is the key link of virus infection, thus block this two The combination of person can effectively suppress the generation of viral disease infection.In recent years, using virus receptor or viral ligand as disease-resistant The target spot of cytotoxic drug, screening antiviral polypeptides medicine and vaccine turn into the focus of research.
Chinese patent discloses CSFV CSFV E2 protein ligands epitope polypeptides and its application (CN2011101126535), its disclosed polypeptid acid sequence is
VHASDERLGPMPCRPKEIGSSAGPVRKTSCTFNYAKTGKNKYYEPRDSYF, its molecular weight is 5.73kDa； And with PK-15 cells as target cell, by the infection of CSFV, harvest, determine the TCID of CSFV50Compare with infection, synthesized Polypeptide and the experiment of the binding tests, viral blocking test and polypeptide blocks CSFV target cell infections of target cell, have screened special Property combination target cell can suppress virus infection part epitope polypeptide, CSFV E2 protein ligands epitopes have been carried out tentatively Positioning.But because the polypeptid acid sequence of above-mentioned patent is more long, part positioning is not accurate enough, is closed in actual application Into high cost, its application is very restricted, therefore being accurately positioned for part epitope polypeptide is particularly important. At present, there is not yet the pinpoint relevant report of E2 protein ligands epitopes.
The content of the invention
In order to solve the above problems, it is an object of the invention to provide CSFV part epitope polypeptide SE242 and its application, The polypeptide can effectively combine PK-15 cells, and the combination of SE242 and PK-15 cells can be blocked by CSFV, it is determined that SE242 is the part epitope of CSFV combination target cells.
To achieve these goals, the technical solution adopted in the present invention is：
CSFV part epitope polypeptide SE242, the amino acid sequence of described polypeptide SE242 is： SAGPVRKTSCTFNYAKTGKNKYYEPRDSYF。
A kind of CSFV part epitope polypeptide SE242 combination PK-15 cells and CFSV blocking SE242 combinations PK-15 are thin Application in born of the same parents' experiment.
A kind of applications of CSFV part epitope polypeptide SE242 in CFSV infection PK-15 cells are suppressed.
A kind of CSFV part epitope polypeptide SE242 is in the medicine or vaccine that suppress CSFV target cell infections is developed Using.
Beneficial effects of the present invention：
1st, small peptide of the present invention design synthesis on SE24 polypeptides, has obtained polypeptide SE242.Polypeptide SE242 and PK-15 The combination of cell and blocking test result show that the average fluorescent strength of polypeptide SE242 combination PK-15 cells is significantly higher than FITC Control group, the average fluorescent strength of CSFV blocking SE242 combination PK-15 cells is significantly lower than binding tests, it was demonstrated that polypeptide SE242 PK-15 cells can be effectively combined, and the combination of SE242 and PK-15 cells can be blocked by CSFV, determine that SE242 is The part epitope of CSFV combination target cells.Meanwhile, SE242 combinations PK-15 cell average fluorescent strengths of the present invention are higher than SE24, quilt CSFV barrier effects are significantly higher than SE24, it was demonstrated that part epitope SE242 specificity is stronger than SE24.
2nd, polypeptid acid sequence disclosed by the invention is shorter, and the part epitope for combining target cell to CSFV is further It is accurately positioned, is greatly reduced than polypeptide cost disclosed in prior art in actual applications.
3rd, the present invention is mutually confirmed by binding tests and blocking test, and CSFV is combined with the virus receptor of PK-15 cells Afterwards, the combination of polypeptide and virus receptor is prevented, and then proves that polypeptide SE242 and CSFV are combined in same cells acceptor.Prompting The polypeptide is the ideal medicament target for preventing and treating swine fever, is that further exploitation and development of new antiviral drugs and vaccine are established Fixed basis.Simultaneously for the preventing and treating of swine fever provides new approach.
Brief description of the drawings
Fig. 1 is the CSFV (200 ×) of immunocytochemistry staining technique detection culture breeding.In figure, A is infection CSFV PK-15 cells；B is blank cell.
Fig. 2 is the LC-MS/MS collection of illustrative plates of polypeptide SE242.Abscissa represents the mass-to-charge ratio value (m/z) of ion, and ordinate is represented The intensity (most strong ion intensity of flow is 100%) of ion stream.
Fig. 3 is combined and blocking test result for polypeptide SE242 with PK-15 cells.In figure, A is compareed for FITC；B is polypeptide SE242 is combined with PK-15 cells；C is the combination that CSFV blocks polypeptide SE242 and PK-15 cell.
Specific embodiment
Specific embodiment of the invention is described in further detail with reference to embodiments.
