CN116655802B

CN116655802B - Fusion protein of melioidosis bacterium Hcp1 protein, monoclonal antibody, hybridoma cell strain and application

Info

Publication number: CN116655802B
Application number: CN202310600510.1A
Authority: CN
Inventors: 李倩; 南栋琪; 刘文正; 饶承龙; 闫晶敏; 赵光强; 毛旭虎
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2023-05-25
Filing date: 2023-05-25
Publication date: 2023-12-12
Anticipated expiration: 2043-05-25
Also published as: CN116655802A

Abstract

The invention provides a recognition epitope of a meliotic bacteria Hcp1 protein, and further prepares and obtains fusion protein, hybridoma cells and monoclonal antibodies, and the obtained monoclonal antibodies only recognize and bind a special epitope of the meliotic bacteria Hcp1 protein, have high specificity, are not easy to cross react with other proteins, and are more efficient for distinguishing and identifying strains of the same genus.

Description

Fusion protein of melioidosis bacterium Hcp1 protein, monoclonal antibody, hybridoma cell strain and application

Technical Field

The invention relates to the field of biology, in particular to a melilotus gangrene bacterium Hcp1 protein fusion protein, a monoclonal antibody, a hybridoma cell strain and application thereof.

Background

The meliosis Boehringer-Hold bacteria (Burkholderia pseudomallei, B.pseudomullei) are short-term meliosis bacteria, widely exist in river channels and mud lands in tropical and subtropical areas, are gram-negative short bacillus with B-type biological threat factors and I-type biological terrorist warfare agents, cause serious and fatal meliosis disease due to infection, and have important social significance for strengthening prevention, diagnosis and treatment of the meliosis bacteria. Research shows that in the infection pathogenic process of the meliotic bacteria, the hemolysin regulatory protein (hemolysin coregulated protein, hcp1) serving as structural protein and effector of a VI type secretion system (T6 SS-1) can be shifted into host cells, mediate the membrane fusion of the host cells, participate in the formation of polynuclear megacells, promote the spread of the meliotic bacteria among the host cells and cause the occurrence and development of infection. In addition, hcp1 stimulates the host to produce specific antibody, and has been studied as a characteristic marker for serological diagnosis of melioidosis infection, so Hcp1 is expected to become a molecular target for melioidosis infection diagnosis and a key target for antibody drug development.

The meliotic bacteria Hcp1 consists of 169 amino acid residues, is highly conserved in the meliotic bacteria, has a plurality of antigen epitopes, can stimulate organisms to generate neutralizing antibodies, and is particularly important for diagnosing meliotic bacteria infection by acquiring accurate antigen epitopes. The existing research is mainly directed against the whole bacterial protein antibody of the melioidosis bacteria or the polyclonal antibody of the Hcp1, the epitope recognized by the antibody is more and can not be clearly identified, the nonspecific cross reaction is easy to cause, the false positive phenomenon is often generated in the differential diagnosis process, and the anti-interference performance is poor. Zhang Meijuan and the like have found that Hcp1 polyclonal antibodies cross-react with other strains of the genus burkholderia (third army university journal of medicine 2020,42 (23): 2296-2301), and cannot distinguish burkholderia cepacia from burkholderia cepacia of the same species. The Hcp1 monoclonal antibody only recognizes and binds one epitope of a single antigen, has high specificity, is not easy to cross react with other proteins, is more efficient for distinguishing and identifying strains of the same species, and can greatly improve the specificity and sensitivity of detection when used for immunological detection of the melioidosis.

Disclosure of Invention

The invention firstly provides a fusion protein which is formed by coupling a melioidosis bacteria Hcp1 protein and hemocyanin KLH; wherein the amino acid sequence of the melioidosis bacteria Hcp1 protein is SEQ ID NO. 5.

