CN116555201B - Monoclonal antibody specifically binding mycoplasma ovipneumoniae pdhD recombinant protein and application thereof - Google Patents
Monoclonal antibody specifically binding mycoplasma ovipneumoniae pdhD recombinant protein and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of cell engineering, and discloses a monoclonal antibody specifically combined with MopdhD recombinant protein and application thereof, wherein a hybridoma cell strain Mo-F2 secreting the monoclonal antibody specifically combined with MopdhD recombinant protein is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C202332 in 3-15 of 2023. Monoclonal antibodies that specifically bind MopdhD recombinant proteins bind specifically to Mo pdhD protein and compete with Mo positive serum for binding to pdhD protein. The competitive ELISA kit for detecting the Mo serum antibody is prepared by taking MopdhD recombinant protein as an antigen and taking a monoclonal antibody specifically combined with MopdhD recombinant protein as a competitive antibody. The ELISA antibody detection kit has higher specificity and sensitivity than an indirect hemagglutination kit for detecting Mo serum antibodies.
Description
Technical Field
The invention belongs to the technical field of cell engineering, and particularly relates to a monoclonal antibody specifically combined with MopdhD (dihydrolipoic acid dehydrogenase) recombinant protein and application thereof.
Background
At present, mycoplasma ovipneumoniae (Mycoplasmaovipneumoniae, mo) is one of the etiologies that cause mycoplasma ovipneumoniae. The clinical manifestations of the sick sheep are asthma, cough, fever, progressive wasting, chest and pleural serous and cellulosic inflammation. Mo is mainly present in respiratory tract, pathological tissue and chest exudates of sheep, and is mainly transmitted through air, so that the sheep with disease resistance can spread pathogen, and secretion can be continuously discharged for months or even more than years. Infected sheep are easy to be infected by other pathogens, the flock of sheep is extremely harmful, serious and even dead, and great economic loss is caused for domestic and foreign sheep breeding industry at present. Therefore, the prevention and control of the disease have very important significance.
Mo often has mixed infection with other pathogens and has antigenic cross-section with other mycoplasma infecting sheep, which increases the serological diagnostic difficulty of Mo. At present, a serological detection technology realizing commercialization is an Indirect Hemagglutination (IHA) kit, wherein Mo whole bacteria are used as antigens in the kit, and the kit has insufficient specificity among species and has different cross reactions with positive serum of mycoplasma of infected goats such as mycoplasma caprae pneumonitis subspecies, mycoplasma caprae subspecies, mycoplasma filiformis subspecies and the like. Some targets with specificity superior to that of Mo whole-bacteria antigens are reported in the literature, including proteins such as P60, P113, hsp70, enolase and the like, but sensitivity is lower than that of the Mo whole-bacteria antigens, and false positives can be detected by the target antigens when positive serum of other mycoplasma such as mycoplasma caprae pneumonitis subspecies, mycoplasma caprae subspecies and mycoplasma filiformis subspecies is higher in cost effectiveness. Because of the above technical drawbacks, none of the proteins based on P60, P113, hsp70, enolase, etc. have been developed for successful commercial kits.
Through the above analysis, the problems and defects existing in the prior art are as follows: in the current commercialized serological detection technology, an Indirect Hemagglutination (IHA) kit takes Mo whole bacteria as an antigen, has insufficient specificity among species, and has different cross reactions with positive serum of mycoplasma of infected goats such as mycoplasma caprae pneumonic subspecies, mycoplasma caprae subspecies, mycoplasma filiformis subspecies and the like. Proteins such as P60, P113, hsp70, enolase are slightly less sensitive as antigens and do not completely overcome cross-reactivity with other mycoplasma caprae positive sera. To overcome this technical disadvantage, serological techniques based on specific antigen targets have to be developed in order to achieve specific serological detection and diagnosis of Mo.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a monoclonal antibody specifically combined with MopdhD recombinant protein and application thereof, in particular relates to a monoclonal antibody specifically combined with mycoplasma ovipneumoniae pdhD recombinant protein, a hybridoma cell strain secreting the monoclonal antibody and application of the monoclonal antibody.
The invention is realized in that a MopdhD recombinant protein, a MopdhD recombinant protein preparation method comprises the following steps: the MopdhD gene sequence is obtained on NCBI, pET-28a plasmid is used as an expression vector, escherichia coli BL21 (DE 3) is used as an expression bacterium, and 0.1mM IPTG is used for inducing the expression of a target protein; then, the thalli are crushed by ultrasonic waves, and the supernatant is taken to obtain soluble expressed pdhD recombinant protein; purifying by using a Ni-NTAAgarose column to obtain the purified pdhD recombinant protein.
Further, mopdhD has the nucleotide sequence of SEQ ID NO 1 as the nucleotide sequence accession No. JFAD01000018.1 and MopdhD; the TGA in SEQ ID NO.1 is mutated into TGG, and the codon sequence is optimized, so that the nucleotide sequence of the S2, S2 gene is SEQ ID NO. 2.
Another object of the present invention is to provide a hybridoma cell line Mo-F2 secreting the monoclonal antibody specifically binding to MopdhD recombinant protein obtained by performing the MopdhD recombinant protein screening.
