CN105254725B - A kind of preparation and application of anti-Cbk1 monoclonal antibody - Google Patents
A kind of preparation and application of anti-Cbk1 monoclonal antibody Download PDFInfo
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- CN105254725B CN105254725B CN201410333699.3A CN201410333699A CN105254725B CN 105254725 B CN105254725 B CN 105254725B CN 201410333699 A CN201410333699 A CN 201410333699A CN 105254725 B CN105254725 B CN 105254725B
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Abstract
The present invention relates to bioengineering technical fields, a kind of present invention provides antifungal cell wall protein Cbk1 monoclonal antibody, is used to prepare the monoclonal antibody polypeptide, the hybridoma cell strain for secreting the monoclonal antibody and the monoclonal antibody, the polypeptide for being used to prepare anti-Cbk1 monoclonal antibody, hybridoma cell strain are in the application in the reagent or kit for preparing Western Blot or immunohistochemistry detection infection by Candida albicans and the application in preparation prevention or treatment infection by Candida albicans drug.
Description
Technical field
The present invention relates to bioengineering technical fields, and in particular to a kind of anti-Cbk1 monoclonal antibody and the list
Application of the clonal antibody in being infected for antifungal.
Background technique
Cell wall protein Cbk1 (Cell wall Biosynthesis Kinase) is the budding of candida albicans yeast state cell
It is proliferated indispensable gene, is the indispensable gene of bacterial strain polarization growth, CBK1 gene delection bacterium is limited because going out bud fission, and double-core is presented；
Meanwhile Cbk1 albumen is also candida albicans hyphal growth indispensable gene, improve bacterial strain to cell membrane and cell wall damage stress
Tolerance, CBK1 gene delection bacterium can not form mycelia, pathogenicity decline, to cell membranes such as Congo red, fluorescence is white, hygromycin
And the sensibility of cell wall damage stimulation significantly increases.Cbk1 albumen be positioned at candida albicans yeast state cell budding neck ring and
Mycelia tip adjusts yeast state cell with Mob2 albumen jointly and goes out bud fission and mycelia growth.Due to Cbk1 albumen not there is only
In yeast state cell surface, and it is positioned at hyphal surface, moreover it is possible to promote candida albicans cell Proliferation, mycelia to be formed, Yi Jiying
To extraneous various stimulations, therefore, Cbk1 albumen is a very promising antimycotic target based on Antybody therapy.
It there is no the document report in relation to anti-Cbk1 monoclonal antibody at present, also nonreactive Cbk1 monoclonal antibody is used for anti-invasion
The document report of the infection of property candida albicans.
Summary of the invention
The purpose of the present invention is to provide a kind of antifungal cell wall protein Cbk1 monoclonal antibody, it is used to prepare this
The application of the polypeptide of monoclonal antibody, the hybridoma cell strain for secreting the monoclonal antibody and the monoclonal antibody.
The first aspect of the present invention, being to provide one kind, to be used to prepare antifungal cell wall protein (Cbk1) monoclonal anti-
The polypeptide of body, amino acid sequence such as SEQ ID NO：Shown in 1 or 2.
DSYMDEDTSE(SEQ ID NO：1)
PVQQHPQFQP(SEQ ID NO：2)
The second aspect of the present invention is to provide a kind of anti-Cbk1 monoclonal antibody, be by deposit number be CCTCC No：
C201481 or CCTCC No：The hybridoma cell strain of C201482, which is secreted, to be generated.
The third aspect of the present invention, is to provide two strain of hybridoma strains, and deposit number is CCTCC No：C201481
Or CCTCC No：C201482.
The step of the fourth aspect of the present invention, is to provide a kind of preparation method of anti-Cbk1 monoclonal antibody, this method is such as
Under：
A) synthetic amino acid array such as SEQ ID NO：Polypeptide shown in 1 or 2；
B) aforementioned polypeptides are imported into expression vector, express and purified, as immunogen immune mouse；
C) splenocyte of immunized mice is taken to merge；
D) cell clone for going out synthesis polypeptide reacting positive through multi-turns screen, the hybridoma as anti-Cbk1 monoclonal antibody
Cell strain CCTCC No：C201481 or CCTCC No：C201482；
E) by producing ascites antibody in mouse Inoculation hybridoma, ascites is purified, anti-Cbk1 is obtained
Monoclonal antibody.