Embodiment 1
The present invention carries out many index measure to Classical Swine Fever Virus Shimen Strain (cell toxicant) used, to determine this plant of Strain Shimen (cell toxicant) CSFV disclosure satisfy that use requirement of the invention.
1st, the ELISA titrations of CSFV
Classical Swine Fever Virus Shimen Strain (cell toxicant) is infected into cultured individual layer PK-15 cells, after 37 DEG C are cultivated through 48-56h Virus is harvested, 3 smudge cellses of multigelation crack out virus, and 4 DEG C, 3000rpm centrifugation 10min discard precipitation, and supernatant is Propagative viruses liquid.Detected with CSFV antigen detection kit (SERELISA HCV Ag Mono Indirect products) and received The virus liquid for obtaining.By virus liquid 1:50、1:100、1:200、1:400 times of dilutions, each dilution factor does 6 repetitions, detection knot Fruit is shown in Table 1.With with negative control OD450The ratio of value be 2.2 times and total value >=0.2 dilution factor be virus potency, from table As can be seen that the potency of virus liquid is 1:200.
Table 1 detects the potency of CSFV
Note："+" represents the positive, and "-" represents feminine gender.
2nd, the TCID of CSFV50Determine
Detect the appeal of viral cultures, i.e. virulence.The measure of virus virulence has 2 kinds of methods, and a kind of method is to use Experimental animal determines median lethal dose (LD50) or half egg infectious amount (ELD is determined in chicken embryo50)；Another method be Half cell culture infective amount (TCD is determined on histocyte50Also known as TCID50)。TCID50Reed-Muench Liang Shi can be used Method, interpolation method, Karber methods are calculated.The present invention calculates the TCID of propagative viruses using the fixed viral method of PK-15 cells dilution50, 2 are the results are shown in Table, is 10 in virus liquid dilution factor-1、10-2、10-3When, 4 hole cells of inoculation all infect, and dilution factor is 10-4It is, There are 2 holes to infect, dilution factor is 10-5、10-6When all do not infect, normal cell as blank all do not infect.According to upper State result of the test and calculate TCID using Reed-Muench Liang Shi methods50。
Table 2 fixes the TCID that the viral method of PK-15 cells dilution calculates CSFV50
Logarithm in this experiment higher than 50% viral dilution is -3, and distance proportion is 0.5, the difference between dilution factor logarithm For -1.
LogTCID50=0.5 × (- 1)+(- 3)=- 3.5
TCID50=10-3.5, implication is that virus liquid is diluted into 103.5Times, 100 μ L virus liquids are inoculated with per hole, half can be made thin Born of the same parents infect.
3rd, the measure of CSFV infection multiplicities (MOI)
Infection multiplicity or infection are than (multiplicity of infection)：Refer to and infect virus in a system The ratio between cell number and TCS.By 2 × TCID50After CSFV infection PK-15 cells, detected with immunocytochemistry staining technique The CSFV of breeding is cultivated, brownish red (Figure 1A) is shown after CSFV infection PK-15 cells, normal control PK-15 cells do not develop the color and (scheme 1B).The TCS of infection cell and 96 porocyte culture plates per hole is counted when calculating MOI, result of the test is then analyzed.Carefully When born of the same parents count, a drop cell suspension is taken on cell counting count board, four block plaids in clinodiagonal are calculated under inverted microscope In TCS, computing formula is：
Per hole TCS=n/4 × extension rate × 104(n is that the cell of four block plaids is total to × cell liquid milliliter number Number).
As a result：The every hole cell average infected in 96 porocyte culture plates is 150；Four block plaids of tally it is thin Born of the same parents' sum is 280, is 0.1ml per hole cell liquid milliliter number, and extension rate is 10-1Times, it is computed learning the total of every hole cell Number is 7.0 × 103It is individual, so the MOI of culture propagative viruses is：1:47.
4th, conclusion
By the ELISA potency, the TCID that determine CSFV50With infection multiplicity (MOI), it was confirmed that this plant of Strain Shimen (cell toxicant) The need for CSFV can meet present invention research.