Further, the method for screening hybridoma cell lines secreting monoclonal antibodies that specifically bind to MopdhD recombinant proteins comprises: mixing MopdhD recombinant protein and Freund's complete adjuvant according to a volume ratio of 1:1, and performing primary mouse immunization; immunization is carried out every 2 weeks, and the volume ratio of MopdhD recombinant protein to Freund's incomplete adjuvant is 1:1; total immunization was carried out for 4 times, and the antigen doses were 100 mug/dose; obtaining mouse serum 2 weeks after the 4 th immunization, using MopdhD recombinant protein as antigen, detecting the titer of the mouse serum by indirect ELISA, and judging positive by P/N being more than or equal to 2.1, wherein the titer reaches more than 1:50000, and the immunization is qualified; obtaining spleen cells of an immunized mouse, and fusing the spleen cells with SP2/0 myeloma cells in a fusion ratio of 5:1; and screening the hybridoma cell lines which are fused successfully, and screening the hybridoma cell lines which secrete the specific monoclonal antibodies.
Furthermore, the hybridoma cell strain Mo-F2 secreting the monoclonal antibody specifically combined with MopdhD recombinant protein is preserved in China center for type culture Collection (CCTCCNO: C202332) at 3-15 of 2023.
Another object of the present invention is to provide a hybridoma cell line Mo-F2 which secretes the monoclonal antibody specifically binding to MopdhD recombinant proteins, and a monoclonal antibody which secretes the cell line and specifically binds to MopdhD recombinant proteins.
Further, monoclonal antibodies that specifically bind MopdhD recombinant proteins specifically bind MopdhD protein and compete with Mo positive serum for binding pdhD protein.
Further, the method for preparing the monoclonal antibody specifically binding to MopdhD recombinant proteins comprises the following steps: injecting the hybridoma cell strain CCTCCNO: C202332 into BALB/C female mice in the abdominal cavity to obtain BALB/C female mice ascites, centrifuging, collecting intermediate layer pale yellow ascites, purifying and obtaining the monoclonal antibody specifically combined with MopdhD.
The invention also aims to provide a competitive ELISA kit for detecting the Mo serum antibody, which is prepared by taking the MopdhD recombinant protein as an antigen and taking the monoclonal antibody which secretes specific binding MopdhD recombinant protein as a competitive antibody.
Further, the competition ELISA kit for detecting Mo serum antibodies comprises:
(1) 2 ELISA plates coated with MopdhD recombinant proteins, 96 wells/block, and preserving at 4deg.C;
(2) 25 times PBST concentrate 1 bottle, 60 mL/bottle;
(3) Standard positive serum 1 tube, 0.6 mL/tube, 4 ℃ storage;
(4) Standard negative serum 1 tube, 0.6 mL/tube, 4 ℃ storage;
(5) 1 bottle of working solution of the mouse monoclonal antibody, 12 mL/bottle and 4 ℃ for preservation;
(6) HRP-marked goat anti-mouse secondary antibody working solution 1 bottle, 25 mL/bottle and 4 ℃ storage;
(7) TMB substrate color development liquid 1 bottle, 25 mL/bottle, 4 ℃ preservation;
(8) 1 bottle of stop solution, 15 mL/bottle, and preserving at 4 ℃;
(9) 2 sealing plate films;
(10) 1 part of a specification.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
First, aiming at the technical problems in the prior art and the difficulty of solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:
The invention relates to a monoclonal antibody specifically combined with MopdhD protein (dihydrolipoic acid dehydrogenase) and a hybridoma cell strain Mo-F2 secreting the monoclonal antibody, wherein the cell strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C202332 in 3-15 of 2023. The invention also provides a preparation method of the mycoplasma ovipneumoniae pdhD recombinant protein, a method for preparing a competitive ELISA antibody detection kit by using the pdhD recombinant protein and a monoclonal antibody thereof, and the prepared kit.
The invention relates to a competition ELISA kit for detecting mycoplasma ovipneumoniae (mycoplasmaovipneumoniae, mo) serum antibodies, a preparation method of pdhD recombinant antigen used in the kit, the recombinant antigen, a monoclonal antibody specifically binding MopdhD protein and a hybridoma cell strain Mo-F2 secreting the monoclonal antibody, wherein the preservation number of the cell strain is CCTCCNO: C202332. The monoclonal antibody secreted by the CCTCCNO: C202332 cell strain only reacts with MophdD protein, but does not cross react with other mycoplasma infected goats, and has extremely high specificity; the kit uses a competition ELISA technology, and uses a mouse monoclonal antibody specifically combined with the pdhD recombinant protein as a competition antibody, so that serum antibodies of various animal sources can be detected, and the species limitation of a sample source is avoided. In addition, the kit has good repeatability, higher coincidence rate with an indirect hemagglutination kit and long storage period. The technical requirements of the kit in operation are relatively loose, and the kit can be widely popularized and used in production and is applied to Mo epidemiological investigation and vaccine immunity level monitoring.
Meanwhile, the advantages of the invention also include the following aspects:
(1) According to the invention, a soluble expressed MopdhD protein is used as an antigen to immunize a mouse, spleen cells of the mouse are fused with myeloma SP2/0 cells, a hybridoma cell strain Mo-F2 secreting a specific monoclonal antibody is screened, and the preservation number is CCTCCNO: C202332.
(2) The CCTCCNO: C202332 cell strain is inoculated into the abdominal cavity of a mouse, the mouse ascites purified antibody is collected, the purified monoclonal antibody specifically combined with MopdhD is obtained, the monoclonal antibody does not react with mycoplasma caprae pneumonitis subspecies, mycoplasma caprae subspecies and filamentous mycoplasma caprae subspecies, and the specificity of the reaction is ensured.
