Hybridoma cell line secreting monoclonal antibody neutralizing CPA toxin and its application

Abstract

The present invention belongs to the field of biotechnology, and relates to a hybridoma cell strain secreting a monoclonal antibody of Clostridium perfringens CPA with neutralizing activity and its application. The present invention uses natural CPA as an immune antigen and a non-toxic CPA mutant as a coating antigen for the first time, and screens a hybridoma cell strain secreting a monoclonal antibody with neutralizing activity. The hybridoma cell strain can stably and efficiently secrete a monoclonal antibody with neutralizing activity, and can achieve large-scale batch production, thereby providing a material basis for the detection and treatment of CPA antigen / antibody in the later stage.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a hybridoma cell strain of Clostridium perfringens CPA monoclonal antibody secreting neutralizing activity and application thereof. Background Art

[0002] Clostridium perfringens (CP) is a Gram-positive, spore-forming anaerobic bacillus, which is thin and straight with blunt ends under microscopic examination. In addition to infecting animals, C. perfringens is also a prevalent foodborne zoonosis pathogen. The exotoxins discovered so far include α, β1, β2, ε, ι, θ, γ, δ, λ, η, μ, κ, ν, etc. (Sun Yu, Wang Xiaoying, Dong Hao, et al. Research progress on Clostridium perfringens exotoxin genes and related vaccines [J]. Chinese Journal of Herbivorous Animal Science, 2016, 36(02): 58-62.), among which α toxin, β toxin, ε toxin, τ toxin, enterotoxin (CPE) and necrotic enteritis B-like toxin (NetB) are the main pathogenic exotoxins, which are encoded by cpa, cpb, etx, iap, cpe and NetB genes respectively. Clostridium perfringens is divided into seven pathotypes (AG) based on the carriage of six toxins. Type A produces α toxin, type B produces α, β and ε toxins, type C produces α and β toxins, type D produces α and ε toxins, type E produces α and ι toxins, type F produces α and CPE toxins, and type G produces α and NetB toxins. All five toxin types (A to E) of Clostridium perfringens carry and express α toxin (Uzal FA, Vidal JE, McClane BA, et al. Clostridium Perfringens Toxins Involved in Mammalian [J]. Open Toxinology 2018, 10(5): 212; Uzal FA, Vidal JE, McClane BA, et al. Clostridium Perfringens Toxins Involved in Mammalian [J]. Open Toxinology 2018, 10(5): 212.). Animals die quickly after being infected with Clostridium perfringens, with a general mortality rate of 5%-30%, causing serious losses to the aquaculture industry (Uzal FA, Vidal JE, McClane BA, et al. Clostridium Perfringens Toxins Involved in Mammalian [J]. Open Toxinology 2018, 10(5): 212.).