The fifth aspect of the present invention is to provide the above-mentioned polypeptide for being used to prepare anti-Cbk1 monoclonal antibody and is preparing
The reagent or the application in kit of Western Blot or immunohistochemistry detection infection by Candida albicans.
And above-mentioned anti-Cbk1 monoclonal antibody detects candida albicans sense in preparation Western Blot or immunohistochemistry
The reagent of dye or the application in kit.
And above-mentioned hybridoma cell strain is preparing Western Blot or immunohistochemistry detection infection by Candida albicans
Application in reagent or kit.
Further, the present invention also provides the above-mentioned polypeptides for being used to prepare anti-Cbk1 monoclonal antibody prevents in preparation
Or the application in treatment infection by Candida albicans drug.
And application of the above-mentioned anti-Cbk1 monoclonal antibody in preparation prevention or treatment infection by Candida albicans drug.
And application of the above-mentioned hybridoma cell strain in preparation prophylactic treatment infection by Candida albicans drug.
The specific technical solution of the present invention is as follows：
1. being directed to encoder block overall amino acid sequence (the GENBANK ID of anti-Cbk1：3634838) it designs and synthesizes more than 16
Peptide, sequence are respectively：
Aforementioned polypeptides are directed respectively into expression vector.
2. the identification of positive colony, is extracted and expression bacterium converts.
3. a large amount of inducing expression productions.
4.Ni column affinity purification obtains proteantigen for being immunized by purifying.
5. female BAl BIc/c the healthy mice 3 for being 8~12 weeks by the antigen mixed immunity age of mouse successfully obtained.It takes small
The spleen of 3 mouse is mixed and is merged by mouse tachysynthesis mode.
6. going out the cell clone of reacting positive through multi-turns screen, the hybridoma as the anti-Cbk1 protein monoclonal antibody of anti-mouse
Cell strain 3553-1hz-6M456/4B7_111202,3553-1hz-6M456/1J4_111128.
7. ascites is purified, it is anti-to obtain anti-mouse by producing ascites antibody in mouse Inoculation hybridoma
The monoclonal antibody of Cbk1.
The present invention provides the monoclonal antibody of anti-Cbk1, which identifies Cbk1 protein amino acid sequence respectively
DSYMDEDTSE and PVQQHPQFQP.This two polypeptides are that 16 above-mentioned polypeptide expression are immunized after purification, what final choice arrived
Generate the targeted epitope of the higher antibody of potency ratio.
The method that the acquisition of monoclonal antibody of the invention uses the art routine, i.e., by inscribed in Mice Body
Kind hybridoma simultaneously generates ascites antibody, and taking-up ascites is purified and obtains.
Monoclonal antibody prepared by the present invention is a kind of immunoglobulin, can specifically bind the albumen of candida albicans.
The present invention also provides the monoclonal antibodies of above-mentioned anti-Cbk1, hybridoma cell strain answering in detection candida albicans
With, in particular to preparing application in Western Blot or immunohistochemistry detection reagent or kit.The present invention is anti-
The clinical anti-fungal infection immunization therapy application of Cbk1 provides new thinking.
Detailed description of the invention
Fig. 1 is the figure of monoclonal antibody Western Blot detection candida albicans albumen.
Fig. 2 is Immunofluorescence test figure, and wherein A is monoclonal antibody immunity fluorescence detection and candida albicans yeast state cell
In conjunction with figure；B is the figure of monoclonal antibody immunity fluorescence detection Yu candida albicans hyphal state cell combination.
Fig. 3 is the knot with the anti-Cbk1 monoclonal antibody of ELISA method detection and fungi living cells and cell wall protein and cytoplasm protein
Curve graph is closed, wherein A is the binding curve figure with ELISA method detection anti-Cbk1 monoclonal antibody and fungi living cells；B is to use ELISA method
Detect the binding curve figure of anti-Cbk1 monoclonal antibody and cell wall protein；C is with the anti-Cbk1 monoclonal antibody of ELISA method detection and cytoplasm protein
Binding curve figure.