(3) The kit provided by the invention takes MopdhD as an antigen, the prepared specific monoclonal antibody is taken as a competitive antibody, and the Mo competition ELISA antibody detection kit is prepared, so that the kit has higher specificity and sensitivity than the indirect hemagglutination kit for detecting the Mo serum antibody at present.
(4) The kit provided by the invention uses the monoclonal antibody of MopdhD recombinant protein as a competitive antibody, can detect serum antibodies of various animal sources, and avoids the species limitation of sample sources.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
The invention provides a method for preparing MopdhD recombinant protein, and the MopdhD protein has higher sensitivity in detecting the corresponding antibody. Through gray scale scanning analysis, the purity of MopdhD recombinant protein prepared by the invention is more than 90%.
The invention also provides a preparation method of MopdhD monoclonal antibody, firstly injecting liquid paraffin into the abdominal cavity of a mouse, inoculating CCTCCNO: C202332 cell strain into the abdominal cavity of a BALB/C female mouse after 1-2 weeks, collecting ascites when the abdominal cavity of the mouse is obviously enlarged, centrifuging, collecting intermediate layer pale yellow ascites, and purifying to obtain the monoclonal antibody. The monoclonal antibody prepared by the invention can specifically bind to MopdhD protein and can competitively bind to pdhD protein with Mo positive serum.
Thirdly, as inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:
(1) The expected benefits and commercial values after the technical scheme of the invention is converted are as follows:
The commercialized Mo competition ELISA antibody detection kit can evaluate the immune effect of Mo inactivated vaccine used in the current sheep raising industry and can also carry out serological investigation on sheep which are not immunized with the Mo vaccine. The product is required in the sheep industry at present, and the estimated sales after commercialization is 100 ten thousand yuan/year.
(2) The technical scheme of the invention fills the technical blank in the domestic and foreign industries:
The invention applies competition ELISA technique and assembles the kit. The kit takes a murine monoclonal antibody as a competitive antibody, the monoclonal antibody specifically reacts with MopdhD, but does not react with mycoplasma caprae pneumonitis subspecies, mycoplasma caprae subspecies and filamentous mycoplasma caprae subspecies, and has very good specificity. At present, no related products exist at home and abroad, and the technical blank at home and abroad is filled.
(3) The technical scheme of the invention solves the technical problems that people are always desirous of solving but are not successful all the time:
Diagnostic reagents for sensitive, specific detection of Mo serum antibodies have been a highly desirable product in the sheep industry, but no ideal related product has been commercialized. The technical scheme of the invention solves the technical problems which are always desired to be solved but are not successful in the sheep industry production.
(4) The technical scheme of the invention overcomes the technical bias:
the technical scheme of the invention is a competitive ELISA technology, and the murine monoclonal antibody is used as a competitive antibody, so that the method is easy for mass preparation and production. The technology can realize high-flux detection, and related professionals can operate according to the specification, so that the technology has universality.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for preparing a monoclonal antibody specifically binding MopdhD recombinant proteins provided in an embodiment of the present invention;
FIG. 2A is a schematic representation of the expression of a pdhD recombinant protein provided by an embodiment of the present invention; m: protein Marker relative molecular mass standard; 1: non-induced bacteria; 2 to 7: inducing supernatant for 2h, 4h, 6h, 8h, 10h and 12 h; 8: pET-28a no-load bacteria;
FIG. 2B is a schematic diagram of a screen for final concentrations of IPTG-inducers according to the examples of the present invention; m: protein Marker relative molecular mass standard; 1: non-induced bacteria; 2 to 4: the final concentration of IPTG inducer is 0.1mM, 0.05mM and 0.5mM in sequence;
FIG. 2C is a schematic diagram of purification of recombinant pdhD protein provided by an embodiment of the present invention; m: PM2510 relative molecular mass standard; 1 to 9: crushing supernatant, flow-through solution, 10mM imidazole eluent, 20mM imidazole eluent, 50mM imidazole eluent, 100mM imidazole eluent, 200mM imidazole eluent, 500mM imidazole eluent and 1M imidazole eluent;
FIG. 3 is a schematic diagram of purification of MopdhD recombinant protein monoclonal antibodies provided in an embodiment of the present invention; m: protein Marker relative molecular mass standard; 1: purified pdhD recombinant protein monoclonal antibodies.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In view of the problems of the prior art, the present invention provides a monoclonal antibody specifically binding to MopdhD recombinant proteins and applications thereof, and the present invention is described in detail below with reference to the accompanying drawings.
The embodiment of the invention provides MopdhD recombinant protein and a preparation method of MopdhD recombinant protein, which comprises the following steps: the MopdhD gene sequence is obtained on NCBI, the pdhD gene is located between 18332bp and 201667 bp, the whole length of the gene sequence is 1836bp, and the marker is S1; mutating TGA in the pdhD gene sequence to TGG, optimizing the codon sequence, and marking as S2; synthesizing an S2 gene sequence, cloning pET-28a plasmid by utilizing BamHI and XhoI restriction enzymes, and transforming positive recombinant plasmid into competent escherichia coli BL21DE3 to obtain recombinant expression strain BL21-28a-pdhD; culturing BL21-28a-pdhD strain at 37 deg.C and 200r/min to OD 600 value of 0.6-0.8, and inducing with 0.1mM IPTG for 6 hr; centrifuging the induced bacterial liquid for precipitation, washing the precipitate with PBS (phosphate buffered saline) with pH of 7.4, and re-suspending; crushing thalli by ultrasonic wave, centrifuging, taking supernatant to obtain soluble expressed pdhD recombinant protein; purifying by using a Ni-NTAAgarose column to obtain the purified pdhD recombinant protein.