[0003] Alpha toxin is the most important exotoxin among all toxin genes of Clostridium perfringens. It can be produced by five types of bacteria, namely A, B, C, D, and E. Alpha toxin is the most important virulence factor of type A CP and the first bacterial protein found to have both enzymatic activity and toxin properties (Kiu R, Hall LJ. An update on the human and animal enteric pathogen Clostridium perfringens[J]. Emerg Microbes Infect, 2018, 7(1):1-15.). Alpha toxin (CPA) is conserved in all types of Clostridium perfringens. Although there are 1.3% nucleotide and 1.2% amino acid sequence differences between different strains, the differences in these nucleotides and encoded amino acids do not affect the activity of the alpha toxin itself. In terms of pathology, alpha toxin can affect the migration of immune cells such as neutrophils to infected tissues, so it can potentially reduce the clearance of pathogens at the site of infection. Alpha toxin can also cause vasoconstriction, reduce blood supply to tissues, and thus create a hypoxic environment that is conducive to the growth of CP. In addition, the inflammatory cascade reaction in metabolism (arachidonic acid and protein kinase C) triggered by α-toxin is the main cause of cell death (Zhao Pinnan, Luo Longlong. Research progress on the pathogenic mechanism of Clostridium perfringens and its therapeutic antibodies [J]. Chinese Journal of Pharmacology and Toxicology, 2024, 38(04): 304-313.). Type A Clostridium perfringens is widely present in different animals and can cause diseases in many animals and humans. Clostridium perfringens can cause hemorrhagic necrotic enteritis in chickens and other animals and is a potentially fatal gastrointestinal pathogen. The main characteristics of infection with this bacterium are rapid course of disease, sudden decrease in feed intake, depression, drooping wings, diarrhea, decreased weight gain rate, lethargy, ruffled feathers, loss of appetite, diarrhea, and even bloody feces, which have caused huge economic losses to the poultry industry (Zhang T, Zhang W, Ai D, et al. Prevalence and characterization of Clostridium perfringens in broiler chickens and retail chicken meat in central China[J]. Anaerobe, 2018, 54: 100- 103.).Type A Clostridium perfringens can also cause diarrhea and enterotoxemia in cattle, sheep, and goats, which are characterized by high mortality, sudden death, and necrotic hemorrhagic lesions in the small intestine, causing serious economic losses to the global livestock industry (Gharieb R, Saad M, Abdallah K, et al. Insights on toxin genotyping, virulence, antibiogram profiling, biofilm formation and efficacy of disinfectants on biofilms of Clostridium perfringens isolated from poultry, animals and humans [J]. Journal of Applied Microbiology, 2021, 130 (3): 819-831.). The enterotoxins produced by some strains of type A Clostridium perfringens can cause food poisoning, and some strains can cause gas gangrene and gastrointestinal diseases in humans. CPA has a strong toxicity, and its LD50 for mice can reach 3 µg / kg. Since there is no specific drug, once animals become ill, they often die before they can be treated. Therefore, prevention and treatment of diseases caused by CPA are particularly important.

[0004] At present, traditional natural toxin inactivated vaccines are usually used for immunization prevention for animals such as cattle, sheep and rabbits, but there is a lack of relevant toxin detection and treatment methods. Due to the complex antigen composition and low effective content of natural toxin inactivated vaccines, it is easy to cause side effects in animals, resulting in it being unable to be used for prevention in animals such as dogs and pigs. Existing studies have shown that the preparation of monoclonal antibodies can provide a basis for the establishment of treatment and diagnosis of certain toxins. The key to the preparation of monoclonal antibodies is the immunogen and the coating antigen, but the preparation, purification and inactivation of natural toxins are complicated and have high biosafety risks. However, Goossens et al. found that recombinant CPA still has a certain toxicity. Summary of the invention

[0005] Based on the selection of natural toxins, the present invention constructs a recombinant protein expressing 6 amino acid mutations, establishes an indirect ELISA detection method, and screens a hybridoma cell line that secretes a monoclonal antibody with neutralizing activity, thereby providing a material basis for the later detection and treatment of CPA antigen / antibody.

[0006] In the first aspect, the present invention provides a biological sequence encoding Clostridium perfringens CPA, wherein the biological sequence is an α-toxin gene fragment with 6 artificially introduced amino acid mutations, wherein the 6 amino acid mutations of the biological sequence are aspartic acid at positions 56 and 130 mutated to glycine, tyrosine at positions 275, 307 and 331 mutated to phenylalanine, and aspartic acid at position 336 mutated to asparagine, and the nucleotide sequence thereof is shown in SEQ ID NO: 1.

[0007] In a second aspect, the present invention provides a recombinant protein, the coding sequence of which is as shown in the biological sequence SEQ ID NO: 1 described in the first aspect: it is a recombinant protein secreted by competent cells after encoding a synthetic gene fragment and constructed into a vector, and is transformed into competent cells, and is named rCPA m6 .

[0008] Furthermore, the synthetic gene fragment includes codon optimization of the CPA encoding gene of type C Clostridium perfringens and addition of a tag protein.

[0009] Furthermore, the codon optimization refers to optimization according to the preferred codons of Escherichia coli.

[0010] Furthermore, the adding of the tag protein refers to adding a 6×His tag protein to the C-terminus of the tandem gene.

[0011] Furthermore, the vector is a prokaryotic expression vector, preferably a pET30a vector.

