Pertussis recombinant protein and monoclonal antibody and application thereof

Abstract

The invention discloses a pertussis recombinant protein and a monoclonal antibody and application thereof.The pertussis recombinant protein is synthesized by selecting an S1 subunit of pertussis PT and a part of sequences of FHA protein, the pertussis recombinant protein is expressed through a prokaryotic expression system and purified, the protein serves as an antigen to immunize a mouse, and the pertussis recombinant protein is obtained. Positive hybridoma cell strains 3B10, 3D9 and 5F7 capable of stably secreting the antibody are obtained by adopting a hybridoma technology, the preservation numbers are respectively CCTCC NO: C202567, CCTCC NO: C202568 and CCTCC NO: C202569, and the obtained monoclonal antibody has relatively high affinity and detection sensitivity to pertussis PT and FHA recombinant proteins, is used for detecting a bordetella pertussis antigen, is strong in specificity and high in sensitivity, can be used for instant detection, and can be applied to clinical application. And the method has a relatively high application value.

Description

Technical Field

[0001] The present invention belongs to the field of rapid detection technology, and specifically relates to a new recombinant protein. The recombinant protein is used to prepare anti-pertussis monoclonal antibodies and is applied to the specific instant detection of pertussis. Background Art

[0002] Whooping cough is an acute respiratory infectious disease caused by Bordetella pertussis (BP). The bacteria is mainly transmitted through airborne droplets. After adhering to the ciliated epithelial cells of the infected person's respiratory tract, it begins to multiply rapidly and spreads and colonizes in the lower respiratory tract. The toxins produced by the bacteria can cause local epithelial cell damage. One to two weeks after infection, secretions increase, leading to a series of symptoms such as sneezing, vomiting, high-pitched spasmodic coughs, and fatigue. The cough symptoms can last for 10 weeks or even longer, hence the name whooping cough.

[0003] Pertussis was the leading cause of illness and death among children in the first half of the 20th century. The subsequent use of whole-cell pertussis vaccine has greatly controlled the morbidity and mortality of pertussis. However, due to the polymorphism of the pertussis antigen gene and antigenic drift, the incidence of pertussis has shown a slow and steady rebound growth trend in recent years. Many countries with high vaccine coverage around the world have seen a "re-emergence of pertussis", with the peak age of onset shifting from infants and young children to adolescents and adults, who have become the main sources of infection for infant pertussis.

[0004] Currently, the WHO's diagnostic criteria for pertussis generally include clinical manifestations, epidemiology, and laboratory testing. Clinical manifestations include a cough lasting more than two weeks with paroxysmal, spasmodic coughs accompanied by a "cock-crow"-like inspiratory roar. Epidemiology primarily relies on contact with a confirmed case and onset of the disease during the incubation period. Laboratory testing primarily involves Bordetella pertussis culture, serum-specific antibody testing, and nucleic acid testing. Bacterial culture and nucleic acid testing require early detection and are highly sensitive, while serum antibody testing excludes individuals with a recent history of vaccination. Currently, pertussis diagnosis in China is primarily clinical, with limited medical facilities capable of laboratory diagnosis. Due to regional differences in economic and medical development, the incidence of pertussis varies significantly across my country. This, to some extent, reflects the level of research and attention paid to pertussis, rather than the true incidence and distribution. Lagging diagnostic standards, clinicians' limited understanding of the disease, which prevents early identification of patients, and limited laboratory testing capacity at medical institutions, which prevents them from providing supplementary diagnosis, lead to potential underdiagnosis, underreporting, misdiagnosis, and false positives in clinical practice.

[0005] In view of the current status of pertussis diagnosis and detection, there is an urgent need for more timely and accurate diagnostic methods. Using point-of-care testing (POCT) technology to detect diseases on-site is becoming a key factor in overcoming the crisis and saving lives. POCT has many advantages such as simple portability, low cost, sensitivity, and rapidity. It is widely used in the early diagnosis and monitoring of respiratory viruses. However, compared with other respiratory viruses, there is relatively less research on pertussis, and there is still great room for development in related raw materials such as monoclonal antibodies and point-of-care testing technology.