The MopdhD gene sequence provided by the embodiment of the invention is JFAD01000018.1, the nucleotide sequence of the Mo pdhD gene is SEQ ID NO. 1, and the nucleotide sequence of the S2 gene is SEQ ID NO. 2.
The embodiment of the invention provides a hybridoma cell strain Mo-F2 which is obtained by implementing MopdhD recombinant protein screening and secretes monoclonal antibody specifically combined with MopdhD recombinant protein.
The screening method of the hybridoma cell strain secreting the monoclonal antibody specifically combined with MopdhD recombinant proteins provided by the embodiment of the invention comprises the following steps: mixing MopdhD recombinant protein and Freund's complete adjuvant according to a volume ratio of 1:1, and performing primary mouse immunization; immunization is carried out every 2 weeks, and the volume ratio of MopdhD recombinant protein to Freund's incomplete adjuvant is 1:1; total immunization was carried out for 4 times, and the antigen doses were 100 mug/dose; obtaining mouse serum 2 weeks after the 4 th immunization, using MopdhD recombinant protein as antigen, detecting the titer of the mouse serum by indirect ELISA, and judging positive by P/N being more than or equal to 2.1, wherein the titer reaches more than 1:50000, and the immunization is qualified; obtaining spleen cells of an immunized mouse, and fusing the spleen cells with SP2/0 myeloma cells in a fusion ratio of 5:1; and screening the hybridoma cell lines which are fused successfully, and screening the hybridoma cell lines which secrete the specific monoclonal antibodies.
The hybridoma cell strain Mo-F2 secreting the monoclonal antibody specifically combined with MopdhD recombinant protein provided by the embodiment of the invention is preserved in the China center for type culture Collection in 3 months of 2023, and addresses are Wuhan City, university of Wuhan in China; the preservation number is CCTCCNO C202332, and the hybridoma cell strain Mo-F2HybridomacelllineMo-F2 is scientifically described; the cultures were received from the collection at 2023, 3 and 15 days and registered. According to your request, the storage is continued for five years from this day on after receiving a request to provide a culture sample before expiration. Viability of the cultures the present collection was tested at day 20 of 3, 2023 and the result was survival.
The embodiment of the invention provides a monoclonal antibody which is obtained by implementing the secretion of a hybridoma cell strain which is used for secreting the monoclonal antibody specifically combined with MopdhD recombinant proteins and specifically combined with MopdhD recombinant proteins.
The monoclonal antibody specifically combined with MopdhD recombinant protein provided by the embodiment of the invention is specifically combined with Mo pdhD protein and competitively combined with Mo positive serum.
As shown in FIG. 1, the preparation method of the monoclonal antibody specifically binding MopdhD recombinant protein provided by the embodiment of the invention comprises the following steps:
s101, inoculating hybridoma cell strain CCTCCNO: C202332;
s102, obtaining ascites of a BALB/c female mouse, centrifuging, and collecting pale yellow ascites of an intermediate layer;
s103, purifying the ascites to obtain the monoclonal antibody specifically bound with MopdhD.
The embodiment of the invention provides a competitive ELISA kit for detecting Mo serum antibodies, which is prepared by taking MopdhD recombinant protein as an antigen and a monoclonal antibody secreting specific binding MopdhD recombinant protein as a competitive antibody.
The competitive ELISA kit for detecting Mo serum antibody provided by the embodiment of the invention comprises:
(1) 2 ELISA plates (96 holes/block) coated with MopdhD recombinant antigens and stored at 4 ℃;
(2) 25-fold PBST concentrate 1 bottle (60 mL/bottle);
(3) Standard positive serum 1 tube (0.6 mL/tube), stored at 4 ℃;
(4) Standard negative serum 1 tube (0.6 mL/tube), stored at 4 ℃;
(5) 1 bottle (12 mL/bottle) of working solution of the mouse monoclonal antibody, and preserving at 4 ℃;
(6) HRP-labeled goat anti-mouse secondary antibody working solution 1 bottle (25 mL/bottle) is stored at 4 ℃;
(7) TMB substrate color development liquid 1 bottle (25 mL/bottle), 4 ℃ storage;
(8) 1 bottle of stop solution (15 mL/bottle), and preserving at 4 ℃;
(9) 2 sealing plate films;
(10) 1 part of a specification.
In order to prove the inventive and technical value of the technical solution of the present invention, this section is an application example on specific products or related technologies of the claim technical solution.
Compared with an indirect hemagglutination kit, the technical scheme of the invention has higher sensitivity and specificity when detecting Mo serum antibodies, and the application examples are as follows:
Sensitivity: 2 sheep of about 6 months of age were selected and immunized with inactivated Mo3-3 strain in a 1:1 mixture with Freund's complete adjuvant at an antigen dose of 500. Mu.g/dose. Serum was collected 2 weeks after immunization, 1 time every 1 week apart, and 3 times in total. The serum samples were tested separately using the indirect hemagglutination kit and the competition ELISA technique of the invention, and the results showed that: 3 times of collected serum is detected to be positive by the competition ELISA technology; the indirect hemagglutination kit only detects the serum collected for the first time as positive, the serum titer is 1:8, and the serum detection results collected for the second time and the third time are negative, which indicates that the sensitivity of the competition ELISA technology is higher than that of the indirect hemagglutination kit.