In one embodiment according to the invention, the melioidosis Hcp1 protein is prepared by a process comprising the steps of:

the genome of the melioidosis bacteria BPC006 (NCBI Reference Sequence:NC_ 018529.1) is used as a template, the nucleotide sequences SEQ ID NO:6 (Hcp 1-F:5'-ATGGATCCATGCTGGCCGGAATATATCTC-3' (BamHI)) and SEQ ID NO:7 (Hcp 1-R:5'-CCAAGCTTTCAGCCATTCGTCCAGTTTGCG-3' (HindIII)) are used as upstream and downstream primers, the Hcp1 gene is obtained through PCR amplification, and then BamHI and HindIII double enzyme digestion and T4 DNA ligase are used for connection, so that an induction expression vector pET-28a-Hcp1 is constructed, and the induction expression vector pET-28a-Hcp1 is transformed into host bacteria to express the melioidosis bacteria Hcp1 protein; preferably, the Hcp1 protein is purified by His Trap affinity chromatography; the expression system of the recombinant protein is one of a mammalian protein expression system, an insect protein expression system, an maternal protein expression system, an escherichia coli protein expression system, an algae protein expression system and the like, and preferably the expression system is the escherichia coli protein expression system. The colibacillus protein expression system has more than 10 expression host bacteria such as BL21 (DE 3), origami B (DE 3), BSR (DE 3) and the like, and preferably the host bacteria are colibacillus BL21 (DE 3).

The present invention further provides a composition for use as an antigen comprising the fusion protein described above; preferably, an adjuvant is also included; preferably, the adjuvant is selected from one of Freund's complete adjuvant and AD11.10 adjuvant.

In another aspect of the present invention, there is provided a method for preparing a hybridoma cell, comprising:

1) Immunizing a mouse with the composition of claim 3;

2) Taking spleen cells of a mouse with neutralization activity, and fusing the spleen cells with SP2/0 cells to obtain fused cells;

3) Screening to obtain hybridoma cells producing monoclonal antibodies recognizing the melioidosis Hcp1 protein or fragments thereof; preferably, the melitemium Hcp1 protein or fragment thereof is a peptide fragment comprising at least the amino acid sequence of SEQ ID NO. 8 (DTTGGDLTELLFTYKFEKVL).

Further, step 3) the screening is achieved by a method comprising the steps of:

culturing the fused cells for about 48 hours, transferring the fused cells into HAT semi-solid selective medium for culturing for 10 days, and selecting monoclonal cell colonies to 96-well plates for further culturing for 7 days; then, cell culture supernatants in 96-well plates were evaluated by an indirect ELISA method, and hybridoma cell lines capable of secreting monoclonal antibodies recognizing the Hcp1 protein of the melioidosis were screened out positive with P/N > 2.1.

The invention further provides the hybridoma cell obtained by the preparation method, wherein the preservation number of the hybridoma cell is CCTCC NO: C202382, and the preservation date is 2023.4.25.

The invention also provides a monoclonal antibody comprising a heavy chain variable region with an amino acid sequence of SEQ ID NO. 1 and/or a light chain variable region with an amino acid sequence of SEQ ID NO. 2.

In one embodiment according to the present invention, the heavy chain variable region of the mab has a nucleotide sequence shown in SEQ ID No.3 and the light chain variable region of the mab has a nucleotide sequence shown in SEQ ID No. 4; preferably, the monoclonal antibody is of the IgG1 subtype of kappa-type light chain, and the Fc segment amino acid sequence of the monoclonal antibody is SEQ ID NO.9.

The invention further provides application of the monoclonal antibody in preparation of a reagent for detecting or preventing infection of melioidosis bacteria.

In yet another aspect of the invention there is provided an agent for detecting or preventing infection by a melioidosis bacterium comprising a monoclonal antibody as described above.