[0006] Monoclonal antibodies play a crucial role in the establishment of pathogen diagnostic methods. Currently, various countries are actively researching the antigen components of pertussis, including pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), tracheal cytotoxin (TCT), etc. Among them, PT is a specific antigen of Bordetella pertussis and plays a key role in the occurrence of pertussis. It can promote the adhesion of Bordetella pertussis to respiratory ciliated epithelial cells, causing them to degenerate and necrosis, resulting in an increase in lymphocytes and affecting the normal secretion of insulin, etc.; FHA is a serotoxin secreted by Bordetella pertussis, which has hemagglutination activity and participates in the activity of Bordetella pertussis adhering to host cells. It is also a key virulence factor. Summary of the Invention

[0007] The object of the present invention is to provide a recombinant protein, and use this recombinant protein to prepare a highly specific and sensitive anti-pertussis monoclonal antibody, and establish a rapid and effective colloidal gold technology for detecting pertussis using this antibody, hoping to fill the gap in point-of-care testing for pertussis through this method and provide more options for the diagnosis and monitoring of pertussis in clinical practice.

[0008] The technical solution adopted by the present invention is:

[0009] A pertussis recombinant protein, the amino acid sequence of the pertussis recombinant protein is shown in SEQ ID NO:1.

[0010] The pertussis recombinant protein of the present invention is composed of the S1 subunit of pertussis PT and a partial sequence of FHA. This recombinant protein has strong antigenicity and is a key virulence factor of pertussis.

[0011] Pertussis PT is an A-B type toxin composed of 6 subunits. The S1 subunit is the site of PT toxicity. It has ADP-ribosyltransferase activity, can catalyze the ADP-ribosylation of GTP-binding regulatory proteins, and is related to most of the biological activities of the PT molecule. FHA is a surface protein molecule with an adhesion function, with a molecular weight of about 220 kDa. It has strong immunogenicity, can assist pertussis in adhering to monocytes, macrophages and other cells, inhibit the phagocytosis of macrophages, and plays a promoting role in the pathogenesis of pertussis. Since the FHA sequence is relatively long, in the present invention, through screening, a part of the FHA sequence was intercepted and combined with the S1 subunit of PT to form a fusion protein.

[0012] Furthermore, the present invention also provides a coding gene for the pertussis recombinant protein, and the nucleotide sequence of the coding gene is shown in SEQ ID NO:2.

[0013] The pertussis recombinant protein of the present invention can be induced and expressed through a prokaryotic expression system to obtain the said pertussis recombinant protein.

[0014] Furthermore, the present invention also provides a recombinant vector containing the coding gene for the pertussis recombinant protein.

[0015] Furthermore, in the said recombinant vector, a DNA sequence encoding a 6×HisTag tag is added to the carboxyl terminus of the peptide chain of the pertussis recombinant protein. The purpose of adding the DNA sequence encoding the 6×His Tag tag is to facilitate affinity chromatography purification and specific detection.

[0016] The said recombinant vector contains a polynucleotide operably linked to a control sequence suitable for expression in a host cell.

[0017] Preferably, the expression vector of the recombinant vector is pET-28a(+).

[0018] The said recombinant vector can be synthesized by inserting the nucleotide sequence of the coding gene of the pertussis recombinant protein with a 6×His Tag tag added into the expression vector pET-28a(+), and is denoted as pET-28a-PT-FHA.

[0019] The present invention also provides a recombinant genetic engineering bacterium expressing the pertussis recombinant protein, which is obtained by transforming the said recombinant vector into a host bacterium. [[ID=]26]

[0020] The said host bacterium is usually Escherichia coli E.coli BL21(DE3).

[0021] Furthermore, the said pertussis recombinant protein can be obtained by the following method:

[0022] Construct a recombinant vector containing the coding gene of the pertussis recombinant protein, transform the recombinant vector into a host bacterium, induce and culture the obtained recombinant genetically engineered bacterium, separate the bacterial cells containing the pertussis recombinant protein from the culture solution, and purify the protein after cell disruption to obtain the pertussis recombinant protein.

[0023] The present invention also provides a method for preparing the pertussis recombinant protein, and the method comprises the following steps;

[0024] Construct a recombinant vector containing the coding gene of the pertussis recombinant protein, transform the recombinant vector into a host bacterium, induce and culture the obtained recombinant genetically engineered bacterium, separate the bacterial cells containing the pertussis recombinant protein from the culture solution, ultrasonically disrupt the bacterial cells, purify the protein by affinity chromatography to obtain inclusion body protein, and perform dialysis renaturation on the inclusion body protein to prepare the pertussis recombinant protein.

[0025] The present invention also provides the application of the pertussis recombinant protein in the preparation of anti-pertussis monoclonal antibodies.

[0026] Furthermore, the present invention provides an anti-pertussis monoclonal antibody, which is obtained by immunizing mice with the pertussis recombinant protein and using the hybridoma technology.