Embodiment 2, synthesis polypeptide
Research shows, polypeptide SE24 (amino acid sequences： CVHASDERLGPMPCRPKEIGSSAGPVRKTSCTFNYAKTGKNKYYEPRDSYF PK-15 cells) can be effectively combined, can Effectively to suppress CSFV infection PK-15 cells, and CSFV can block SE24 combination PK-15 cells, it is therefore evident that polypeptide SE24 It is CSFV CSFV E2 protein ligands epitope polypeptides.To be further accurately positioned the part epitope information on polypeptide SE24, if 1 small peptide on polypeptide SE24 is counted and has synthesized, the LC-MS/MS collection of illustrative plates of synthetic peptide SE242 is shown in Fig. 2, and sequence is as follows：
SE242：SAGPVRKTSCTFNYAKTGKNKYYEPRDSYF(SEQ ID NO.1)
Embodiment 3, the combination of SE242 and PK-15 cells and blocking test
Well-grown PK15 cells are taken, PBS is washed 3 times, and vitellophag is carried out with 1% trypsase, about digests 1min, is abandoned Fall digestive juice, plus the DMEM containing 10% hyclone is suspended, 1000rpm is centrifuged 5min, thin with the resuspended PK15 of appropriate PBS Born of the same parents, count, adjustment cell concentration to 1.0 × 106Individual/ml, it is standby.
The binding tests of SE242 and PK-15 cells：
By above-mentioned PK15 cell suspensions, (cell concentration is 1.0 × 106Individual/ml), it is placed in EP pipes after being well mixed, often manage In 100 μ l, i.e. cell number reaches 1.0 × 105It is individual.Yu Guanzhong addition polypeptides SE242 (FITC marks) reaches SE242 final concentrations To 0.2mg/ml, while setting FITC positive controls, three repetitions are done, lucifuge acts on 2h on ice, PBS is washed 3 times, washed every time 1000rpm is centrifuged 5min, and last time adds 800 μ l PBS re-suspended cells, flow cytomery to count 10000 after being centrifuged Cell, decision analysis result.
CSFV blocks SE242 and PK15 Cell binding assays：
(cell concentration is 1.0 × 10 to take the above-mentioned PK15 cell suspensions of 1.0ml6Individual/ml), plus 200 μ l TCID50It is 10-3.5 CSFV liquid, be well mixed, 4 DEG C effect 40min.Centrifugation discards CSFV liquid, then is washed 2 times with PBS, finally uses PBS re-suspended cells, It is sub-packed in EP pipes after well mixed, cell number reaches 1.0 × 10 in making every pipe5It is individual.
Yu Guanzhong addition polypeptides SE242 (FITC marks) makes SE242 final concentrations reach 0.2mg/ml, while setting FITC sun Property control, do three repetitions, the 2h of lucifuge effect on ice, PBS washed 3 times, and 1000rpm centrifugation 5min are washed every time, and last time is centrifuged Afterwards plus 800 μ l PBS re-suspended cells, flow cytomery, counts 10000 cells, decision analysis result.
Binding tests and blocking test show that the average fluorescent strength of polypeptide SE242 combination PK-15 cells is significantly higher than FITC control groups, the average fluorescent strength of CSFV blocking SE242 combination PK-15 cells is significantly lower than binding tests (Fig. 3, table 3), Confirm that polypeptide SE242 can effectively combine PK-15 cells, and the combination of SE242 and PK-15 cells can be blocked by CSFV, Determine the part epitope that SE242 is CSFV combination target cells.The further accurate part epitope information of CSFV combination target cells, The medicine or vaccine for suppressing CSFV target cell infections to develop lay the foundation.
The polypeptide SE242 of table 3 is combined and CSFV blocking polypeptide binding tests results with PK-15 cells
Note：The different letters of colleague's data shoulder mark represent significant difference (P ＜ 0.05)；Shoulder mark same letter or without letter Represent difference not significantly (P ＞ 0.05).
Table 4 SE24, SE242 polypeptide is combined and CSFV blocking polypeptide binding tests results contrasts with PK-15 cells
Note：The different letters of colleague's data shoulder mark represent significant difference (P ＜ 0.05)；Shoulder mark same letter or without letter Represent difference not significantly (P ＞ 0.05).
SE24, SE242 and PK15 Cell binding assay fluorescence intensity significant difference, SE24, SE242 as can be seen from Table 4 By CSFV blocking test fluorescence intensity significant differences.SE242 is combined with PK-15 cells and its combination is notable by CSFV barrier effects Higher than SE24.
The combination checking of embodiment 4, other small peptides
Show that SE242 can effectively combine PK-15 cells, and CSFV can by above in association with experiment and blocking test result It is the part epitope information being further accurately positioned on SE24 polypeptides to block SE242 combination PK-15 cells, designs and close Into 3 small peptides on SE24 polypeptides, sequence is as follows：
SE24-1：CVHASDERLGPMPCRPKEIG(SEQ ID NO：2)
SE24-2：PKEIGSSAGPVRKTSCTFNYA(SEQ ID NO：3)
SE24-3：TFNYAKTGKNKYYEPRDSYF(SEQ ID NO：4)
Above-mentioned 3 small peptides are by binding tests repeatedly with PK-15 cells, it was demonstrated that this three small peptides not with PK-15 cells With reference to.