The technical scheme of the invention has the following beneficial effects:

according to the invention, recombinant expressed Hcp1 protein and hemocyanin KLH are coupled into Hcp1-KLH fusion protein, and then a BALB/c mouse is immunized, and a hybridoma cell strain Lmjys2023-13F1 capable of stably passaging and secreting anti-meliotic bacteria Hcp1 monoclonal antibody is obtained through immunization, cell fusion, screening and cloning; the type of the anti-melioidosis bacteria Hcp1 monoclonal antibody secreted by the hybridoma cell strain is the IgG1 subtype of kappa type light chain; the monoclonal antibody for resisting the meliotic bacteria Hcp1 can specifically react with the meliotic bacteria Hcp1, and the ELISA detection titer can reach 1:200000; the monoclonal antibody can be used as a detection reagent of the meliotic bacteria Hcp1, is applied to immunological detection of the meliotic bacteria, has strong anti-interference capability, does not generate cross reaction with bacteria of different species such as pseudomonas aeruginosa, escherichia coli, helicobacter pylori, mycobacterium tuberculosis, staphylococcus aureus, salmonella enteritidis, clostridium nucleatum and the like, does not react with Burkholderia cepacia and Burkholderia thailand of the same species, can specifically detect the meliotic bacteria, and is an antibody reagent for detecting the meliotic bacteria with high quality in immunology.

The hybridoma cells provided by the invention are preserved in China center for type culture collection (address: eight paths of 299 in Wuchang district of Wuhan, hubei province), and the names and the noted identification features of the culture: hybridoma cell strain Lmjys2023-13F1 Hybridoma cell line Lmjys2023-13F1, collection number CCTCC NO: C202382, collection date 2023.4.25.

Drawings

FIG. 1 shows the expression and purification results of recombinant protein rHcp1 of example 1 of the present invention; wherein A is a schematic diagram of an Hcp1 recombinant expression vector pET-28a-Hcp 1; b is coomassie brilliant blue staining to detect the purity of the recombinant expression protein; c is western blotting identification recombinant egg; m is a protein molecular weight standard; 1 is a purified recombinant expressed rHcp1 protein; 2 is a control BSA protein.

FIG. 2 shows the results of mouse immunization procedure and serum antibody titer determination in example 2 of the present invention; wherein A is an immunization flow diagram of a mouse, Y01 is antigen prepared by mixing rHcp1-KLH with CFA adjuvant, and the mouse is immunized subcutaneously on the back; y02 is antigen prepared by mixing rHcp1-KLH and AD11.10 adjuvant, and the antigen is used for immunizing mice through leg muscles; b is ELISA to detect mouse serum titer, the abscissa is mouse serum dilution ratio, and the ordinate is OD value at 450 nm.

FIG. 3 shows the results of ELISA screening for monoclonal hybridoma cell lines in example 2 of the present invention; wherein A is ELISA primary screening positive hybridoma cell strain, 15 ELISA plates are added, H11 holes of each plate are Negative Control (NC), H12 holes are Positive Control (PC), and 13F1, 14G11 and 15D9 are positive detection holes; b is ELISA to review positive cells, the antibody titer of the culture supernatant of the positive cells is detected after 3 passages, cryopreservation and resuscitation, and PC is a positive control; NC is negative control;

FIG. 4 shows the results of preparation and purification of the monoclonal antibody of the abdominal water type 13F1 in example 3 of the present invention; wherein A is a flow chart for preparing 13F1 monoclonal antibody and purifying ascites; b is SDS-PAGE to identify the purity of 13F1 monoclonal antibody, M is protein molecular weight standard; 1 is purified 13F1 mab; 2 is positive control of IgG standard substance; 3 is a BSA negative control;

FIG. 5 shows ELISA detection of monoclonal antibody subtypes and titers in example 4 of the present invention; wherein, the A.ELISA identifies the subtype of the monoclonal antibody 13F1, the abscissa is the different subtypes of the antibody, and the ordinate is the OD value at 450 nm; ELISA determines the titer of the monoclonal antibody 13F1, and the abscissa is the dilution ratio of the purified antibody;

FIG. 6 shows the results of the identification of the antigen linear binding region of 13F1 mab in example 5 of the present invention; wherein, the A.Hcp1 protein shift method truncating strategy, I: first round of truncating construction; II: performing a second round of truncate construction based on the positive region determined in the first round; western blotting verifies that the His-Hcp1 pure protein is positive control in the first round of truncated positive region. C. Fluorescent Western blotting determines an antigen binding region of the monoclonal antibody, green fluorescent marker Hcp1, red fluorescent marker GFP protein;

FIG. 7 shows the result of Western blotting detection of monoclonal antibody in example 4 of the present invention; wherein 1 is B.pseudomullei; 2 is b.thailandensis;3 is B.cepacian;4 is p.aerocinosa; 5 is E.coli;6 is H.pyri; 7 is m. 8 is S.aureus;9 is F.nucleic; 10 is S.enteritidis;11 is rHcp1.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.

