CN105255845B

CN105255845B - Preparation and application of anti-Och 1 monoclonal antibody

Info

Publication number: CN105255845B
Application number: CN201410332277.4A
Authority: CN
Inventors: 阎澜; 张艳霞; 王晓娟; 王丽; 姜远英
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2014-07-14
Filing date: 2014-07-14
Publication date: 2020-03-31
Anticipated expiration: 2034-07-14
Also published as: CN105255845A

Abstract

The invention relates to the technical field of medical bioengineering, and provides an anti-candida albicans cell wall protein Och1 monoclonal antibody, a polypeptide for preparing the monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody, application of the monoclonal antibody, the polypeptide for preparing the anti-Och 1 monoclonal antibody and the hybridoma cell strain in preparation of a reagent or a kit for Western Blot or immunohistochemical detection of candida albicans infection, and application in preparation of a medicine for preventing or treating candida albicans infection.

Description

Preparation and application of anti-Och 1 monoclonal antibody

Technical Field

The invention relates to the technical field of medical bioengineering, in particular to an anti-Och 1 monoclonal antibody and application of the monoclonal antibody in anti-Candida albicans infection.

Background

OCH1 is a gene encoding cell wall α -1, 6-mannose transferase, deletion can block excessive glycosylation on glycoprotein, and is necessary for candida albicans virulence, a specific gene of fungi, therefore, Och1 protein is a promising antifungal target based on antibody therapy.

At present, no literature report about an anti-Och 1 monoclonal antibody exists, and no literature report about the application of the anti-Och 1 monoclonal antibody in resisting infection of invasive candida is available.

Disclosure of Invention

The invention aims to provide an anti-candida albicans cell wall protein Och1 monoclonal antibody, a polypeptide for preparing the monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody and application of the monoclonal antibody.

In a first aspect, the invention provides a polypeptide for preparing an anti-Candida albicans cell wall protein (Och1) monoclonal antibody, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1.

DWKLFTGMEQ(SEQ ID NO:1)

The second aspect of the invention provides an anti-Och 1 monoclonal antibody, which is prepared from the following components in a preservation number of CCTCC No: c201484 hybridoma cell strain is secreted and produced.

The third aspect of the invention provides a hybridoma cell strain with a preservation number of CCTCC No: C201484.

in a fourth aspect of the present invention, there is provided a method for preparing an anti-Och 1 monoclonal antibody, comprising the steps of:

a) the synthetic amino acid sequence is shown as SEQ ID NO: 1;

b) introducing the polypeptide into an expression vector, expressing and purifying to obtain an immunogen immune mouse;

c) taking spleen cells of an immunized mouse for fusion;

d) cell clones positive in synthetic polypeptide reaction are screened in multiple rounds, and the cell clones are used as hybridoma cell strains CCTCC No of the anti-Och 1 monoclonal antibody: c201484;

e) the hybridoma cells are inoculated into a mouse body to produce an ascites antibody, and the ascites is purified to obtain the anti-Och 1 monoclonal antibody.

In the fifth aspect of the invention, the application of the polypeptide for preparing the anti-Och 1 monoclonal antibody in preparing a reagent or a kit for Western Blot or immunohistochemical detection of Candida albicans infection is provided.

And the application of the anti-Och 1 monoclonal antibody in preparing a reagent or a kit for Western Blot or immunohistochemical detection of Candida albicans infection.

And the application of the hybridoma cell strain in preparing a reagent or a kit for Western Blot or immunohistochemical detection of candida albicans infection.

Furthermore, the invention also provides application of the polypeptide for preparing the anti-Och 1 monoclonal antibody in preparing a medicine for preventing or treating candida albicans infection.

And the application of the anti-Och 1 monoclonal antibody in preparing a medicament for preventing or treating candida albicans infection.

And the application of the hybridoma cell strain in preparing a medicine for preventing and treating candida albicans infection.