[0027] Furthermore, immunize mice with the pertussis recombinant protein as an immunogen, use the hybridoma technology to fuse mouse spleen cells with SP2 / 0 cells using PEG, screen for positive hybridoma cells that stably secrete antibodies, inject the positive hybridoma cells into mice to prepare ascites and purify it to obtain a monoclonal antibody that can specifically immunoreact with the pertussis antigen.

[0028] The present invention also provides an anti-pertussis monoclonal antibody secreted by the hybridoma cell line 3B10 with the preservation number CCTCC NO: C202567.

[0029] The present invention also provides an anti-pertussis monoclonal antibody secreted by the hybridoma cell line 3D9 with the preservation number CCTCC NO: C202568.

[0030] The present invention also provides an anti-pertussis monoclonal antibody secreted by the hybridoma cell line 5F7 with the preservation number CCTCC NO: C202569.

[0031] The present invention also provides the hybridoma cell line 3B10 that secretes anti-pertussis monoclonal antibodies, which is preserved in the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan, China, the preservation date being March 9, 2025, and the preservation number being CCTCC NO: C202567.

[0032] The hybridoma cell line 3D9 secreting anti - pertussis monoclonal antibody is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is March 9, 2025, and the deposit number is CCTCC NO: C202568.

[0033] The hybridoma cell line 5F7 secreting anti - pertussis monoclonal antibody is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is March 9, 2025, and the deposit number is CCTCC NO: C202569.

[0034] The present invention also provides a method for preparing an anti - pertussis monoclonal antibody, and the method comprises the following steps:

[0035] (1) Construct a recombinant vector containing the coding gene of the pertussis recombinant protein, transform the recombinant vector into E.coli BL21(DE3) competent cells, induce protein expression using IPTG, break the bacterial cells by ultrasonic disruption, and identify the protein expression form by SDS - PAGE electrophoresis; purify the protein by nickel - column affinity chromatography, and renature the recombinant protein expressed as inclusion bodies by dialysis to obtain the purified pertussis recombinant protein as the immunizing antigen.

[0036] (2) Immunize mice with the above - purified pertussis recombinant protein. After multiple immunizations, select the mouse with the highest antibody titer for booster immunization. Take the spleen cells and SP2 / 0 cells for fusion, and screen out the positive hybridoma cells that can stably secrete antibodies by indirect ELISA and sub - cloning. Inject the positive hybridoma cells into mice to prepare ascites and purify it to obtain the anti - pertussis monoclonal antibody.

[0037] Furthermore, step (2) is preferably:

[0038] Immunize mice with the above - purified pertussis recombinant protein. After three immunizations, measure the antibody titer of mouse serum by indirect ELISA method, and select the mouse with the highest antibody titer for booster immunization.

[0039] Take the spleen of the booster - immunized mouse and fuse it with SP2 / 0 cells using PEG to obtain positive hybridoma cells. Perform multiple rounds of sub - cloning on the positive hybridoma cells to screen out the hybridoma cell line that can stably secrete anti - pertussis antibodies.

[0040] Expand the culture of the positive hybridoma cells and prepare mouse ascites. After collecting the mouse ascites, purify it to obtain the anti - pertussis monoclonal antibody.

[0041] The present invention also provides the application of the anti - pertussis monoclonal antibody in detecting Bordetella pertussis.

[0042] Furthermore, the present invention also provides the use of the anti-pertussis monoclonal antibody in the preparation of a diagnostic reagent for Bordetella pertussis.

[0043] The present invention also provides a diagnostic kit for detecting pertussis antigen, comprising the anti-pertussis monoclonal antibody.

[0044] Furthermore, preferably, the diagnostic kit adopts the double antibody sandwich method.

[0045] Furthermore, in the diagnostic kit, a labeled antibody and a coated antibody are included, and the combination of the labeled antibody and the coated antibody can be any one of the following:

[0046] (1) The coated antibody is the anti-pertussis monoclonal antibody secreted by the hybridoma cell line 5F7 with the preservation number CCTCC NO: C202569; the labeled antibody is the anti-pertussis monoclonal antibody secreted by the hybridoma cell line 3D9 with the preservation number CCTCC NO: C202568;

[0047] (2) The coated antibody is the anti-pertussis monoclonal antibody secreted by the hybridoma cell line 3B10 with the preservation number CCTCC NO: C202567; the labeled antibody is the anti-pertussis monoclonal antibody secreted by the hybridoma cell line 3D9 with the preservation number CCTCC NO: C202568;

[0048] (3) The coated antibody is the anti-pertussis monoclonal antibody secreted by the hybridoma cell line 3B10 with the preservation number CCTCC NO: C202567; the labeled antibody is the anti-pertussis monoclonal antibody secreted by the hybridoma cell line 5F7 with the preservation number CCTCC NO: C202569;

[0049] (4) The coated antibody is the anti-pertussis monoclonal antibody secreted by the hybridoma cell line 3D9 with the preservation number CCTCC NO: C202568; the labeled antibody is the anti-pertussis monoclonal antibody secreted by the hybridoma cell line 5F7 with the preservation number CCTCC NO: C202569.

