CN116554282A - Preparation method and application of monoclonal antibody for identifying swine fever virus type 2 infection - Google Patents
Preparation method and application of monoclonal antibody for identifying swine fever virus type 2 infection Download PDFInfo
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Abstract
The invention relates to an antibody preparation technology for detecting viruses, and aims to provide a monoclonal antibody preparation method for identifying swine fever virus type 2 infection and application thereof. The invention provides CSFV 2 type E2 protein expressed by baculovirus, and the cDNA sequence of the coding region of the protein is shown as SEQ ID NO. 5. Purifying the CSFV 2 type E2 protein, and then applying the purified CSFV 2 type E2 protein to mice immunized by CSFV 2 type strain HZ08 whole virus to realize mixed immunization; preparing a hybridoma cell strain by utilizing a mouse myeloma cell SP2/0 and spleen cells of a mouse after mixed immunization; obtaining a monoclonal cell strain capable of stably secreting a specific antibody through screening and subcloning; subtype determination, IFA identification, immunoblotting identification and epitope identification are carried out on the obtained monoclonal antibody, and the monoclonal antibody capable of blocking an ELISA system is screened. The monoclonal antibody prepared by the invention can be used for distinguishing CSFV epidemic strains from vaccine strains or distinguishing vaccinated animals from naturally infected animals.
Description
Technical Field
The invention relates to an antibody preparation technology for detecting viruses, in particular to a monoclonal antibody preparation method for identifying swine fever virus type 2 infection and application thereof.
Background
Hog cholera (Classical swine fever, CSF) is an important viral disease that infects pigs and wild boars, is a virulent infectious disease that seriously harms the global pig industry caused by hog cholera virus (Classical swine fever virus, CSFV), and has the characteristics of high infectivity and high pathogenicity. Due to the global distribution of swine fever and its huge economic losses to the global pig industry, one of the epidemic diseases that has to be reported is regulated by the world animal health Organization (OIE), which belongs to the second animal epidemic in our country. The disease was first found in ohio in the united states in 1833 and, after the first report, rapidly spread to all parts of the world. The infectious disease is still endemic or sporadic in asia, europe, central america, most countries in south america, and a few africa countries, etc.
CSFV belongs to a member of the Flaviviridae family (Flaviviridae) Pestivirus genus (Pestivirus), and it is recognized that bovine viral diarrhea virus type 1 (BVDV-1), bovine viral diarrhea virus type 2 (BVDV-2), ovine border virus (BDV) belong to the Pestivirus genus as well as CSFV. CSFV is a enveloped single-stranded positive-stranded RNA virus of genomic size about 12.3kb containing a large open reading frame (Open Reading Frame, ORF) flanked by a 5 'non-coding region (5'untranslated region,5 '-UTR) and a 3' non-coding region (3'untranslated region,3' -UTR). The ORF encodes a polyprotein of 3898 amino acids that is cleaved by viral and host cell proteases into 12 mature viral proteins, 4 of which include nucleocapsid protein C and 3 envelope glycoproteins, erns, E1 and E2, and 8 of which include Npro, p7, NS2, NS3, NS4A, NS4B, NS a and NS5B. Each mature protein performs a different function in the life cycle and infection process of classical swine fever virus.
CSFV comprises 3 genotypes and 11 genotypes (1.1,1.2,1.3,1.4,2.1,2.2,2.3,3.1,3.2,3.3 and 3.4). In China, a safe and effective attenuated vaccine (CSFV attenuated C strain) is developed since 1954, a forced inoculation policy of swine fever vaccine is generally implemented, and the large-scale outbreak epidemic of the disease is effectively controlled. The CSFV attenuated C strain is obtained by continuously passaging a CSFV 1 type virulent strain Shimen in rabbit body, and can generate good protection effect on highly pathogenic viruses and provide life-long immunity after being inoculated for 1 time, thus being one of the safest and effective veterinary vaccines.
The broad application of the strain C vaccine successfully controls the epidemic of CSF in China, but under the pressure of the strain C vaccine, the 1.1 subgenotype gradually exits from the epidemic stage since the beginning of 21 st century, and the CSFV strain popular in the main production area of domestic pigs is mainly of subgenomic type 2.1 at present, such as QZ14 strain, HZ08 strain, huN strain, HLJ1 strain and the like. Strain C is recognized as a safe and effective CSF vaccine, but does not have the ability to provide a relevant detection kit for identifying CSFV wild virus infection serum antibodies and vaccine strain C immune serum antibodies, failing to ultimately decontaminate swine fever. The currently popular CSFV subgenomic type 2.1 epidemic strain has low homology with the C strain, and can influence the immunoprotection effect of the C strain vaccine based on the genotype 1. Therefore, establishing an effective rapid identification technology of CSFV epidemic strain infection serum antibodies and C strain immune serum antibodies is an important problem to be solved at present, which further promotes the purification of the swine fever virus.