The specific technical scheme of the invention is as follows:

1. 16 polypeptides were designed and synthesized for the full sequence of amino acids encoding the cassette of anti-Och 1 (GENBANK ID: 3641751), the sequences being:

polypeptide 1 KDDGMPEMEQ (SEQ ID NO:2)

Polypeptide 2 PTSTHKTEYN (SEQ ID NO:3)

Polypeptide 3 VGIDEKSFPK (SEQ ID NO:4)

Polypeptide 4 NQMGAKDSHD (SEQ ID NO:5)

Polypeptide 5 ELELNSNWKE (SEQ ID NO:6)

Polypeptide 6 KKWATIIDWK (SEQ ID NO:7)

Polypeptide 7 EQLYSQVPDV (SEQ ID NO:8)

Polypeptide 8 QTWEDKNPDY (SEQ ID NO:9)

Polypeptide 9 NSPKLQLAKE (SEQ ID NO:10)

Polypeptide 10 DWKLFTGMEQ (SEQ ID NO:1)

Polypeptide 11 PNKKYSLPDE (SEQ ID NO:11)

Polypeptide 12 TRHKKGQLKK (SEQ ID NO:12)

Polypeptide 13 DESTLRQQLS (SEQ ID NO:13)

Polypeptide 14 DEWISNSEMI (SEQ ID NO:14)

Polypeptide 15 KVLGKNEGGD (SEQ ID NO:15)

Polypeptide 16 QFPYDESKPF (SEQ ID NO:16)

The polypeptides are introduced into expression vectors.

2. And identifying positive clones, extracting and transforming expression bacteria.

3. Inducing expression and producing in large quantity.

And 4, carrying out affinity purification on the Ni column, and purifying to obtain the protein antigen for immunization.

5. And (3) successfully obtained antigen mixed immune mice are female BALB/c healthy mice with the age of 8-12 weeks. Spleens of 3 mice were pooled and fused using a mouse tachyphylaxis approach.

6. Cell clones which have positive reaction are screened in multiple rounds and are used as hybridoma cell strains 5737-1RE-4M4/4N 5-111218 of anti-mouse anti-Och 1 protein monoclonal antibodies.

7. The ascites antibody is produced by inoculating hybridoma cells in a mouse body, and the ascites is purified to obtain the monoclonal antibody of the anti-mouse anti-Och 1.

The invention provides a monoclonal antibody of anti-Och 1, which recognizes Och1 protein amino acid sequence DWKLFTGMEQ. The polypeptide is the epitope which is finally selected and generates antibodies with higher titer and is aimed at after 16 polypeptides are expressed and purified.

The monoclonal antibody of the invention is obtained by adopting a conventional method in the technical field, namely, inoculating hybridoma cells in a mouse body and generating an ascites antibody, taking out ascites and purifying.

The monoclonal antibody prepared by the invention is an immunoglobulin and can be specifically combined with the protein of candida albicans.

The invention also provides application of the anti-Och 1 monoclonal antibody and the hybridoma cell strain in detecting candida albicans, in particular application in preparing Western Blot or immunohistochemical detection reagents or kits. The invention provides a new idea for the application of the clinical antifungal infection immunotherapy against Och 1.

Drawings

FIG. 1 is a diagram of detection of Candida albicans protein by monoclonal antibody Western Blot.

FIG. 2 is an immunofluorescence assay format in which A is a monoclonal antibody immunofluorescence assay for binding to Candida albicans yeast cells; and B is a graph of the combination of monoclonal antibody immunofluorescence detection and candida albicans hyphae state.

FIG. 3 is a graph showing the binding of anti-Och 1 monoclonal antibody to live fungal cells, cell wall proteins and cytoplasmic proteins detected by ELISA

FIG. 4 shows Candida albicans at 1X 10⁵Graph of protection of monoclonal antibodies given at different doses at the cell/mouse challenge dose.

FIG. 5 shows Candida albicans at 1X 10⁵Graph of protection against systemic Candida albicans infection at cell/mouse challenge dose given 1mg/kg monoclonal antibody twice. Wherein, a is a mouse survival curve; b is the mouse kidney load at day 60 post challenge.

The hybridoma cell strain is named as 5737-1RE-4M4/4N5_111218, is preserved in China center for type culture Collection (CCTCC for short), has a preservation date of 2014, 6 months and 17 days, and has a preservation number of CCTCC No: C201484.