Specificity: 6 sheep of about 6 months of age are selected and randomly divided into 3 groups, 2 sheep/group, and each group of sheep is immunized with mycoplasma caprae and mycoplasma caprae respectively, wherein the immunization antigen dose is 500 mug/dose. The immunization mode is as follows: mixing the inactivated strain with Freund's complete adjuvant 1:1 for immunization; serum was collected after 2 weeks of interval, 1 time every 1 week of interval, and 3 times in total. The serum samples were tested separately using the indirect hemagglutination kit and the competition ELISA technique of the invention, and the results showed that: 3 times of collected serum is detected as negative by the competition ELISA technology; the indirect hemagglutination kit has positive serum detection results of the first collection, the serum titer is 1:8, and negative serum detection results of the second collection and the third collection, which shows that the specificity of the competition ELISA technology is higher than that of the indirect hemagglutination kit.
Preparation of MopdhD antigen
Specific antigen targets were first screened using Pull-down and LC-MS techniques. According toProteinG the kit instructions comprise that Mo negative serum is firstly adsorbed on magnetic beads which are respectively incubated with supernatants of MoY, FL3 and 3-3 strains after being broken, and then the supernatants are taken, so that nonspecific antigens combined with the negative serum can be eliminated to the greatest extent. Incubating the obtained supernatant with magnetic beads combined with Mo positive serum at 4 ℃ overnight, washing impurities by washingbuffer, taking the magnetic beads, carrying out LC-MS analysis, and screening out target proteins with high conservation in seeds, specificity among seeds and high credibility value. The five aspects Peptides, PSMs, uniquePeptides, area and ScoreSequestHT provided by LC-MS were combined for comprehensive screening, the more peptides matched, the more UniquePeptides, the more reliable the signal and score. By comprehensively ranking the above five aspects MopdhD ranks first, the target protein was determined.
The MopdhD gene sequence is obtained on NCBI, the gene sequence accession number is JFAD01000018.1, the pdhD gene is located between 18332bp and 201667 bp, the total length of the gene sequence is 1836bp, and the marker is S1. TGA in the Mo pdhD gene sequence was mutated to TGG, and the optimized codon sequence was labeled S2. S2 gene sequence is synthesized by Wuhan Jin Kairui biological engineering Co., ltd, bamHI and XhoI restriction endonuclease are utilized to clone into pET-28a plasmid, positive recombinant plasmid is transformed into competent escherichia coli BL21 (DE 3), and recombinant expression bacterium BL21-28a-pdhD is obtained. BL21-28a-pdhD strain was cultured at 37℃and 200r/min until OD 600 was 0.6-0.8, and induced with 0.1mM IPTG for 6 hours. Centrifuging the induced bacterial liquid, washing the precipitate with PBS (phosphate-buffered saline) with pH7.4, re-suspending, then crushing the bacterial body by ultrasonic waves, centrifuging, taking the supernatant to obtain soluble expressed pdhD recombinant protein, and purifying with a Ni-NTAAgarose column to obtain purified pdhD recombinant protein; through gray scanning analysis, the purity is over 90 percent.
The nucleotide sequence of the S1 provided by the embodiment of the invention is as follows:
atgtataaatttaaatttgctgacattggagaaggactccatgaaggagttgtcgcccaaatttacaaaaaagaaggcgaccaagttaatgaaggtgattcgttgttctcagttgaaactgacaaaattaccgctgacataccttcaccaaaaaccggaaaaattgttaaagttttaatgtctgagggtgatacaattcacgttggtcaagaaatttattatattgatgatggatcaggtgatagcgatattcccgaagttaaaactgaagagaaaaaggaagaagaagcaagcggagcaagtgttgttggtgaagtaaaagtaagcaatgatcttttaagttttgattttggtccaaaaaaatcaacagcaaaaccagcaaaaactcctgccccaaaagttgaaaaagcacctgaaaaacctacctcttctgcaaatactggcaaagtttaccaaggtaaaattgaccaagaatttgatgtaattgttatcggttcaggacctggcggttatcttgcagctgctgaggctggtaaaaatggtttatcaactctaattgttgaaaaagaatattgaggtggtgtttgtttaaatgttggctgcattccaacaaaagcaatgttaaaaactgccgaagttcttgattatgtaactcactttagtgactatggtcttgaaggaaaaactgatgtaaaaatttcctgagagaaaatgcaccaacgaaaaactgaagttgttaacaaattagttggtggagttaaggcgattgtaaaatctgcaagagctacaagcatttttggtgaagctaaattcctcggtgcacacgaaatttcagttgaaggtaaagtttatcgtggaaaacacattattcttgcaactggatcacgtgaccgtaaattgaatttacctggttttgaacaaggttaccaatctggaaaaattttaacttcaaaagaagcaattaatcttgaagaaaaaattagttcaattgtaataattggtggcggtgttattggtgttgaatttgcccaaatttttgtcgctgctggagttaaggtaacaattttacaaaatcttccaagacttttggcaaaccttgatggcgaaatttcacaaattattacaaaaaacttagctgaccgtggtgttaatgttgttctaaattctaatattattagctatgaaaatgatagtattgtttacgaacttgacggaaaaagacaacaaattagcgctgataaagttttagtaagtatcggtcgtgagccaaattctgaaggtcttagcgaagttggagttgaacttgatactcgcaaaagtgttatcgttgatgatcaatgcagaacaaatgttgacggagtttatgcaattggtgatcttagtgcaaaagcaatgctagcccatgttgcttaccgtcatgccgttgttgcagtaagtacaataaccggaaaaggtgaaaaatataacgataaaaccgttccagcttgcgtttatactcatcctgaaattgcttcagttggtctaactgaagaacaagcaaaagaacaaggttatgattttgttgttggaaaagctagttttgcccacattggaaaagcaatcgctgccggagatgcccacggatttgctaaattaattgttgataaaaaatatggcgaaattcttggtgctcactttatcggtccagtagctactgatatgatttcagaaattgtcgtttcaatggacgcagaagtaacaattcatgaacttgcagcggcaattcacccacaccctacttatagcgaagtaatttgagaagcagcccgtgctgcacaagcaaaattaaaaagataa