[0050] Generally speaking, for multiple stable hybridoma cell lines secreting anti-pertussis antibodies, the multiple antibodies secreted by them are paired pairwise and used as the labeled antibody and the coated antibody respectively to prepare a diagnostic kit based on the double antibody sandwich method for detecting pertussis recombinant protein and inactivated Bordetella pertussis culture, and the combination mode of the labeled antibody and the coated antibody with the highest sensitivity can be screened out.

[0051] Furthermore, the diagnostic kit can be an enzyme-linked immunosorbent assay kit, a chemiluminescent immunoassay kit, a fluorescence immunoassay kit or a colloidal gold immunoassay kit.

[0052] Preferably, the diagnostic kit is a colloidal gold immunoassay kit, comprising colloidal gold test paper, wherein the colloidal gold test paper is a lateral flow chromatography test paper, and adopts a double antibody sandwich method, comprising a labeled antibody and a coated antibody, wherein the labeled antibody is bound to the colloidal gold particles and fixed on the binding pad of the test paper; the coated antibody is fixed on the detection area (T line) and the control area (C line); the combination of the labeled antibody and the coated antibody can be any one of combinations (1) to (4).

[0053] The present invention has the following beneficial effects: fragments of the two antigens PT and FHA are screened separately to form a fusion protein, the fusion protein is expressed using a prokaryotic expression system, the protein is purified and then used to immunize mice, and hybridoma technology is used to screen three hybridoma cell lines 3B10, 3D9 and 5F7 that can stably secrete anti-pertussis monoclonal antibodies. The obtained monoclonal antibodies have high affinity and detection sensitivity for pertussis PT and FHA recombinant proteins. The pertussis monoclonal antibodies are used to establish a highly sensitive and accurate instant detection kit for detecting Bordetella pertussis antigens. The kit has the characteristics of strong specificity and high sensitivity, can be used for rapid and accurate immunodetection and immunoanalysis of Bordetella pertussis antigens, and lays a foundation for the research, development and promotion of ELISA, colloidal gold test strips and fluorescent immunoassay strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is the vector map of the pertussis pET-28a-PT-FHA recombinant plasmid.

[0055] Figure 2 This is the result diagram of the induced expression of pertussis recombinant protein under different conditions.

[0056] Figure 3 This is the SDS-PAGE electrophoresis result of the supernatant and inclusion body after ultrasonic lysis of the induced expression product.

[0057] Figure 4 This is the SDS-PAGE electrophoresis result of the flow-through of the inclusion body solubilization solution after passing through the nickel column affinity chromatography.

[0058] Figure 5 This is the SDS-PAGE electrophoresis result after the purified protein was concentrated and dialyzed and renatured.

[0059] Figure 6 This is a photograph of well-growing hybridoma cell colonies observed under a microscope on the 6th day after cell fusion.

[0060] Figure 7 This is the SDS-PAGE electrophoresis result of mouse ascites after purification by octanoic acid-saturated sulfuric acid.

[0061] Figure 8Results of detecting pertussis recombinant protein antigen after preparing colloidal gold test strips with 6 antibody combinations.

[0062] Figure 9 Results of detecting blank diluent after preparing colloidal gold test strips with 6 antibody combinations.

[0063] Figure 10 Results of detecting inactivated pertussis bacillus culture after preparing colloidal gold test strips with 6 antibody combinations.

[0064] Figure 11 Results of detecting inactivated pertussis bacillus culture with different dilution multiples for combination 6. Detailed implementation manners

[0065] To make the present invention easier to understand, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Specific experimental methods not mentioned in the following examples are usually carried out according to conventional experimental methods.