Antibodies have the ability to bind to specific antigens and are currently one of the most valuable biological molecules to be studied and developed. The research method and technology using the antibody as a key component are widely applied to the treatment, diagnosis and related research of diseases such as human infectious diseases, tumors and the like. In the fields of prevention and control and research of animal epidemic diseases, monoclonal antibodies play a great role. Monoclonal antibodies (Monoclonal antibody, MAb) have the advantages of high specificity, high uniformity, high stability, easy mass production and the like, and are widely applied to clinical treatment and diagnosis. The hybridoma technology is a classical technology method for preparing monoclonal antibodies, and uses polyethylene glycol, sendai virus or electrofusion and other technologies to fuse B cells sensitized by antigens with myeloma cells, and most hybridoma cells cannot generate antibodies, so that a series of antibody detection such as resistance screening, ELISA, IFA and the like are needed, and subcloning is performed on cell clones positive to the antibody detection in time, so that a hybridoma cell line which can stably proliferate and can secrete homoplasmic specific monoclonal antibodies with high efficiency is obtained. The technical method is simple and easy to operate, and a large number of monoclonal antibodies can be prepared and play an important role in life science research.
MAb has been widely used in diagnosis of swine fever since the first time swine fever virus immunized mice were used in cell culture in 1986 to establish and prepare MAb for swine fever. To date, many monoclonal antibodies against CSFV and their recognition epitopes have been found, but monoclonal antibodies capable of discriminating between circulating strains of CSFV and vaccine strain C have not been reported.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and providing a preparation method and application of a monoclonal antibody for identifying swine fever virus type 2 infection.
In order to solve the technical problems, the invention adopts the following solutions:
a recombinant baculovirus expressed CSFV 2 type E2 protein is provided, and the cDNA sequence of the coding region of the protein is shown as SEQ ID NO. 5.
The invention further provides a preparation method of the CSFV 2 type E2 protein, which comprises the following steps:
(1) Using cDNA of CSFV 2 strain HZ08 as template, using specific primer shown in SEQ ID NO.3 and SEQ ID NO.4 to make amplification so as to obtain E2 protein coding region cDNA sequence;
(2) Cloning of the E2 protein coding region sequence into the modified pFastBac with secretion Signal Peptide (SP) TM On HTB vector, recombinant plasmid pFastBac is obtained TM HTB-SP-E2HZ; transposing the recombinant plasmid to E.coli DH10Bac competent cells to obtain recombinant Bacmid-SP-E2HZ;
(3) Transfecting the recombinant Bacmid-SP-E2HZ into Sf9 cells to obtain recombinant baculovirus; the recombinant baculovirus expressed CSFV type 2E 2 protein, abbreviated as SP-E2HZ protein, was harvested.
As a preferred embodiment of the present invention, in the step (2), bamHI and HindIII are selected as the target gene insertion sites, and pFastBac is amplified using the primers shown in SEQ ID NO.1 and SEQ ID NO.2 TM HTB vectors, after linearization, carry secretion Signal Peptide (SP).
The invention also provides a monoclonal antibody for identifying swine fever virus type 2 infection, wherein the heavy chain variable region amino acid cDNA sequence of the monoclonal antibody is shown as SEQ ID NO.6, and the light chain variable region amino acid cDNA sequence is shown as SEQ ID NO. 7.
The invention further provides a preparation method of the monoclonal antibody, which comprises the following steps:
(1) Purifying the CSFV 2 type E2 protein, and then applying the purified CSFV 2 type E2 protein to mice immunized by CSFV 2 type strain HZ08 whole virus to realize mixed immunization;
(2) Preparing a hybridoma cell strain by utilizing a mouse myeloma cell SP2/0 and spleen cells of a mouse after mixed immunization; obtaining a monoclonal cell strain capable of stably secreting a specific antibody through screening and subcloning;
(3) Subtype determination, IFA identification, immunoblotting identification and epitope identification are carried out on the obtained monoclonal antibody, and the monoclonal antibody capable of blocking an ELISA system is screened.