Fig. 4 is candida albicans with 2 × 105Under cells/ mouse challenge dose, 1mg/kg monoclonal antibody (CCTCC No：
C201481 protective effect figure).
Fig. 5 is candida albicans with 1 × 105Under cells/ mouse challenge dose, 1mg/kg monoclonal antibody (CCTCC No：
C201481 protective effect figure).
Fig. 6 is candida albicans with 1 × 105Under cells/ mouse challenge dose, it is anti-to give 1mg/kg monoclonal once or twice
Body, and once give 2mg/kg monoclonal antibody (CCTCC No：C201481), systemic infection by Candida albicans is protected and is made
With figure.
Fig. 7 is candida albicans with 1 × 106Under cells/ mouse challenge dose, 2mg/kg monoclonal antibody (CCTCC No：
C201481) to the Scavenging activity figure of Mouse Liver kidney enrichment bacterium colony.
Fig. 8 is candida albicans with 1 × 105Under cells/ mouse challenge dose, 2mg/kg monoclonal antibody (CCTCC is given
No：C201481), to the therapeutic effect figure of mice with systemic Candida albicans infection.
Fig. 9 is candida albicans with 1 × 105Under cells/ mouse challenge dose, 4mg/kg monoclonal antibody (CCTCC is given
No：C201481), to the therapeutic effect figure of mice with systemic Candida albicans infection.
Figure 10 is candida albicans with 1 × 105Under cells/ mouse challenge dose, various dose monoclonal antibody (CCTCC is given
No：C201482 protective effect figure).
Hybridoma cell strain of the invention, is named as 3553-1hz-6M456/4B7_111202, is preserved in Chinese Typical Representative training
Support object collection（Wuhan, China Wuhan University）(abbreviation CCTCC), preservation date on June 25th, 2014, deposit number
CCTCC No：C201481.
Hybridoma cell strain of the invention, is named as 3553-1hz-6M456/1J4_111128, is preserved in Chinese Typical Representative training
Support object collection（Wuhan, China Wuhan University）(abbreviation CCTCC), preservation date on July 11st, 2014, deposit number
CCTCC No：C201482.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be described in detail.But the following example should not be regarded as to the present invention
The limitation of range.Experimental method in following embodiments is unless otherwise specified conventional method.
The preparation of the polypeptide sequence selection and polypeptide of the monoclonal antibody of the anti-Cbk1 of embodiment 1.
The selection of polypeptide sequence
Cbk1 albumen has 732 amino acid (GENBANK ID：3634838) it, is entrusted and is ended according to its amino acid sequence information
Than the design of Ma Te biological medicine (Shanghai) Co., Ltd., screens and synthesized following two polypeptide sequences for Peptide systhesis：
DSYMDEDTSE(SEQ ID NO：1)
PVQQHPQFQP(SEQ ID NO：2)
The preparation and purification of the monoclonal antibody of the anti-Cbk1 of embodiment 2.
Ai Bimate biological medicine (Shanghai) Co., Ltd. is entrusted to carry out the preparation and purification of monoclonal antibody.
1. the production of antigen
The full genome of aforementioned polypeptides synthesizes, and gene order refers to candida albicans gene database (Candida Genome
Database).Expression vector establishment；Positive clone identification；Positive colony plasmid is extracted and expression bacterium conversion；A large amount of inducing expressions
Production；Ni column affinity purification；By purifying, proteantigen is obtained for being immunized.
2. the preparation and purification of monoclonal antibody
Female BAl BIc/c the healthy mice 3 for being 8~12 weeks by the antigen mixed immunity age of mouse successfully obtained.Take mouse
The spleen of 3 mouse is mixed and is merged by tachysynthesis mode.
The splenocyte for being immunoreacted best mouse is merged with myeloma cell (SP2/0), fused cell warp
Appropriate dilution is crossed, is placed in 96 well culture plates and cultivates, progress ELISA detection in 10-14 days is cultivated, selects in the high hole of OD value
Cell carries out limiting dilution assay subclone.