[0066] Example 1 Expression and purification of pertussis recombinant protein

[0067] (1) Synthesis of pertussis pET-28a-PT-FHA plasmid

[0068] In the present invention, the S1 subunit sequence of pertussis PT refers to NCBI: WP_019248461.1, and the FHA sequence refers to NCBI: WP_010930610.1. The full sequence of the S1 subunit of PT and a partial sequence of FHA are recombined, specifically as shown in SEQ ID NO:1:

[0069] SEQ ID NO:1:

[0070] GSMRCTRAIRQTARTGWLTWLAILAVTAPVTSPAWADDPPATVYRYDSRPPEDVFQNGFTAWGNNDNVLDHLTGRSCQVGSSNSAFVSTSSSRRYTEVYLEHRMQEAVEAERAGRGTGHFIGYIYEVRADNNFYGAASSYFEYVDTYGDNAGRILAGALATYQSEYLAHRRIPPENIRRVTRVYHNGITGETTTTEYSNARYVSQQTRANPNPYTSRRSVASIVGTLVRIAPVIGACMARQAESSEAMAAWSERAGEAMVLVYYESIAYSFGGGGSGGGGSGGGGSLKNLDLGYQAKPAPTAPPMPKAPELDLRGHTLESAEGRKIFGEYKKLQGEYEKAKMAVQAVEAYGEATRRVHDQLGQRYGKALGGMDAETKEVDGIIQEFAADLRTVYAKQADQATIDAETDKVAQRYKSQIDAVRLQ

[0071] 编码该重组蛋白的DNA序列如SEQ ID NO:2所示:

[0072] SEQ ID NO:2:

[0073]

[0074] The above sequence was inserted into the prokaryotic expression vector pET-28a(+), and a HisTag tag was added to the carboxyl end of the target fragment. The synthesis was entrusted to Nanjing GenScript Company. The synthesized recombinant plasmid was named pET-28a-PT-FHA. The plasmid map is shown in Figure 1 shown.

[0075] (2) Expression of pertussis pET-28a-PT-FHA plasmid

[0076] The pertussis pET-28a-PT-FHA plasmid was transformed into E. coli BL21 (DE3) competent cells, spread on a plate containing kanamycin resistance and cultured overnight. A single colony was picked and inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C overnight. The next day, the bacterial solution was taken out and inoculated into LB liquid medium containing kanamycin resistance at a ratio of 1:100. The bacterial solution was cultured at 37°C with a shaker at 160 rpm / min until its OD 600nm When the value reached 0.6-0.8, IPTG was added at final concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mmol / L, respectively, and induced at 37°C for 5 h. The expression product was centrifuged at 4°C, 12000 rpm / min for 5 min, and the bacterial pellet was collected and washed three times with 0.01 mol / L PBS. The supernatant and the pellet resuspension were then subjected to SDS-PAGE electrophoresis to test the protein expression at different IPTG concentrations ( Figure 2 ). Figure 2 The results showed that the expression level was high at a concentration of 0.8 mmol / L IPTG, so 0.8 mmol / L IPTG was induced for large-scale expression. The expression product was centrifuged according to the above operation, washed with PBS, and then the bacterial pellet was resuspended with cell lysis buffer. It was placed on ice and ultrasonically disrupted (power 220 W, working 5 s, rest 5 s) until the bacteria were no longer viscous. Centrifuged at 4 ° C and 12000 rpm / min for 20 min. The supernatant was the soluble part, and the precipitate was the inclusion body part. The precipitate was resuspended with lysis buffer, and part of the supernatant and inclusion body were taken for SDS-PAGE electrophoresis to determine the expression form of the recombinant protein. The SDS-PAGE electrophoresis results of the supernatant and inclusion body after ultrasonic lysis of the induced expression product are shown in the figure. Figure 3 shown. Figure 3 The SDS-PAGE electrophoresis results showed that a target band appeared at about 46 kDa in both the whole bacteria and inclusion bodies after induction, which was consistent with the theoretical value of the recombinant protein, indicating that the recombinant protein was expressed in E. coli in the form of inclusion bodies.

[0077] (3) Purification and dialysis refolding of pertussis recombinant protein

[0078] The inclusion body suspension was rotated overnight on a shaker at 4°C until completely dissolved, and then purified by nickel column affinity chromatography (the SDS-PAGE electrophoresis result diagram of the flow-through solution after the inclusion body lysate passed through the nickel column by nickel column affinity chromatography is as shown in Figure 4 ), concentrated with an ultrafiltration tube. The purified protein solution contains different concentrations of imidazole and a high concentration of urea. At the same time, the protein expressed by the inclusion body loses its original activity. In order to remove substances such as imidazole and urea and restore the activity of the protein expressed by the inclusion body as much as possible, the concentrated protein solution was dialyzed and refolded, and finally a protein with a relatively high purity was obtained (the SDS-PAGE electrophoresis result diagram after the purified protein was concentrated and dialyzed and refolded is as shown in Figure 5 ).