The invention also provides a blocking ELISA kit for detecting type 2 CSFV infection serum antibodies, which comprises the following components:
SP-E2HZ protein and a pre-coated ELISA plate thereof, 100 ng/hole;
a sample diluent;
a negative control;
a culture solution containing the monoclonal antibody;
HRP-labeled goat anti-mouse IgG;
washing liquid: 0.5% PBST with 10mM PBS and 0.5% Tween 20;
color development liquid: TMB substrate comprising solution A and solution B;
stop solution: 2M H 2 SO 4 。
The invention further provides a use method of the kit, which comprises the following steps:
(1) Returning all the components to the room temperature of 18-25 ℃, and uniformly mixing the reagents by vortex;
(2) Diluting a serum sample to be detected by using a sample diluent according to a ratio of 1:5, adding 50uL of the sample diluent into each hole of a culture plate, and setting a compound hole and a negative control; after incubation at 37℃for 1h or at 4℃overnight, 0.5% PBST was washed 3 times, 5min each time, and patted dry;
(3) Diluting the monoclonal antibody with a sample diluent at a ratio of 1:8000, adding 50uL of the monoclonal antibody into each hole, and incubating for 1h at 37 ℃; washing with 0.5% PBST for 3 times and 5min each time, and drying;
(4) Diluting HRP-labeled goat anti-mouse IgG with sample diluent at 1:10000, adding 50uL per well, and incubating at 37 ℃ for 1h; washing with 0.5% PBST for 3 times and 5min each time, and drying;
(5) Adding 100uL of TMB developing solution which is prepared at present, and mixing the solution A and the solution B in equal volume; placing for 10min at room temperature in dark place, and starting timing after the 1 st hole is added;
(6) Adding 50uL of stop solution into each reaction hole at 10min to stop the reaction, wherein the sequence of adding the stop solution is consistent with that of adding the color development solution;
(7) Measuring the absorbance value of the sample at 450 nm;
(8) The blocking rate of the serum sample to be tested is calculated according to the following formula:
blocking rate (%) = (1-serum OD to be tested) 450nm ) Negative serum OD 450nm ；
(9) And (3) result judgment:
if the blocking rate of the detected sample is more than or equal to 36%, judging that the detected sample is positive, namely that the serum contains CSFV 2-type strain antibodies;
if the blocking rate of the tested sample is less than or equal to 29%, the tested sample is judged to be negative, namely the serum does not contain CSFV 2 type strain antibody;
if the blocking rate of the detected sample is 29-36%, the detected sample is judged to be suspicious.
Compared with the prior art, the invention has the beneficial effects that:
1. the monoclonal antibody prepared by the invention is a precious tool for developing novel diagnosis technology, and can be particularly used for distinguishing CSFV epidemic strains from vaccine strains or distinguishing vaccinated animals from naturally infected animals. This is important for more effective control of CSF epidemic and has great significance for purification of swine fever in pig farms.
2. The preparation method provided by the invention is simple and feasible, and can be used for efficiently screening out the monoclonal antibody for identifying the swine fever virus type 2 infection.
Drawings
FIG. 1 is a recombinant plasmid pFastBac TM Schematic construction of HTB-SP-E2 HZ.
FIG. 2 is a diagram showing Immunofluorescence (IFA) identification of recombinant baculovirus infected with CSFV type 2E 2 protein by Sf9 cells.
Wherein, the left column is CSFV 2 type E2 protein recombinant baculovirus P1 generation virus infected Sf9 cells, and the right column is normal Sf9 cell control.
FIG. 3 shows SDS-PAGE and Western blot to identify P1 and P2 generation CSFV 2 type E2 protein recombinant baculoviruses. Wherein A is SDS-PAGE electrophoresis diagram, B is Western blot identification diagram, and lanes 1-5 are Sf9 cells, P1 generation supernatant, P1 generation cell sediment, P2 generation supernatant and P2 generation cell sediment respectively.
FIG. 4 is a diagram showing SDS-PAGE of purified CSFV 2 type E2 recombinant protein SP-E2 HZ. Lanes 1-4 are, respectively, a Sichuan solution, a 50mM PBS hetero-protein eluent containing 10mM imidazole, a 50mM PBS hetero-protein eluent containing 20mM imidazole, and a 50mM PBS target protein eluent containing 500mM imidazole.
FIG. 5 is a diagram of the serum IFA identification of mice after immunization. Sf9 cells were infected with CSFV 2 type E2 protein recombinant baculovirus. M1-M3 serum is serum collected 35 days after whole virus immunization of mice, negative control is Negative serum (Negative serum) of mice, and positive control is 6 XHis monoclonal antibody and CSFV E2 monoclonal antibody 6D10.
FIG. 6 shows the results of screening hybridoma cell lines for identification ability using the identification IFA method.