[0012] Furthermore, the competent cell is Escherichia coli, preferably Escherichia coli BL21 (DE3).

[0013] In a third aspect, the present invention provides a hybridoma cell strain that secretes anti-Clostridium perfringens CPA monoclonal antibodies, and the accession number of the hybridoma cell strain is: CGMCC NO.46024.

[0014] In a fourth aspect, the present invention provides a method for screening the hybridoma cell line according to the third aspect, the screening method comprising the following steps:

[0015] S1. Immunize mice, determine serum titer by ELISA, and use spleen cells from mice with the highest titer for cell fusion;

[0016] S2. Take the mouse spleen cells screened in S1 and fuse them with myeloma cells;

[0017] S3. Using the recombinant protein described in the second aspect to screen positive clones and subclones, a hybridoma cell line capable of secreting a monoclonal antibody against Clostridium perfringens alpha toxin with neutralizing activity is obtained.

[0018] Further, in step S1, the immunogen is natural CPA.

[0019] Furthermore, the natural CPA refers to the inactivated natural CPA of Clostridium perfringens.

[0020] Furthermore, in step S2, the myeloma cells are SP2 / 0 cells.

[0021] Furthermore, in step S2, the fusion method is a PEG-mediated chemical method.

[0022] Further, in step S3, the positive clone screening refers to the recombinant protein rCPA described in the second aspect. m6 The positive hybridoma cells were screened by indirect ELISA method using coated antigen.

[0023] Furthermore, in step S3, the subclone screening refers to performing a serum neutralization experiment on the cell supernatant of positive hybridoma cells using the prepared Clostridium perfringens natural toxin, and screening hybridoma cell lines with neutralization activity after subcloning.

[0024] In a fifth aspect, the present invention provides an anti-CPA antibody against Clostridium perfringens, wherein the antibody is a monoclonal antibody or a polyclonal antibody; the monoclonal antibody is obtained from the culture fluid of the hybridoma cell line described in the third aspect, or is obtained by inoculating the hybridoma cell line described in the third aspect into the peritoneal cavity of mice to produce ascites; the polyclonal antibody is immunized with inactivated natural CPA in mice and then with the rCPA described in the second aspect. m6 The protein was inoculated into the peritoneal cavity of mice to produce ascites.

[0025] In a sixth aspect, the present invention provides an antibody blocking ELISA detection method for Clostridium perfringens CPA, wherein the primary antibody is the polyclonal antibody described in the fifth aspect, and the secondary antibody is an HRP-labeled monoclonal antibody.

[0026] Furthermore, the HRP-labeled monoclonal antibody is a monoclonal antibody obtained by purifying the ascites of the monoclonal antibody described in the fifth aspect and then coupling and labeling it with HRP.

[0027] Furthermore, the working dilution of the CPA polyclonal antibody was 1:40, and the working concentration of the monoclonal antibody 6B8 was 1:16000.

[0028] In a seventh aspect, the present invention provides a use of the antibody described in the fifth aspect in preparing a reagent for detecting Clostridium perfringens CPA.

[0029] In an eighth aspect, the present invention provides a kit for detecting Clostridium perfringens CPA, wherein the kit comprises the antibody described in the fifth aspect.

[0030] Furthermore, the kit may be one or more of an ELISA detection kit, a colloidal gold detection kit, an immunohistochemistry kit, an immunofluorescence kit and / or an in situ hybridization staining kit.

[0031] Furthermore, the diagnostic method of the kit includes one or more of a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

[0032] The hybridoma cell line secreting anti-CPA monoclonal antibodies in the present invention has been provided with a biological deposit to the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The name of the deposit is mouse hybridoma cell, the deposit number is CGMCCNO.46024, and the deposit date is August 1, 2024.

[0033] Beneficial Effects

[0034] 1. The present invention obtains for the first time a safe 6-amino acid mutant rCPAm6 with a high soluble expression ratio.