[0079] Example ② Preparation of Monoclonal Antibody Against Pertussis Recombinant Protein

[0080] (1) Immunization of Balb / c Mice

[0081] The purified pertussis recombinant protein was mixed with Freund's complete adjuvant at a volume ratio of 1:1 and emulsified. The mice were immunized by multiple subcutaneous injections in the back. The immunization dose was 50 μg / mouse. Immunization was carried out once every 3 weeks for a total of 3 times. The immunization dose and route for the second and third immunizations were the same as those for the first immunization, but the adjuvant was changed to Freund's incomplete adjuvant. Seven days after the third immunization, the mice were bled from the tip of the tail, and the serum was separated and the antibody titer of the immunized mouse serum was measured by indirect ELISA. The mouse with the highest titer was intraperitoneally boosted 3 days before cell fusion. The protein for boosting immunization did not add adjuvant, and the immunization dose was 100 μg / mouse.

[0082] (2) Screening, Identification and Subcloning of Positive Hybridoma Cells

[0083] 1) Preparation of SP2 / 0 Cells

[0084] Before fusion, the SP2 / 0 cells were expanded in culture to ensure that there were enough cells for fusion requirements. Take the SP2 / 0 cells in good growth state in logarithmic growth phase. After blowing down the cells with a Pasteur pipette, collect them in a 50 mL centrifuge tube, centrifuge at 1000 rpm for 10 min, discard the supernatant, and finally resuspend the cells with RPMI-1640 medium. After cell counting, place them in an incubator at 37°C and 5% CO2 for standby.

[0085] 2) Preparation of Feeder Cells

[0086] Eight-week-old Balb / c mice were sacrificed by cervical dislocation, immersed in 75% alcohol for 5 - 10 min, and then transferred to a biosafety cabinet for supine fixation. The abdominal skin of the mice was lifted with forceps, and a small incision was made perpendicular to the mid-abdominal line with scissors. The skin was separated along the mid-abdominal line to expose the entire abdominal cavity. The culture medium was aspirated with a syringe and injected into the abdominal cavity of the mice. The abdominal cavity of the mice was gently kneaded to fully mix the culture medium in the abdominal cavity. The mixed liquid in the abdomen was aspirated, and the process was repeated 3 times. The three abdominal lavage fluids were combined and centrifuged at 1000 rpm / min for 10 min. The supernatant was discarded, and cell counting was performed. The cells were resuspended in complete HAT medium, and the cell number was adjusted to 1×10 5 cells / mL. The cells were mixed evenly and evenly plated into a 96-well plate, 100 μL / well, and transferred to an incubator at 37°C and 5% CO2 for standby.

[0087] 3) Preparation of splenocytes

[0088] The mice after booster immunization were sacrificed by eye blood collection, and then their cervical vertebrae were dislocated and they were immersed in 75% alcohol for 10 min. They were transferred to a biosafety cabinet and fixed in the supine position. The abdominal cavity of the mice was opened, the location of the spleen was found and carefully removed. A petri dish containing RPMI-1640 medium was prepared, and a 70-μm cell strainer was placed on the petri dish. After the mouse spleen was removed, fat and connective tissue were first removed, and it was cut into pieces and placed in the strainer. It was ground with the plunger of a syringe. After that, the cell strainer was rinsed several times with RPMI-1640 medium to obtain as many splenocytes as possible; the filtrate was transferred to a 50-mL centrifuge tube and filtered again. It was centrifuged at 800 rpm for 10 min, and the supernatant was discarded. Then the cells were resuspended with RPMI-1640 medium, and the washing was repeated once. The splenocytes were resuspended with 10 mL of RPMI-1640 medium, and the suspension was taken for counting and standby.