FIG. 7 results of subtype identification of monoclonal antibodies.
FIG. 8 is a characterization of a monoclonal hybridoma cell line. Wherein A is the reactivity of IFA identification antibodies 6A12 and 11B8 with CSFV strains, and B is the reactivity of Western blot identification antibodies 6A12 and 11B8 with CSFV E2;
FIG. 9 is epitope identification of monoclonal antibodies 6A12 and 11B 8. Wherein A is the epitope identification result of the monoclonal antibody 6A12, and B is the epitope identification result of the monoclonal antibody 11B 8.
FIG. 10 is an alignment of the amino acid sequences of E2 proteins of CSFV 2 and vaccine strain C in NCBI database from different sources based on epitope intervals of MAb 6A12 and 11B 8.
FIG. 11 shows the results of Western blot identification of the reactivity of monoclonal antibodies 6A12, 11B8 with CSFV type 2E 2 prokaryotic protein HZE and its muteins (i.e., identification of the key amino acid sites of monoclonal antibodies 6A12 and 11B8 for identification of CSFV type 2E 2 protein and vaccine strain C E2 protein). Wherein A is the key amino acid site identified by monoclonal antibody 6A12, and B is the key amino acid site identified by monoclonal antibody 11B 8.
FIG. 12 shows the results of the optimization of blocking ELISA antigen coating concentration and monoclonal antibody dilution ratio.
Fig. 13 is a preliminary attempt to establish a blocking ELISA based on optimized antigen coating concentration and mab dilution ratio.
FIG. 14 shows the results of blocking ELISA specific assays for serum from different sources: QZ14-1 to QZ14-2 and HZ08-1 to HZ08-2 are CSFV wild strain infected pig serum, BVDV1-1 to BVDV1-2 are BVDV1 type infected pig serum, BVDV2-1 to BVDV2-2 are BVDV2 type infected pig serum, and C-1 to C-3 are CSFV vaccine C strain immunized pig serum. Wherein A is an indirect ELISA for detecting the reactivity of serum with CSFV 2 type E2, BVDV E2 or vaccine strain C E2 protein. B is blocking ELISA to detect serum reactivity with CSFV type 2E 2 protein.
Detailed Description
It should be noted that the contents of each virus, reagent, instrument, operation method, etc. mentioned in the present invention are all the prior art. Those skilled in the art can implement this in light of the present disclosure and legal source acquisition and in combination with mastering skills.
The CSFV 2 strain HZ08 is a microbial material obtained by the applicant, the login information of the microbial material in an NCBI (national center for biotechnology information ) database is GenBank:FJ582642.1, and the public and the society can obtain the microbial material through legal approaches according to related information.
The implementation of the present invention will be described in detail below with reference to specific examples.
1. Immune antigen preparation
Amplification of CSFV 2-type strain HZ08
1. Culturing PK-15 cells
Resuscitates 1 PK-15 cell to T25 square bottle for culture, wherein the culture medium is DMEM medium containing 6% Fetal Bovine Serum (FBS) and 1% anti-biological-anti-biological (100X), and the culture environment is 37deg.C, 5% CO 2 . When the cells were confluent with monolayers and in good condition, T25 square flask cells were passaged in the normal manner. That is, the cell culture solution was discarded, and the cells were washed 3 times with 2mL of Hanks solution pre-heated at 37 ℃; then adding 2mL of 0.25% pancreatin to digest the cells, and discarding the digestive juice when the cells are subjected to circular shrinkage separation; the digestion was stopped by adding 5mL of cell culture medium and the cells were blown down to give the cells a single state. After cell digestion, 1mL of the cell suspension is taken and inoculated into a new T25 square bottle, cell culture solution is added to 5mL, and the cell state is observed every 12 h.
CSFV amplification
Continuously transferring 2 generations until PK-15 cells grow stably, then separating the cells into 2T 75 square bottles for culture, discarding a primary cell culture solution when the cell intersection degree reaches about 70%, and washing the cells for 3 times by Hanks solution. The virus solution of CSFV type 2 strain HZ08 thawed from a-80℃refrigerator was diluted 5-fold and added to a T75 square bottle at 5 mL/bottle. After incubation for 2h at 37℃the virus solution was discarded and 10mL of DMEM medium containing 2% FBS and 1% anti-animal-anti-animal (100X) was supplemented. Observing every 24 hours, and when the color of the culture medium in the T75 square bottle is changed into orange, putting the T75 square bottle in a refrigerator at the temperature of-80 ℃ for repeated freezing and thawing for 3 times. Pouring the culture medium containing virus and cell debris into a 50mL centrifuge tube, centrifuging at 4 ℃ and 4000rpm for 15min, removing cell debris in the virus liquid, collecting supernatant, subpackaging into a 1.5mL centrifuge tube, 500 uL/tube, and preserving at-80 ℃ in a refrigerator.