The nucleotide sequence of the S2 provided by the embodiment of the invention is as follows:
atgtacaagttcaagttcgcagatattggcgaaggcctgcatgaaggcgttgtggcccagatttataaaaaagaaggcgatcaggtgaatgaaggtgatagtctgtttagtgttgaaaccgataaaattaccgcagatattccgagcccgaaaccggcaaaattgttaaagttctgatgagcgaaggtgataccattcatgtgggtcaggaaatttattatatcgatgatggcagcggcgatagcgatattccggaagttaaaaccgaagaaaaaaaagaagaggaagcaagtggcgcaagcgttgtgggcgaagtgaaagttagcaatgatctgctgagttttgattttggtccgaaaaaaagtaccgccaaaccggcaaagaccccggccccgaaagtggaaaaagcaccggaaaaaccgaccagcagtgccaataccggcaaagtttatcagggcaaaattgatcaggaatttgatgttattgtgatcggcagtggcccgggcggctatctggccgcagctgaagccggcaaaaatggtctgagtaccctgattgtggaaaaagaatattggggtggcgtgtgtctgaatgtgggctgcattccgaccaaagccatgctgaaaaccgcagaagttctggattatgttacccattttagtgattatggtctggaaggtaaaaccgatgtgaaaattagctgggaaaaaatgcatcagcgcaaaaccgaagttgtgaataaactggttggtggtgttaaagccattgttaaaagtgcacgcgcaaccagtatttttggcgaagcaaaatttctgggtgcccatgaaattagcgttgaaggcaaagtgtatcgtggcaaacatattattctggccaccggtagccgtgatcgtaaactgaatctgccgggttttgaacagggttatcagagcggcaaaattctgaccagcaaagaagccattaatctggaagaaaaaatcagtagcatcgttattatcggtggcggtgttattggtgttgaatttgcacagatttttgtggcagcaggcgtgaa
agttaccattctgcagaatctgccgcgcctgctggcaaatctggatggtgaaattagtcagatta
ttaccaaaaacctggcagatcgtggcgttaatgttgttctgaatagcaatattatcagctatgaaaacgacagcattgtttatgaactggatggtaaacgccagcagattagtgcagataaagttctggttagtattggtcgtgaaccgaatagcgaaggcctgagtgaagttggcgtggaactggatacccgtaaaagcgttattgtggatgatcagtgccgtaccaatgtggatggtgtgtatgccattggcgatctgagcgccaaagccatgttagcccatgtggcatatcgtcatgccgtggttgcagtgagtaccattaccggcaaaggtgaaaaatataatgataaaaccgtgccggcatgtgtgtatacccatccggaaattgcaagcgttggcctgaccgaagaacaggccaaagaacagggttacgattttgttgttggtaaagcaagttttgcccatattggtaaagccattgcagccggcgatgcccatggttttgcaaaactgattgttgataaaaagtacggcgaaattctgggcgcccattttattggcccggtggcaaccgatatgattagcgaaattgttgtgagcatggatgccgaagtgaccattcatgaactggccgcagccattcatccgcatccgacctatagcgaagttatttgggaagccgcccgcgccgcccaggcaaaactgaaacgttaa
(II) MopdhD preparation of monoclonal antibodies
5 BALB/c female mice with the age of 6-8 weeks are selected, and the pdhD recombinant protein is mixed with Freund's complete adjuvant 1:1 for the first immunization; immunization is carried out every 2 weeks in the follow-up period, the used adjuvant is Freund's incomplete adjuvant, and the volume ratio of the pdhD recombinant protein to Freund's incomplete adjuvant is 1:1; total immunization was 4 times, and the antigen dose was 100. Mu.g/dose. Serum of the mice is collected 2 weeks after 4 th immunization, pdhD recombinant protein is used as antigen, the titer of the serum of the mice is detected by indirect ELISA, and the positive is judged by P/N being more than or equal to 2.1, and the titer reaches more than 1:50000, so that the mice are qualified by immunization. Spleen cells of the immunized mice are taken and fused with SP2/0 myeloma cells in a fusion ratio of 5:1, and the fused cells are subjected to half-liquid exchange and full-liquid exchange on the 7 th day and the 10 th day after fusion respectively. On the third day after total pipetting, hybridoma cells secreting anti-Mo pdhD protein antibodies were screened by indirect ELISA. And (3) performing secondary screening on the positive hole full liquid exchange obtained in the primary screening. And (3) performing expanded culture and freezing storage on the holes which are still positive in the second screening, simultaneously continuously performing 3-5 subcloning until the positive rate is 100%, and freezing the positive cell strain in a liquid nitrogen tank.
BALB/C mice were sensitized by intraperitoneal injection of 1mL of autoclaved liquid paraffin, and after 7 days, 1.0X10 6 well-grown positive hybridoma cells were intraperitoneally injected. The abdominal cavity change of the mice is observed every day, and when the mice are inflated to be inconvenient to move, ascites is collected. The ascites obtained by the invention is purified by a ProteinG purification column of Thermo, and then is subjected to SDS-PAGE to observe the purity, and the concentration is measured and stored at-80 ℃ for standby.