[0035] 2. The present invention uses natural CPA as an immune antigen for the first time, uses a non-toxic CPA mutant (rCPAm6) as a coating antigen, and combines the serum neutralization test of Clostridium perfringens natural toxin to screen for neutralizing resistant monoclonal antibody hybridoma cell lines and establish a subsequent ELISA detection method; the method of using the above two different forms of toxins to screen monoclonal antibody hybridoma cell lines has the following advantages:

[0036] (1) Using a small amount of natural CPA as an immune antigen ensures that the natural conformation of CPA is maintained to the greatest extent;

[0037] (2) Using the non-toxic CPA mutant rCPAm6 with a purity of more than 90% as the coating antigen for screening of positive clones and subclones and the establishment of subsequent ELISA detection methods, it not only ensures the purity of the coating antigen, but also avoids the cumbersome purification steps and biosafety risks of natural CPA of Clostridium perfringens;

[0038] (3) Using the natural toxin of Clostridium perfringens to conduct a serum neutralization test of monoclonal antibodies, a monoclonal antibody hybridoma cell line with neutralization resistance to natural CPA was obtained.

[0039] 3. Obtain a hybridoma cell line that secretes a neutralization-resistant Clostridium perfringens CPA monoclonal antibody. The hybridoma cell line can stably and efficiently secrete a neutralizing monoclonal antibody and can be mass-produced. The monoclonal antibody can neutralize the natural toxin of Clostridium perfringens in vitro in mice, and its neutralization titer can reach 1 mouse MLD / 0.1mL, thus providing important materials for the preparation of animal-specific drugs for the clinical prevention and treatment of Clostridium perfringens disease.

[0040] 4. For the first time, an antibody blocking ELISA detection method for CPA was established using a neutralizing-resistant monoclonal antibody against Clostridium perfringens. This method has the characteristics of simple sample operation, low cost, rapid reaction, and strong specificity. It can not only provide a reference for the diagnosis of Clostridium perfringens disease, but also provide a basis for the screening of Clostridium perfringens antibody-negative animals and the study of alternative methods for testing the efficacy of related vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Survival curve for rCPAm6 virulence assay.

[0042] Figure 2 SDS-PAGE identification of rCPAm6. Note: M, Marker; 1, 0.5 mg / ml BSA; 2, whole bacteria after ultrasound; 3, supernatant after ultrasound; 4, precipitate after ultrasound. DETAILED DESCRIPTION

[0043] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0044] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0045] Example 1 Expression and purification of rCPAm6

[0046] According to the coding gene sequence of CPA, an α-toxin gene fragment with 6 amino acid mutations (aspartic acid at positions 56 and 130 mutated to glycine, tyrosine at positions 275, 307 and 331 mutated to phenylalanine, and aspartic acid at position 336 mutated to asparagine) was artificially introduced, and its nucleotide sequence is shown in SEQ ID NO: 1. At the same time, a 6×His tag coding gene was added to the C-terminus of the recombinant fusion protein. The above gene sequence was optimized according to the preferred codons of Escherichia coli to synthesize GCPA m6GCPA was transformed into m6 Constructed into pET30a vector to obtain the prokaryotic expression vector pET30a-GCPA m6 . pET30a-GCPA m6 The vector was transformed into E. coli BL21 (DE3) competent cells for induction expression and purification, and finally the recombinant protein rCPA with a purity of about 90% and a concentration of 1.2 mg / mL was obtained. m6 The toxicity of the protein was tested using ICR mice.

[0047] The results are as follows Figure 1 As shown, when rCPA m6 When the inoculation dose was 100 μg, all mice were alive and had no adverse reactions, while natural CPA of one mouse MLD caused 5 / 5 deaths of mice within 1 hour of inoculation. m6 It was non-toxic in mice and was determined to be a non-toxic recombinant fusion protein.

[0048] Example 2 Screening of hybridoma cell lines secreting neutralizing Clostridium perfringens CPA monoclonal antibodies and preparation of monoclonal antibody ascites

[0049] 1. Immunization of BALB / c mice and determination of antibody titer

[0050] Female BALB / c mice were immunized with inactivated natural CPA as the immunogen by multiple subcutaneous injections. Immunization was performed every 14 days. 7-10 days after the third immunization, an appropriate amount of blood was collected from the eye sockets and the serum titer was determined by indirect ELISA. If the serum titer reached the fusion requirement (the titer was higher than 12800), the immunization was strengthened three days before the fusion, and the rCPAm6 protein was directly injected intraperitoneally. 3-5 days after the last immunization, the spleen cells of the mouse with the highest titer (1:25600) were taken for cell fusion.