Detailed Description

The present invention will be described in detail below with reference to examples and the accompanying drawings. The following examples should not be construed as limiting the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified.

Example 1 selection of polypeptide sequence and preparation of polypeptide of monoclonal antibody against Och1

Selection of polypeptide sequences

Och1 has 385 amino acids, and according to the amino acid sequence information, Eibemat biomedicine (Shanghai) Limited company is entrusted to design, screen and synthesize a polypeptide sequence for polypeptide synthesis: DWKLFTGMEQ

Example 2 preparation and purification of monoclonal antibody against Och1

Abiramate biomedicine (Shanghai) Co., Ltd is entrusted to the preparation and purification of monoclonal antibodies.

1. Production of antigens

The polypeptide is synthesized by whole gene, and the gene sequence is referred to Candida gene database (Candida genome database). Constructing an expression vector; positive clone identification; extracting positive clone plasmid and converting expression bacteria; large-scale inducible expression production; affinity purification by a Ni column; and purifying to obtain the protein antigen for immunization.

2. Preparation and purification of monoclonal antibodies

And (3) successfully obtained antigen mixed immune mice are female BALB/c healthy mice with the age of 8-12 weeks. Spleens of 3 mice were pooled and fused using a mouse tachyphylaxis approach.

Spleen cells of mice with the best immunoreaction are fused with myeloma cells (SP2/0), the fused cells are diluted appropriately, are cultured in a 96-well culture plate for 10-14 days to carry out ELISA detection, and cells in wells with high OD values are selected to carry out subcloning by a limiting dilution method.

The specific method comprises the following steps: the cells in limiting dilution were cultured in 96-well plates, and when clones grew to 1/6 in full wells, the monoclonal and polyclonal were labeled and the monoclonal was tested by ELISA. After ELISA detection, the monoclonal with the highest OD value is subjected to limiting dilution and is inoculated into a 96-well plate for secondary subcloning in the method, and the process is repeated for a plurality of times until the positive well rate is 100%, and the monoclonal is considered to be the monoclonal. I.e., a cell line that we generally believe was successful in establishing a strain. The positive monoclonal obtained by screening is expanded and cultured, and the number of cells is 1-2 multiplied by 10⁶Freezing and storing in a tube. Meanwhile, collecting cells and arranging ascites preparation.

The cell strain is used for preparing ascites by adopting a mouse abdominal cavity inoculation method, the ascites is collected for 10-14 days, and ELISA is used for detecting whether the ascites is successfully prepared. After completion of ascites, ascites was purified by Protein G column.

Example 3 identification and detection applications of monoclonal antibodies against Och1

1. Western Blot detection application of monoclonal antibody

Performing SDS-PAGE protein electrophoresis on the denatured candida albicans cell wall protein sample; and after electrophoresis is finished, performing membrane transfer by a semidry method. Adding sealing liquid and sealing at room temperature for at least 1 h; washing the membrane with PBST washing solution for 5 times, using monoclonal antibody as primary antibody, and incubating overnight at 4 ℃; after the incubation is finished, washing the membrane for 5 times by using PBST washing liquid, adding a second Antibody DYLightTM 800-laboratory Antibody to MouseIgG (H + L)1:10000 for dilution, then incubating for 2H at room temperature, and slightly oscillating in the incubation process; the membranes were washed 5 times with PBST wash and NC membranes were scanned with an Odyssey infrared imaging system. The results are shown in FIG. 1. The result shows that the monoclonal antibody can specifically recognize the cell wall protein of the candida albicans.