(III) construction of MopdhD Competition ELISA antibody detection method
Determining that the optimal coating concentration of MopdhD recombinant proteins is 600ng/mL and 100 mu L/hole through a chessboard titration test; serum is original multiple, 50 mu L/hole; the dilution factor of the monoclonal antibody is 1:10000, 50 mu L/hole, and the incubation time is 30min; dilution of HRP-labeled goat anti-mouse antibody was 1:20000, 100. Mu.L/well, incubation time was 30min. The TMB of the substrate solution is 100 mu L/hole, and the incubation time is 15min; the stop solution was 50. Mu.L/well and OD 450 was measured immediately after addition.
The antigen coating program is as follows: the frozen antigen at-75℃was thawed, diluted to 500ng/mL with a pH9.6 carbonate buffer, and the ELISA plate was added at 100. Mu.L/well and transferred to 4℃overnight. The antigen solution was thrown off the next day, after washing, blocked with 5% nonfat milk powder, 100. Mu.L/well, incubated at 37℃for 30min, and washed again. Then adding protein stabilizer, 100 mu L/hole, incubating for 10min at room temperature, spin-drying, and blowing in an ultra-clean bench for 1h. Finally, the mixture is put into a packaging bag and vacuumized.
The preparation method of the standard positive serum in the kit comprises the following steps: selecting sheep about 6 months old, mixing inactivated Mo3-3 strain with Freund's complete adjuvant 1:1, and performing primary immunization; after an interval of 2 weeks, performing secondary immunization by mixing the inactivated Mo3-3 strain with Freund's incomplete adjuvant 1:1; after a further 2 weeks interval, a third immunization was performed with inactivated Mo3-3 strain mixed with Freund's incomplete adjuvant 1:1; the dose of the immunizing antigen is 500 mug/dose. Negative sera were healthy sheep sera that were neither immunized with Mo vaccine nor infected with Mo.
The HRP-labeled goat anti-mouse antibody in the detection kit is purchased from Sigma company; the monoclonal antibody diluent, the enzyme-labeled antibody diluent, the TMB color development liquid, the stop solution and the 50-time concentrated washing liquid are all purchased from Lanzhou animal research biological limited company of China academy of agricultural sciences.
(IV) MopdhD Competition ELISA antibody detection kit
The various reagents are assembled into a competition ELISA kit for detecting MopdhD serum antibodies after completion. Suggested kits include: 2 ELISA plates coated with MopdhD recombinant antigens (96 wells/block); 25-fold PBST concentrate 1 bottle (60 mL/bottle); standard positive serum 1 tube (0.6 mL/tube); standard negative serum 1 tube (0.6 mL/tube); monoclonal antibody working solution 1 bottle (12 mL/bottle); HRP-labeled goat anti-mouse secondary antibody working solution 1 bottle (25 mL/bottle); TMB substrate color development 1 bottle (25 mL/bottle); stop solution 1 bottle (15 mL/bottle); 2 sealing plate films; 1 part of a specification. The kit is preserved at 4 ℃ for 6 months. 180 serum can be detected by the kit.
(Fifth) method of Using the kit example
1. The kit was removed from the refrigerator at 4 ℃ and equilibrated to room temperature. The 25-fold wash was diluted to 1-fold use concentration with double distilled water.
2. And taking out the ELISA plate, determining the required ELISA plate according to the quantity of serum to be detected, and placing the rest plates into a packaging bag for storage at 4 ℃. The unpacked microplate is preferably used up within 10 days.
3. Sample adding: 2 wells of standard positive serum and standard negative serum respectively, 50 μl/well; the serum sample to be tested was added to 1 well, 50. Mu.L/well.
4. Monoclonal antibody working solution, 50 μl/well was added.
5. Incubation: sealing the ELISA plate with a sealing plate film, and incubating in a 37 ℃ incubator for 30min.
6. Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, and discarding. This was repeated 4 times and the swatter dried.
7. HRP-labeled goat anti-mouse secondary antibody working solution is added to 100 mu L/well.
8. Incubation: the operation is the same as in step 5.
9. Washing: the operation is the same as in step 6.
10. Color development: TMB color development solution is added, 100 mu L/hole is added, and color development is carried out at 37 ℃ in a dark place for 5min.
11. And (3) terminating: stop solution, 50. Mu.L/well, was added.
12. And (3) measuring: the absorbance (OD 450 value) of each well was measured immediately with a microplate reader at a wavelength of 450 nm.
13. Result determination
① OD 450 values of the negative and positive control wells and each serum well to be detected on the same ELISA plate are calculated, and inhibition rates (PI) of standard positive and negative serum and serum to be detected are calculated according to the following formula. Pi= (1-sample OD 450 mean/standard negative serum OD 450 mean) ×100%.
② The PI of the standard positive serum is more than 70%, the OD 450 of the standard negative serum is more than 1.0, the test is true, otherwise, the test result is invalid.
③ The PI of the serum sample to be tested is more than or equal to 50%, and the sample is positive; PI <50%, negative.
(VI) sensitivity, specificity, repeatability, compliance rate and shelf life determination of the kit
Mo positive serum with antibody titer of 1:32 was tested by indirect hemagglutination kit at 5 dilutions of 1:10, 1:20, 1:40, 1:80, 1:160, samples of each dilution gradient were tested with the kit of the invention, and the lowest antibody titer detectable by the kit was tested. The result shows that the positive serum can still be detected when diluted to 1:80, which indicates that the kit has higher sensitivity.