[0051] 2. Cell fusion and screening of hybridoma cell lines

[0052] Myeloma cells SP2 / 0 with good growth status were chemically fused with the spleen cells of the mouse with the highest titer detected by ELISA using PEG1450. m6Positive hybridoma cells were screened by the indirect ELISA method of coated antigen, and the cell supernatant of positive hybridoma cells was subjected to serum neutralization experiment with the prepared natural toxin of Clostridium perfringens. After 3-5 subclonings by limiting dilution method, 4 hybridoma cell lines of Clostridium perfringens CPA with neutralizing activity were obtained, which were named 6E3, 6B8, 10A11, and 13D10, respectively. The indirect ELISA titer of cell supernatant can reach 1:3200 or above. The four monoclonal antibodies were detected by subclass identification kit, and their subtypes were determined to be IgG1, IgG1, IgG2a, and IgG1, respectively. According to the provisions of the third part of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the culture supernatants of four cells were neutralized with natural CPA toxin and injected into mice through the tail vein.

[0053] The results showed that 100% (5 / 5) of the mice injected with the cell supernatant of hybridoma cell 6B8 and the toxin neutralizer survived, while the mice injected with the cell supernatant of the other three strains and the toxin neutralizer all died, indicating that the 6B8 cell supernatant has neutralizing activity.

[0054] 3. Preparation of ascites containing CPA monoclonal antibodies with neutralizing activity

[0055] Healthy multiparous BALB / c mice aged 6 to 8 months were selected and sensitized by intraperitoneal injection of Freund's incomplete adjuvant. Hybridoma cells were injected 7 days later. The above-mentioned hybridoma cells 6B8 and SP2 / 0 cells in the logarithmic growth phase were collected, centrifuged, washed and resuspended in DMEM culture medium, and the cell density was adjusted to 10 6 After 7-14 days, when the mouse abdomen is obviously swollen, use a sterile needle to pierce the swollen part of the mouse abdomen to make the ascites flow out or drip out; collect the ascites, centrifuge, mark as 6B8 and myeloma cell SP2 / 0 ascites control (S), and store at -20℃ for later use.

[0056] Example 3 Purification and detection of monoclonal ascites

[0057] Protein A (GE Healthcare 17-5079-01) affinity column purification method was used for purification. The purification steps are as follows:

[0058] Sample pretreatment: Ascites was diluted 1:3 with coupling buffer (20 mM sodium phosphate buffer, pH 7.0), and fat, cell residues and small particulate matter were removed after centrifugation.

[0059] Equilibration: Equilibrate the column with 5-10 column volumes of coupling buffer, maintaining a flow rate of 2 s / drop.

[0060] Loading: Use a syringe to inject the sample into the upper interface of the column, collect the effluent, and maintain a flow rate of 4s / drop.

[0061] Washing: Pass 5 column volumes of coupling buffer through the column, maintaining a flow rate of 2s / drop.

[0062] Elution: Elute the antibody with 5 column volumes of elution buffer (0.1 M sodium citrate buffer, pH 9.0), collect, and maintain a flow rate of 4 s / drop.

[0063] Sample detection: The purified monoclonal antibody was identified by SDS-PAGE.

[0064] The results are as follows Figure 2 As shown in the figure, a monoclonal antibody with a purity of 91% was obtained by purification using a Protein A affinity column; the concentration of the monoclonal antibody was determined using the BCA method, and its concentration could reach 2.8 mg / mL.

[0065] Example 4 HRP labeling of monoclonal antibodies

[0066] Mix the antibody and activated HRP: Take 0.1 mg of antibody, add 25 ul of reagent B, and mix well with a pipette tip.

[0067] Add 2ul of coupling catalyst solution A to the mixture of antibody and HRP enzyme, mix well with a pipette tip or vortex.