The specific method is as follows：By the cell culture of limiting dilution into 96 orifice plates, when the 1/6 of clonal growth to complete opening,
Monoclonal and polyclonal is marked, ELISA detection is carried out to monoclonal.It is after ELISA detection that the highest monoclonal of OD value is limited again
It being as above subcloned described in method again in dilution 96 orifice plates of access, this process is repeated several times, until positive hole ratio is 100%, I
Think this for monoclonal.What i.e. we had been generally acknowledged that builds the successful cell strain of strain.The positive monoclonal that screening is obtained expands
Big culture, cell number press 1-2 × 106/ pipe is frozen.It is collected simultaneously cell and arranges ascites preparation.
Cell strain prepares ascites, 10-14 days collection ascites using mice celiac inoculation, and whether ELISA detection ascites is made
Standby success.Protein G column purification ascites is used after the completion of ascites.
The identification and detection application of the monoclonal antibody of the anti-Cbk1 of embodiment 3.
1. the Western Blot of monoclonal antibody detects application
The candida albicans cell wall protein sample of denaturation carries out SDS-PAGE protein electrophoresis；After electrophoresis, carry out half-dried
Method transferring film.Confining liquid is added to close at least 1h in room temperature；PBST washing lotion is washed film 5 times, uses monoclonal antibody as primary antibody, 4 DEG C of incubations
Overnight；It is washed film 5 times after incubation with PBST washing lotion, adds secondary antibody DYLightTM800-Labeled Antibody to Mouse
IgG(H+L)1:It is incubated at room temperature 2h after 10000 dilutions, slight oscillation during incubation；It is washed film 5 times with PBST washing lotion, NC film is used
The scanning of Odyssey infrared imaging system.The result is shown in Figure 1.The result shows that the monoclonal antibody more can specifically identify white beads
The cell wall protein of bacterium.
The Immunofluorescence test application of 2 monoclonal antibodies
Fungi yeast state cell and mycelia state cell concentration are adjusted to a certain concentration, PBS is cleaned 3 times, abandons supernatant, is added
1%BSA solution 1ml, room temperature close 1h.Supernatant is abandoned in centrifugation, and PBS is washed 3 times.Anti- CBK1 antibody (being diluted with PBS), control group is added
PBS is added.On vertical rotary instrument, 4 DEG C of incubation 2h.Supernatant is abandoned in centrifugation, and PBS is washed 3 times.The fluorescence secondary antibody of addition FITC label, 4
DEG C be incubated for 1h.Supernatant is abandoned in centrifugation, and PBS is washed 3 times, and PBS is resuspended.It takes 7 μ l bacterium solutions drop on glass slide, uses laser confocal microscope
Fluorescence is observed under 488nm exciting light.Fig. 2 .A is monoclonal antibody immunity fluorescence detection and candida albicans yeast state cell combination
Figure, B is the figure of monoclonal antibody immunity fluorescence detection Yu candida albicans hyphal state cell combination
The result shows that the monoclonal antibody more can specifically identify candida albicans.
The ELISA of 3 monoclonal antibodies detects application
96 hole elisa plates every hole inoculation 0.5 μ g candida albicans SC5314 cell wall extraction albumen, or inoculation 1 ×
106The candida albicans living cells of cells/ml, 4 DEG C of coatings overnight, are washed 3 times, each 5min is closed, often with confining liquid with PBST
Hole 200 μ l, 37 DEG C of incubation 2h；PBST is washed 3 times, each 5min；Anti- Cbk1 the antibody 100 μ l, antibody myc of different dilutions is added
As negative control, 37 DEG C of incubation 2h；PBST is washed 3 times, each 5min；It is separately added into the secondary antibody IgG100 of HRP conjugated
μ l, 37 DEG C of incubation 1h；PBST is washed 5 times, each 5min；The TMB of 200 μ l, room temperature are protected from light at colour developing 10min, 370nm and survey its extinction
Degree shows；The 2M H2SO4 of 50 μ l terminates reaction, its absorbance is surveyed at 450nm.As a result see that Fig. 3 shows with the anti-Cbk1 of ELISA
The combination of monoclonal antibody and candida albicans living cells and cell wall protein and cytoplasm protein.