After SE24 polypeptide designs are synthesized 1 small peptide SE242 by the present invention, the small peptide is still combined with target cell, and in knot Close experiment and virus blocking binding tests show that the effect that SE242 is combined with target cell and its combination is blocked by CSFV is obvious Higher than SE24.Prove that polypeptide SE242 of the invention has specificity and accuracy well.Further SE24 polypeptide designs are closed Into after 3 small peptides, 3 small peptides are not combined with target cell, illustrate that being more accurately positioned CSFV part epitopes needs further screening Research.
The optimal embodiment of the present invention is the foregoing is only, for a person skilled in the art, the present invention can have Various modifications and variations.All any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., all should It is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Henan Science and Technology College
<120>CSFV part epitope polypeptide SE242 and its application
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> PRT
<213>Artificial sequence
<400> 1
Ser Ala Gly Pro Val Arg Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys
1 5 10 15
Thr Gly Lys Asn Lys Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe
20 25 30
<210> 2
<211> 20
<212> PRT
<213>Artificial sequence
<400> 2
Cys Val His Ala Ser Asp Glu Arg Leu Gly Pro Met Pro Cys Arg Pro
1 5 10 15
Lys Glu Ile Gly
20
<210> 3
<211> 21
<212> PRT
<213>Artificial sequence
<400> 3
Pro Lys Glu Ile Gly Ser Ser Ala Gly Pro Val Arg Lys Thr Ser Cys
1 5 10 15
Thr Phe Asn Tyr Ala
20
<210> 4
<211> 20
<212> PRT
<213>Artificial sequence
<400> 4
Thr Phe Asn Tyr Ala Lys Thr Gly Lys Asn Lys Tyr Tyr Glu Pro Arg
1 5 10 15
Asp Ser Tyr Phe
20

Claims (4)

1. CSFV part epitope polypeptide SE242, it is characterised in that the amino acid sequence of described polypeptide SE242 is： SAGPVRKTSCTFNYAKTGKNKYYEPRDSYF。
2. a kind of CSFV part epitope polypeptide SE242 combination PK-15 cells and CFSV block polypeptide SE242 combinations PK-15 Application in test cell line.
3. applications of a kind of CSFV part epitope polypeptide SE242 in CFSV infection PK-15 cells are suppressed.
4. a kind of CSFV part epitope polypeptide SE242 develop suppress CSFV target cell infections medicine or vaccine in should With.
CN201710213707.4A 2017-04-01 2017-04-01 Hog cholera virus ligand epitope polypeptide SE242 and application thereof Expired - Fee Related CN106928327B (en)

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CN1338310A (en) * 2000-08-10 2002-03-06 清华大学 Epitope vaccine of hog cholera virus and its preparing process
CN102268079A (en) * 2011-05-03 2011-12-07 新乡学院 (Classical swine fever virus) CSFV E2 protein ligand epitope peptide and application thereof
CN106188250A (en) * 2016-03-25 2016-12-07 武汉科前生物股份有限公司 The epitope simulative peptide of a kind of CSFV E 2 protein and preparation method and application
CN106456741A (en) * 2014-05-23 2017-02-22 勃林格殷格翰动物保健有限公司 Recombinant classical swine fever virus (csfv) comprising substitution in the tav epitope of the e2 protein

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Publication number Priority date Publication date Assignee Title
CN1338310A (en) * 2000-08-10 2002-03-06 清华大学 Epitope vaccine of hog cholera virus and its preparing process
CN102268079A (en) * 2011-05-03 2011-12-07 新乡学院 (Classical swine fever virus) CSFV E2 protein ligand epitope peptide and application thereof
CN106456741A (en) * 2014-05-23 2017-02-22 勃林格殷格翰动物保健有限公司 Recombinant classical swine fever virus (csfv) comprising substitution in the tav epitope of the e2 protein
CN106188250A (en) * 2016-03-25 2016-12-07 武汉科前生物股份有限公司 The epitope simulative peptide of a kind of CSFV E 2 protein and preparation method and application

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108084250A (en) * 2017-11-10 2018-05-29 浙江美保龙生物技术有限公司 One species specificity inhibits micromolecule polypeptide and its application of swine fever virus multiplication
CN108084250B (en) * 2017-11-10 2021-07-06 浙江美保龙生物技术有限公司 Small molecular polypeptide for specifically inhibiting hog cholera virus proliferation and application thereof

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