[0089] 4) Cell fusion

[0090] Mix SP2 / 0 cells and spleen cells at a ratio of 1:5 to 1:10 by count, centrifuge at 800 rpm for 10 min, discard the supernatant, gently tap the bottom of the centrifuge tube to loosen the cells, place the centrifuge tube containing the cells in a beaker with warm water at 40 °C, take out the preheated PEG1450, add 1 mL of PEG1450 dropwise with a Pasteur pipette, finish adding within 45 s, gently shake while adding, and gently stir with a dropper for 1 min after adding; slowly add 10 mL of RPMI-1640 medium, add 1 drop every 2 s for the first 30 s, add 1 drop every 1 s for the next 30 s, add 2 mL at the 2nd min, and add 3 mL at the 3rd min, and add until 10 mL of RPMI-1640 culture medium is added at such a speed; place the centrifuge tube containing the cells in an incubator at 37 °C and let it stand for 10 min, then centrifuge at 800 rpm for 10 min and discard the supernatant; add 40 mL of HAT complete medium to the tube to resuspend the cells, gently pipette and mix well. For each 96-well plate seeded with cells, add another 10 mL of HAT complete medium until 6 plates are seeded. Add all the cells to the 96-well plates seeded with feeder cells the previous day, 100 μL per well, and culture in an incubator at 37 °C, 5% CO2, and saturated humidity.

[0091] 5) Screening and identification of hybridoma cells

[0092] Replenish the HAT medium 5 days later, observe the cell growth under a microscope 5 - 7 days later and mark the single-cell clusters (a photo of the well-grown hybridoma cell colonies observed under a microscope on the 6th day after cell fusion is shown as Figure 6 shown), aspirate the culture medium completely around the 8th day, change the medium with HAT culture medium, when the cells grow to 1 / 4 - 1 / 2 of the entire well bottom, aspirate the cell supernatant of each well for indirect ELISA to screen positive wells. When the OD of the positive control (P value) 450nm > 1.0 and the OD of the negative control (N value) 450nm < 0.2, the experimental conditions are established. Calculate the P / N value. When the P / N value ≥ 2.1, it is judged as positive, and when the P / N value < 2.1, it is judged as negative.

[0093] 6) Subcloning

[0094] Positive wells were screened by indirect ELISA, and positive wells with high P / N values and good growth status were marked. Subcloning was then performed using the limiting dilution method. Feeder cells were prepared one day before subcloning. Cell clumps in the positive wells were gently blown away. The cell suspension was diluted 10-fold and counted. The cell density was adjusted to 10 cells / mL and added to a 96-well plate containing feeder cells at 100 μL / well. The plate was cultured in a 37°C, 5% CO2 incubator. After 3-5 days, the cell growth was observed and the wells with only single cell clusters were marked for subsequent subcloning. After the cell colonies grew to a certain size, the supernatant was collected for indirect ELISA detection, and then the cell clusters with high P / N values were selected for further subcloning until the positive rate of the clone wells reached 100%. Generally, 2 to 3 subclonings were required. Finally, three hybridoma cell lines that could stably secrete antibodies were obtained and named hybridoma cell lines 3B10, 3D9 and 5F7, respectively. They were deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit date of March 9, 2025, and deposit numbers of CCTCC NO: C202567, CCTCC NO: C202568 and CCTCC NO: C202569, respectively.

[0095] 7) Expansion culture and cryopreservation of hybridoma cells

[0096] Positive hybridoma cells were expanded from the cell plate to cell culture flasks. When the cell density in the flask reached over 80% and the cells were in logarithmic growth, the culture medium was discarded and cells attached to the flask wall were blown off with fresh culture medium. The precipitate was centrifuged at 1500 rpm for 10 min, the supernatant was discarded, and the pellet was resuspended in culture medium containing 10% DMSO. The pellets were aliquoted into cryovials at 1 mL / tube and stored in liquid nitrogen for long-term storage at -80°C overnight.

[0097] (3) Preparation of monoclonal antibody ascites

[0098] Prepare several 8-10 week old Balb / c mice and high pressure liquid paraffin in advance. Each mouse was sensitized by intraperitoneal injection of 500 μL liquid paraffin. Seven days after the injection of liquid paraffin, each mouse was intraperitoneally injected with hybridoma cells in the logarithmic growth phase. Each mouse was injected with 1.0×10 6 After 7-10 days, when the abdomen of the mouse is obviously bulging, collect the ascites. Centrifuge at 4500 rpm / min, 4°C for 15 min, and collect the supernatant.