The monoclonal antibody of the anti-Cbk1 of embodiment 4. is directed to the protection of infection by Candida albicans
1) Cbk1 antibody is protected in advance with 1mg/kg, gives candida albicans SC53142 × 10 after 2h respectively5/ only, 1 × 105/
Only attack mouse, after 40d, mouse survival rate is respectively 50%, 60% or more.As a result see Fig. 4, Fig. 5.The result shows that with invading
The reduction of attacking property candida albicans challenge dose, Cbk1 antibody gradually increase the protective effect of aggressive monilial infection mouse.
2) antibody is protected in advance with 1mg/kg, candida albicans SC53141 × 10 is given after 2h5/ only attack mouse, after 5d again
Secondary 1mg/kg is protected, after 30d, mouse survival rate 80%.Or only protect Cbk1 antibody in advance with 2mg/kg, it is given respectively after 2h
Candida albicans SC53141 × 105/ only, and after 30d, mouse survival rate 50%.As a result see Fig. 6, the results showed that prompt is repeatedly given anti-
Physical efficiency improves infecting mouse survival rate.
3) Cbk1 antibody is protected in advance with 2mg/kg/ mouse tail vein injection, given after 2h candida albicans SC53141 ×
106/ mouse is only attacked, liver kidney carries bacterium amount and substantially reduces after 48.As a result see Fig. 7, prompt Cbk1 antibody can be used not only for non-lethal
The protection of dosage infection, and also have good scavenging effect for high dose invasion monilial infection.
4) candida albicans SC53141 × 10 are first given5/ attack mouse causes aggressive monilial infection, antibody after 2h
It is treated with 2mg/kg, after 30d, as a result mouse survival rate 30% is shown in Fig. 8；If antibody is protected with 4mg/kg, mouse survival rate is increased
It is 40%.As a result see Fig. 9, prompt Cbk1 antibody to can be used not only for prevention & protection, and can be used to treat aggressive candida albicans sense
Contaminate mouse.
5) Cbk1 antibody is protected with 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg in advance respectively, gives white thought after 2h respectively
Pearl bacterium SC53141 × 105Cells/ only attacks mouse, and mouse survival rate is respectively 40%, 40%, 80%, 60% after 40d, knot
Fruit sees Figure 10.The result shows that Cbk1 antibody has protective effect to aggressive monilial infection mouse.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (6)
1. it is CCTCC No that a kind of anti-Cbk1 monoclonal antibody, which is by deposit number,：C201481 or CCTCC No：C201482's
Hybridoma cell strain secretion generates.
2. a kind of hybridoma cell strain, deposit number is CCTCC No：C201481 or CCTCC No：C201482.
3. a kind of anti-Cbk1 monoclonal antibody as described in claim 1 is white in preparation Western Blot or immunohistochemistry detection
The reagent of monilial infection or the application in kit.
4. a kind of hybridoma cell strain as claimed in claim 2 detects white beads in preparation Western Blot or immunohistochemistry
Application in the reagent or kit of bacterium infection.
5. a kind of anti-Cbk1 monoclonal antibody as described in claim 1 is preparing the application in antifungal infection medicine.
6. a kind of hybridoma cell strain as claimed in claim 2 is preparing the application in antifungal infection medicine.
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| CN1948335A (en) * | 2005-10-14 | 2007-04-18 | 中国科学院上海生命科学研究院 | Candida albicans mycellium regulating and controlling factor gene and its use |
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| CN1948335A (en) * | 2005-10-14 | 2007-04-18 | 中国科学院上海生命科学研究院 | Candida albicans mycellium regulating and controlling factor gene and its use |
Non-Patent Citations (2)
| Title |
|---|
| A Monoclonal Antibody Directed against a Candida albicans Cell Wall Mannoprotein Exerts Three Anti-C. albicans Activities;María D. Moragues;《INFECTION AND IMMUNITY》;20030930;第5273-5279页 * |
| NCBI Reference Sequence:XP_723591;NCBI;《NCBI》;20080528;第1页 * |
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