The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The following examples are only illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 preparation of the haemolysin Conditioning protein Hcp1 antigen

In this example, recombinant rHcp1 protein was expressed and purified by the experimental method disclosed in Zhang Meijuan et al (third et al, university of medical science, 2020,42 (23): 2296-2301). Namely, using the melioidosis bacteria BPC006 genome (NCBI Reference Sequence:NC_ 018529.1) as a template, and Hcp1-F:5' -ATGGATCCATGCTGGCCGGAATATATCTC-3' (BamHI) and Hcp1-R:5' -CCAAGCTTTCAGCCATTCGTCCAGTTTGCG-3' (HindIII) is an upstream primer and a downstream primer, PCR amplification is carried out on the Hcp1 gene, then BamHI and HindIII double enzyme digestion T4 DNA ligase is used for connection to construct an induced expression vector pET-28a-Hcp1, after His Trap affinity chromatography is carried out on the recombinant expressed protein, as shown in figure 1, recombinant protein rHcp1 with the purity of more than 98% and being identified by a purified tag protein antibody is obtained, and then the recombinant protein rHcp1 is mixed with hemocyanin KLH (purchased from Wuhan Yitai technology Co., ltd.) and EDC reagent (purchased from Shanghai Biyun biotechnology Co., ltd.) is added to enable Hcp1 and KLH to be obtainedImmunogens Y01 and Y02 were prepared by coupling amino and carboxyl groups as fusion proteins, and mixing the fusion proteins with Freund's complete adjuvant CFA (available from Sigma) and AD11.10 adjuvant (available from Addison, st.).

Example 2 establishment of hybridoma cell lines

1. Immunized mice

The Hcp1-KLH fusion protein is used as immunogen, and 3 female BALB/c mice with the age of 5-6 weeks are immunized by subcutaneous back and leg intramuscular injection, wherein the first boost immunization is separated from the first immunization by 2 weeks, and then the immunization is performed every 1 week. Primary immunization was performed by subcutaneous injection of 100 μg/dose of immunogen Y01 in combination with intramuscular injection of 100 μg/dose of immunogen Y02; the first boost using 100 μg/g of immunogen Y02 intramuscular injection, the second boost using 100 μg/g of immunogen Y01 back subcutaneous injection and 100 μg/g of immunogen Y02 intramuscular injection, the third boost using 100 μg/g of immunogen Y02 intramuscular injection, tail-breaking blood sampling 1 week after the third boost, diluting serum at 1:10 fold ratio starting from 1:100, coating ELISA plates with purified recombinant Hcp1 protein, ELISA detection of serum antibody titers, as in FIG. 2, selecting Mouse 3 with higher serum antibody titers, boosting with Y02 antigen 3 days prior to cell fusion.

2. Cell fusion and screening

Spleen cells from mice were mixed with SP2/0 cells (from Shanghai department of sciences cell bank, TCM 18) at a ratio of 10:1, and then slowly warmed up PEG was added and fused in a 37℃water bath for 25min. Cells after completion of the fusion were cultured for about 48 hours and transferred to HAT (H-Hypoxan Hypoxanthine, A-Aminopterin Aminopterin, T-Thymidine Thymidine) semi-solid selective medium for 10 days, and colonies of the monoclonal cells were selected and cultured in 96-well plates for additional 7 days. Cell culture supernatants (after 1:1 dilution) in 96-well plates were evaluated by indirect ELISA, and hybridoma cell lines capable of secreting monoclonal antibodies recognizing Hcp1 protein were screened positive for P/N > 2.1. Transferring and enlarging culture of positive hybridoma cells to a 48-well plate, continuing to culture for 2 days, taking cell supernatant, and enlarging culture and freezing storage after re-screening and verification by an indirect ELISA method. As shown in FIG. 3, 1416 monoclonal cells were knocked down, and three hybridoma cell lines 13F1, 14G11 and 15D9 were selected, and a subsequent study was performed on the basis of 13F1, with a collection number of CCTCC NO: C202382.