2 immunofluorescence detection application of monoclonal antibody

Adjusting the concentration of fungus yeast cells and mycelial cells to a certain concentration, washing with PBS for 3 times, discarding the supernatant, adding 1ml of 1% BSA solution, and sealing at room temperature for 1 h. The supernatant was discarded by centrifugation and washed 3 times with PBS. anti-Och 1 antibody (diluted with PBS) was added, and PBS was added to the control group. Incubate for 2h at 4 ℃ on a vertical rotator. The supernatant was discarded by centrifugation and washed 3 times with PBS. FITC-labeled fluorescent secondary antibody was added and incubated at 4 ℃ for 1 h. The supernatant was discarded by centrifugation, washed 3 times with PBS, and resuspended in PBS. 7 μ l of the bacteria were dropped on a glass slide, and fluorescence was observed with a confocal laser microscope under 488nm excitation light. FIG. 2.A is a graph showing binding of monoclonal antibody immunofluorescence assay to Candida albicans yeast cells, and B is a graph showing binding of monoclonal antibody immunofluorescence assay to Candida albicans hyphal cells

The result shows that the monoclonal antibody can specifically recognize the candida albicans.

3 ELISA detection application of monoclonal antibody

Inoculating 0.5 μ g Candida albicans SC5314 cell wall extract protein per 96-well enzyme linked plate, or inoculating 1 × 10⁶cells/ml live Candida albicans cells, coating overnight at 4 ℃, washing for 3 times by PBST, 5min each time, sealing by sealing liquid, incubating at 37 ℃ for 2h, wherein each hole is 200 mu l; PBST washing for 3 times, 5min each time; adding 100 mu l of anti-Och 1 antibody with different dilutions, taking the antibody myc as a negative control, and incubating for 2h at 37 ℃; PBST washing for 3 times, 5min each time; adding 100 mu l of HRP conjugated secondary antibody IgG respectively, and incubating for 1h at 37 ℃; PBST washing 5 times, each time for 5 min; developing 200 μ l TMB at room temperature in dark for 10min, and measuring absorbance at 370 nm; the reaction was stopped with 50. mu.l of 2M H2SO4 and the absorbance was measured at 450 nm. The results are shown in FIG. 3, which shows the binding of the anti-Och 1 monoclonal antibody to live cells and cell wall and cytoplasmic proteins of Candida albicans by ELISA.

Example 4 protection of anti-Och 1 monoclonal antibodies against Candida albicans infection

1) The Och1 antibody was pre-protected at 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, respectively, and 2h later, Candida albicans SC 53141X 10⁵The survival rates of the cells/challenged mice after 40d were 20%, 40% and 40%, respectively, and the results are shown in FIG. 4. The results show that Oc isThe h1 antibody has a certain protective effect on mice infected by invasive candida.

2) The antibody was pre-protected at 1mg/kg and 2h later Candida albicans SC 53141X 10⁵Mice were challenged once, protected again at 1mg/kg after 5d, and mice survived 40% after 60d, the results are shown in FIG. 5A. The results indicate that multiple administrations of the Och1 antibody can improve the survival rate of infected mice. After 60d, mice were sacrificed by dislocation and the number of colonies enriched in kidney colonies for each group of mice is shown in fig. 5B. The results show that the Och1 antibody has a remarkable clearing effect on kidney-enriched colonies of mice infected by invasive candida.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A polypeptide for preparing an anti-Och 1 monoclonal antibody, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO:1 is shown in the figure.

2. An anti-Och 1 monoclonal antibody is prepared from the antibody with the preservation number of CCTCC No: c201484 hybridoma cell strain is secreted and produced.

3. A hybridoma cell strain with a preservation number of CCTCC No: C201484.

4. use of the polypeptide of claim 1 for preparing an anti-Och 1 monoclonal antibody in preparation of a reagent or a kit for Western Blot or immunohistochemical detection of Candida albicans infection.

5. Use of the anti-Och 1 monoclonal antibody of claim 2 in the preparation of a reagent or kit for Western Blot or immunohistochemical detection of Candida albicans infection.

6. The use of the hybridoma cell line of claim 3 in the preparation of a reagent or kit for Western Blot or immunohistochemical detection of Candida albicans infection.

7. Use of the polypeptide of claim 1 for preparing an anti-Och 1 monoclonal antibody in the preparation of a medicament for resisting Candida albicans infection.

8. Use of the anti-Och 1 monoclonal antibody of claim 2 in the preparation of a medicament for the treatment of Candida albicans infection.

9. The use of the hybridoma cell line of claim 3 in the preparation of a medicament for treating candida albicans infection.