[0068] Allow to react at room temperature for 2 hours, or overnight at 4°C. The coupled antibody can be used immediately or stored at low temperature.

[0069] Example 5 Establishment of a blocking ELISA detection method for Clostridium perfringens CPA antibody based on monoclonal antibody 6B8

[0070] 1. Preparation of anti-Clostridium perfringens CPA polyclonal antibodies:

[0071] Using inactivated natural CPA as the immunogen, female BALB / c mice were immunized subcutaneously once every 14 days. 7 to 10 days after the third immunization, an appropriate amount of blood was collected from the eye sockets and the serum titer was determined by indirect ELISA. If the serum titer reached the fusion requirement (titer higher than 12,800), blood could be collected.

[0072] 2. Determination of the conditions for a blocking ELISA detection method for Clostridium perfringens CPA antibodies

[0073] Screening of optimal working concentrations of mouse CPA polyclonal and monoclonal antibodies:

[0074] The purified rCPA was washed with PBS m6Dilute to 2 ug / ml, 100 μL / well, block overnight at 4°C, wash with PBST, add PBST solution containing 5% skim milk powder as blocking solution, 200 μL / well, block at 37°C for 2 h;

[0075] The plate was washed with PBST solution, patted dry, and stored at -20°C for later use.

[0076] Take out the coated ELISA plate, place it at room temperature for 30 min, add the obtained anti-Clostridium perfringens CPA polyclonal antibody, use PBS as the diluent, and the dilution ratios are original solution, 1:5, 1:10, 1:20, 1:40 and 1:80, 100 μL / well, and add negative serum of corresponding dilution ratio at the same time, incubate at 37°C for 1 h.

[0077] Wash with PBST solution, add purified HRP-labeled monoclonal antibody 6B8, use PBS as the diluent, dilute 2-fold from 1:2000 to 1:64000, 100 μL / well, and incubate at 37°C for 30 min.

[0078] Wash with PBST solution and pat the ELISA plate dry.

[0079] Finally, add 50 μL / well of soluble TMB substrate colorimetric solution and react for 10 min at room temperature in the dark. 2 SO 4 The solution was used to terminate the reaction and the absorbance was read at 450 nm.

[0080] The OD value of the negative control well was selected to be close to 1.0, and the dilution corresponding to the positive serum blocking rate ≥50% was the optimal working concentration.

[0081] The working dilution of CPA polyclonal antibody was finally determined to be 1:40, and the working concentration of monoclonal antibody 6B8 was 1:16000.

Claims

1. A biological sequence encoding Clostridium perfringens CPA, the biological sequence is an alpha toxin gene fragment with 6 amino acid mutations artificially introduced, the 6 amino acid mutations of the biological sequence are aspartic acid at positions 56 and 130 mutated to glycine, tyrosine at positions 275, 307 and 331 mutated to phenylalanine, and aspartic acid at position 336 mutated to asparagine, and its nucleotide sequence is shown in SEQ ID NO:

1.

2. A recombinant protein, the recombinant protein is encoded by the biological sequence of claim 1: it is a recombinant protein secreted by the competent cells after the gene fragment encoded by the biological sequence of claim 1 is constructed into a vector and transformed into competent cells, and is named rCPA m6 .

3. The recombinant protein according to claim 2, wherein the synthetic gene fragment comprises codon optimization of the CPA encoding gene of Clostridium perfringens type A and addition of a tag protein.

4. The recombinant protein according to claim 3, wherein the codon optimization refers to optimization according to the preferred codons of Escherichia coli.

5. The recombinant protein according to claim 3, wherein the added tag protein refers to adding a 6×His tag protein to the C-terminus of the tandem gene. The recombinant protein according to claim 2 , wherein the vector is a prokaryotic expression vector. The recombinant protein according to claim 6 , wherein the prokaryotic expression vector is a pET30a vector. The recombinant protein according to claim 2 , wherein the competent cells are Escherichia coli.

9. The recombinant protein according to claim 8, wherein the Escherichia coli is Escherichia coli BL21.

Images