EXAMPLE 3 preparation of monoclonal antibodies by ascites Induction

1. Preparation and purification of a web mAb

Positive hybridoma 13F1 (-10) ⁷ And) injecting into the abdominal cavity of 8-week-old female BALB/c mice pre-injected with IFA adjuvant, and after the mice had distended in the abdomen (7-10 days), extracting the ascites from the mice and centrifuging, collecting the supernatant, purifying the antibody from the ascites by using HiTrap rProtein A FF affinity chromatography column (purchased from GE company) according to instructions. As shown in FIG. 4, the purity of the antibody was identified by SDS-PAGE, and the molecular weights thereof were about 55kDa and 25kDa, respectively, which were consistent with the molecular weight of the heavy and light chains of the antibody, and the protein bands with other bands disappeared, indicating that the ascites type monoclonal antibody of 13F1 had good purification effect. The concentration measured by the Bradford method was 8.22mg/mL, which was 2mL in total. Purified antibodies were stored at 20 ℃.

Example 413 identification of ascites-type Hcp1 monoclonal antibodies to F1

Determination of 1.13F1 ascites type Hcp1 monoclonal antibody titer

The titer of the antibody secreted by the hybridoma cells 13F1 was determined by an indirect ELISA method. Diluting the recombined expressed Hcp1 protein to 1 mug/mL, coating an ELISA plate, adding 100 mug/hole, and reacting at 4 ℃ overnight; plates were washed 3 times with PBS solution, blocked 2h with 5% nonfat dry milk at room temperature, and washed 5 times with PBS solution. Adding gradient diluted purified antibodies into an ELISA plate and coated Hcp1 protein respectively to react for 1h at room temperature, washing the plate for 3 times by using a PBS solution, then performing patting drying, adding 1:2000 diluted goat anti-mouse IgG (Fc) secondary antibody (purchased from jackson lmmuno Research company), performing reaction for 1h at room temperature, washing the plate for 5 times by using the PBS solution, performing patting drying, adding TMB (purchased from Beijing Bunge company) to perform color development, and performing reaction for 20min under the conditions of light shielding and room temperature; then 50. Mu.L of stop solution was added, and after mixing, the OD450 value was read on an ELISA reader. As shown in FIG. 5, the results indicate that the monoclonal antibody titer of 13F1 reaches more than 1:200000.

Identification of 2.13F1 Fushui type Hcp1 monoclonal antibody subtype

The subtype of the monoclonal antibody was determined using a mouse monoclonal antibody subtype identification ELISA kit (Wuhan, proteintech). According to the use instruction of the kit, diluting positive hybridoma cell culture supernatant by 1:100, adding a detection sample hole, sequentially adding an enzyme-labeled secondary antibody, a chromogenic substrate and a stop solution, and reading an OD450 value on an enzyme-labeled instrument. Interpretation of the results: the monoclonal antibody is only in a sample Kong Xianse of one subtype, and other subtypes are not developed, so that the antibody to be detected is the subtype corresponding to the development hole. As shown in FIG. 5, the results show that 13F1 ascites type Hcp1 mab is the IgG1 subtype of kappa type light chain.

Example 5 identification of antigen binding epitopes of 13F1 ascites type Hcp1 monoclonal antibody

1.13F1 monoclonal antibody linear epitope mapping to 50 Amino Acids (AA)

According to the Hcp1 sequence, a step-by-step method is adopted to design a truncated sequence of two sections of overlapping Hcp1-ab and Hcp1-bc, and one-step trial verification is carried out. As shown in fig. 6A, specifically: covering the full-length Amino Acid (AA) sequence of the Hcp1, overlapping 59 AA in the middle, and constructing the Hcp1 ^1-114 、Hcp1 ^56-169 And Hcp1 ^56-114 Three truncated expressed pET-GST vectors are used for transforming E.coli BL21 competent cells, inducing expression, and verifying by using 13F1 monoclonal antibody as a primary antibody and Western blotting. As shown in FIG. 6B, the mAb recognizes 3 truncated peptide fragments, indicating that the 13F1 monoclonal antibody recognizes an epitope between positions 56-114 of amino acids Hcp1.