[0099] (4) Purification of monoclonal antibodies

[0100] Purify mouse ascites by the caprylic acid - saturated ammonium sulfate method. Add 0.06 M acetic acid - sodium acetate buffer (pH around 4.5) with a volume three times that of the ascites to the ascites while stirring. Add caprylic acid at room temperature (at a ratio of 33 μL / mL), react at room temperature for 30 min, and invert the mixture up and down from time to time during this period. Then let it stand at 4°C for 2 h to allow the impurity proteins to precipitate fully. Centrifuge at 8000 rmp for 30 min at 4°C, retain the supernatant, filter the supernatant through filter paper, add 1 / 10 volume of 10×PBS (pH = 7.4), mix well, add an equal volume of saturated ammonium sulfate solution (calculate the amount of ammonium sulfate required according to a concentration of 0.2778 g / mL), let it stand at 4°C for 1 - 2 h, centrifuge at 12000 rpm for 10 - 20 min, discard the supernatant, and retain the precipitate. Dissolve the precipitate in 1×PBS and dialyze overnight at 4°C, changing the dialysis fluid 3 - 5 times. Take a part of the antibody for SDS - PAGE gel electrophoresis, and the results are as Figure 7 shown. The purified monoclonal antibodies 3B10, 3D9, and 5F7 were obtained from the three hybridoma cell lines 3B10, 3D9, and 5F7 respectively.

[0101] Example 3 Application of Anti - Pertussis Monoclonal Antibody

[0102] Using three antibodies as labeled antibodies and coating antibodies respectively, and pairing them in pairs, 6 combinations can be obtained, as shown in Table 1.

[0103] Table 1: Different Antibody Pairings

[0104] Number 1 2 3 4 5 6 Coated antibody 3B10 3B10 3D9 3D9 5F7 5F7 Labeled antibody 3D9 5F7 3B10 5F7 3B10 3D9

[0105] Prepare colloidal gold test strips using 6 antibody combinations respectively. The labeled antibody binds to colloidal gold particles and is fixed on the conjugate pad of the test strip; the coating antibody is fixed on the test area (T line) and the control area (C line). Use the prepared colloidal gold detection reagents to detect PT - FHA recombinant antigen and blank diluent respectively, and at the same time detect inactivated Bordetella pertussis cultures to prove that the antibody pairs can detect Bordetella pertussis. The results of detecting pertussis recombinant protein by 6 groups of antibody pairings are as Figure 8 shown. The T line of pairing 6 is the most obvious, the T line of pairing 1 is also relatively obvious, and the T lines of pairings 2 and 4 are also clearly shown.

[0106] The result diagram of detecting blank diluent after preparing colloidal gold test strips with 6 antibody pairings is as Figure 9 shown. The results show good specificity and no T line is shown.

[0107] The result diagram of detecting inactivated Bordetella pertussis cultures after preparing colloidal gold test strips with 6 antibody pairings is as Figure 10 shown. The T line of pairing 6 is the clearest and has higher sensitivity, and the T lines of pairings 1, 2, and 4 are also relatively clear.

[0108] Therefore, combination 6 was selected as the optimal antibody combination method.

[0109] The minimum detection limit of inactivated Bordetella pertussis culture was explored with combination 6. The result graph of detecting inactivated Bordetella pertussis cultures with different dilution multiples using combination 6 is as Figure 11 shown Figure 11 It shows that the detection limit can be as low as 1:3000, and the original concentration of the inactivated Bordetella pertussis culture is 15 IU / mL. Therefore, the detection limit can be calculated to be as low as 0.005 IU / mL.

Claims

1. A pertussis recombinant protein, characterized in that The amino acid sequence of the pertussis recombinant protein is shown in SEQ ID NO:

1.

2. The coding gene of the pertussis recombinant protein according to claim 1, characterized in that The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

3. The hybridoma cell line 3B10 secreting anti-pertussis monoclonal antibody is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is March 9, 2025, and the deposit number is CCTCC NO: C202567.

4. The hybridoma cell line 3D9 secreting anti-pertussis monoclonal antibody is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is March 9, 2025, and the deposit number is CCTCC NO: C202568.

5. The hybridoma cell line 5F7 secreting anti-pertussis monoclonal antibody is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is March 9, 2025, and the deposit number is CCTCC NO: C202569.

6. An anti-pertussis monoclonal antibody is secreted by the hybridoma cell line 3B10 with the deposit number CCTCC NO: C202567 as described in claim 3.

7. An anti-pertussis monoclonal antibody is secreted by the hybridoma cell line 3D9 with the deposit number CCTCC NO: C202568 as described in claim 4.

8. An anti-pertussis monoclonal antibody is secreted by the hybridoma cell line 5F7 with the deposit number CCTCC NO: C202569 as described in claim 5.

9. The application of the anti-pertussis monoclonal antibody as described in any one of claims 6 to 8 in detecting Bordetella pertussis.

10. A diagnostic kit for detecting Bordetella pertussis, comprising the anti-pertussis monoclonal antibody as described in any one of claims 6 to 8.

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