The kit is used for detecting positive serum of mycoplasma caprae and mycoplasma caprae, positive serum of mycoplasma caprae and filamentous mycoplasma caprae, and positive serum of escherichia coli and salmonella, and the results are negative, so that the kit has good specificity, and the pdhD antigen used by the kit has no cross reaction with positive serum of other mycoplasma and common bacteria (escherichia coli and salmonella) of infected goats.
Taking 20 parts of Mo positive serum and 20 parts of Mo negative serum, repeatedly detecting for three times by using the same batch of kit under the same conditions of different time, and judging that the results are identical; detecting the serum by using 3 batches of kits, wherein the judgment results are identical; these results demonstrate that the kits are better reproducible.
And (3) detecting 75 parts of positive serum and 45 parts of negative serum identified by the indirect hemagglutination kit by using the kit, and verifying the coincidence rate of the kit and the indirect hemagglutination kit. The results showed that the compliance of the kit to the indirect hemagglutination kit was 92.5% (111/120).
The same 20 positive and 20 negative sera were tested every 2 weeks with the kits prepared in the same batch. The results show that the detection results of the kit stored for 6 months are the same for the same serum, and the storage life of the kit is at least 6 months.
Seventh, the kit detects the serum sample in the field
A total of 400 sheep serum samples from 8 farms were tested using the kit of the invention and the test results are shown in Table 1.
TABLE 1 detection of serum samples in the field with the kit and the indirect hemagglutination kit of the present invention
The detection results show that the kit and the indirect hemagglutination kit are used for respectively detecting 360 parts of field serum, the coincidence rate of the two methods is 93 percent (286+86/400), and the kit can completely replace the indirect hemagglutination kit to detect Mo serum antibodies and is popularized and applied in production.
FIG. 2AMopdhD schematic representation of recombinant protein expression. M: protein Marker relative molecular mass standard; 1: non-induced bacteria; 2 to 7: inducing for 2h, 4h, 6h, 8h, 10h and 12h respectively, centrifuging the induced recombinant expression bacteria BL21-28a-pdhD bacterial liquid, washing the precipitate with PBS (phosphate-buffered saline) with pH7.4, re-suspending, then crushing the bacterial cells by ultrasonic waves, centrifuging, and taking the supernatant to obtain the soluble expressed pdhD recombinant protein. 8: an expression bacterium carrying the pET-28a plasmid.
FIG. 2BIPTG A schematic screen of final inducer concentration. M: protein Marker relative molecular mass standard; 1: the uninduced recombinant expression bacterium BL21-28a-pdhD;2 to 4: recombinant expression bacteria BL21-28a-pdhD was induced with IPTG at a final concentration of 0.1mM, 0.05mM, and 0.5mM, and the pdhD protein was obtained after 6 hours of induction.
FIG. 2 schematic purification of CMo recombinant pdhD protein. Inducing recombinant expression bacteria BL21-28a-pdhD, centrifuging bacterial liquid for precipitation, washing the bacterial liquid for 2 times by PBS, re-suspending the bacterial liquid, crushing the bacterial liquid by ultrasonic waves, centrifuging, and taking the supernatant to obtain soluble expression pdhD protein. Purification was performed using a Ni-NTAAgarose column, and the procedure was followed as described to obtain purified pdhD recombinant protein. M: PM2510 relative molecular mass standard; 1 to 9: the method comprises the steps of sequentially crushing thalli, passing through column, eluting with 10mM imidazole, eluting with 20mM imidazole, eluting with 50mM imidazole, eluting with 100mM imidazole, eluting with 200mM imidazole, eluting with 500mM imidazole and eluting with 1M imidazole.
FIG. 3MopdhD schematic representation of purification of recombinant protein monoclonal antibodies. BALB/C mice were sensitized by intraperitoneal injection of 1mL of autoclaved liquid paraffin, and after 7 days, 1.0X10 6 positive hybridoma cells CCTCCNO: C202332 were intraperitoneally injected with good growth. The abdominal cavity change of the mice is observed every day, when the mice are inflated to be inconvenient to move, the ascites is collected, the obtained ascites is purified by a ProteinG purification column of Thermo, and the operation steps are carried out according to the specification. M: protein Marker relative molecular mass standard; 1: purified pdhD recombinant protein monoclonal antibodies.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (2)
1. The hybridoma cell strain Mo-F2 secreting the monoclonal antibody specifically binding MopdhD recombinant protein is characterized in that the hybridoma cell strain secreting the monoclonal antibody specifically binding MopdhD recombinant protein is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C202332 at 3-15 of 2023.
2. A monoclonal antibody that specifically binds to MopdhD recombinant protein secreted by the hybridoma cell line secreting the monoclonal antibody that specifically binds to MopdhD recombinant protein of claim 1.
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| CN101519447A (en) * | 2009-04-08 | 2009-09-02 | 江苏省农业科学院 | Anti-rabbit hemorrhagic disease virus VP60 albumen monoclonal antibody |
| CN105602925A (en) * | 2016-01-26 | 2016-05-25 | 中国科学院天津工业生物技术研究所 | Non-classical secretory protein and application thereof to protein secretory expression |
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| CN101519447A (en) * | 2009-04-08 | 2009-09-02 | 江苏省农业科学院 | Anti-rabbit hemorrhagic disease virus VP60 albumen monoclonal antibody |
| CN105602925A (en) * | 2016-01-26 | 2016-05-25 | 中国科学院天津工业生物技术研究所 | Non-classical secretory protein and application thereof to protein secretory expression |
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