2. Linear epitope mapping of monoclonal antibodies to 20 amino acids

Positive segment Hcp1 as described in step 1 of example 5 ^56-114 Repeating the above method to design truncated body, overlapping 20 AA between two overlapped truncated sequences, and constructing Hcp ^156-9 4 and Hcp ^175-114 And converting E.coli BL21 competent cells by using two truncated pET-GST vectors, inducing expression, taking a 13F1 monoclonal antibody and a rabbit GST tag antibody as primary antibodies, taking a rabbit anti-mouse IgG green fluorescent antibody and a mouse anti-rabbit IgG red fluorescent antibody as secondary antibodies, carrying out verification by using fluorescent Western blotting, and finally preliminarily determining an antigen binding region of the Hcp1 monoclonal antibody. As shown in FIG. 6C, monoclonal antibody 13F1 is only anti-HCP 1 (75-114)In addition, a single green fluorescent band exists at the theoretical molecular weight, the positions of the bands incubated with the corresponding GST tag antibody are completely overlapped, the 56-94 truncations which are not reacted are eliminated, and the monoclonal antibody 13F1 can specifically recognize the 95 th-114 th amino acid of the Hcp1.

3. Monoclonal antibody specificity and application Effect

The specificity of monoclonal antibodies was identified by Western blotting by collecting whole bacterial proteins from Pseudomonas aeruginosa (P.aeromonas), escherichia coli (E.coli), helicobacter pylori (H.pyri), mycobacterium tuberculosis (M.tuberculosis), staphylococcus aureus (S.aureus), salmonella enteritidis (S.enteritidis), fusobacterium nucleatum (F.nucleic), and Burkholderia cepacia (B.cepacia) and Burkholderia thailand (B.thailand) of the same species. The result shows that the Hcp1 monoclonal antibody provided by the invention does not cross react with bacteria of different species such as pseudomonas aeruginosa, escherichia coli, helicobacter pylori, mycobacterium tuberculosis, staphylococcus aureus, salmonella enteritidis, clostridium nucleatum and the like, does not react with Burkholderia cepacia and Burkholderia thailand of the same species, and has strong specificity in the aspect of immunological detection of the meldonium.

In conclusion, the invention provides the anti-meliotic bacteria hemolysin regulatory protein Hcp1 monoclonal antibody, the cell strain and the preparation method thereof, and the monoclonal antibody can be used as a meliotic bacteria Hcp1 detection reagent and applied to immunological detection of meliotic bacteria, has strong anti-interference capability, does not cross react with bacteria of different species such as pseudomonas aeruginosa, escherichia coli, helicobacter pylori, mycobacterium tuberculosis, staphylococcus aureus, salmonella enteritidis, clostridium nucleatum and the like, does not react with Burkholderia cepacia and Burkholderia thailand of the same species, and has strong specificity in immunological detection of the meliotic bacteria.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Claims

1. A hybridoma cell is characterized in that the preservation number is CCTCC NO: C202382.

2. A monoclonal antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID No. 1 and a light chain variable region having the amino acid sequence of SEQ ID No. 2.

3. The monoclonal antibody of claim 2, wherein the DNA sequence encoding the heavy chain variable region of the monoclonal antibody is the nucleotide sequence set forth in SEQ ID No.3 and the DNA sequence encoding the light chain variable region of the monoclonal antibody is the nucleotide sequence set forth in SEQ ID No. 4.

4. The monoclonal antibody of claim 3, wherein the monoclonal antibody is of the IgG1 subtype of kappa light chain and the Fc fragment of the monoclonal antibody has the amino acid sequence of SEQ ID No.9.

5. Use of a monoclonal antibody according to any one of claims 2-4 for the preparation of a reagent for detecting infection with melioidosis.

6. An agent for detecting a melioidosis infection comprising the monoclonal antibody of any one of claims 2-4.