CSFV viral titer assay
Will grow wellThe well PK-15 cells were digested and inoculated into 96-well cell culture plates at 100 uL/well. When the cell intersection degree reaches about 70%, the original cell culture solution is discarded, the cells are washed 3 times by Hanks liquid, 10 times of gradient diluted virus liquid is added, 100 uL/hole is added, and each dilution is inoculated with a longitudinal row of 8 holes, and 7 dilutions are continued. The dilution method is as follows: preparing 7 1.5mL centrifuge tubes, adding 900uL serum-free DMEM medium into each tube, adding 100uL virus stock solution into the 1 st tube, mixing, sucking 100uL, adding the 2 nd tube, mixing, and so on to make 7 dilutions (10 -1 ～10 -7 ). After incubation for 1h at 37℃the virus solution was discarded and the Hanks solution was washed 3 times and 100uL of DMEM medium containing 2% FBS and 1% anti-animal (100X) was added to each well. After 72h virus titer was determined by indirect immunofluorescence (Indirect immunofluorescence assay, IFA) as follows: discarding the cell culture solution in the 96-well cell culture plate, washing with PBS for 2 times, adding 100 uL-20deg.C precooled 80% acetone into each well, and fixing at-20deg.C for more than 30 min; discarding cold acetone, washing with PBS for 2 times, and drying; 50uL of swine fever E2 monoclonal antibody hybridoma cell supernatant (monoclonal antibody 6D 10) diluted in a ratio of 1:1000 (the diluent is PBS) is respectively added into the holes, and the mixture is incubated for 1h at 37 ℃; discarding the primary antibody, washing with PBS for 2 times, and drying; 50uL of FITC-labeled goat anti-mouse IgG diluted 1:1000 was added to each well and incubated at 37℃for 1h in the absence of light. The secondary antibody was discarded and observed under an inverted fluorescence microscope. The virus titer was calculated according to the Karber method as follows:
LgTCID 50 ＝L-d(s-0.5)。
L: logarithm of highest dilution
D: differences between log dilutions
s: sum of positive well ratios
Preparation of (II) CSFV 2 type E2HZ eukaryotic protein
Construction of CSFV 2 type E2HZ protein eukaryotic expression vector
The envelope protein E2 of the swine fever virus is a main immunogenic protein and can induce the organism to produce high-level neutralizing antibodies. Because of the excellent ability of MAbs to recognize differences in virus antigen structure, the present invention has been directed to the development of MAbs with the ability to discriminate between the CSFV vaccine strain C, E2 protein and the gene type 2.1 epidemic strain E2 protein, for the purpose of researchThe study of the antigenic structure and antigenic evolution of CSFV E2 provides a new tool. To this end, the invention firstly clones the E2 protein coding region sequence of the strain HZ08 of CSFV 2 into a modified pFastBac with secretion Signal Peptide (SP) TM Construction of recombinant plasmid pFastBac on HTB vector TM HTB-SP-E2HZ was constructed as shown in FIG. 1. The recombinant plasmid is transposed to E.coli DH10Bac receptor bacteria to obtain recombinant Bacmid-SP-E2HZ.
(1) Vector linearization and CSFV 2 strain E2 protein coding region amplification
BamHI and HindIII were selected as target gene insertion sites, and primers shown in SEQ ID NO.1 and SEQ ID NO.2 were designed for amplifying pFastBac TM HTB vector, linearizes it.
PK-15 cell infection 200TCID 50 After 48h of CSFV type 2HZ 08 strain, viral RNA was extracted using a total RNA rapid extraction kit, the specific steps being carried out with reference to the RNA extraction kit protocol. After reverse transcription into the corresponding cDNA, an amplification primer of the CSFV 2 type strain E2 protein gene HZ08-E2 (abbreviated as E2 HZ) is designed. And (3) recovering the PCR product by using a Norpran gum recovery kit, wherein specific operation steps are shown in the specification, and determining the concentration of the product by using a Nano Drop.
The corresponding primers used in the amplification of the above vectors and fragments are shown in the following table.
TABLE 1 primers for PCR
(2)pFastBac TM Construction of HTB-SP-E2HZ recombinant plasmid
Vector pFastBac to be linearized TM The HTB and the fragment of interest E2HZ were ligated using a one-step cloning enzyme. The connection system is shown in Table 2, the reaction conditions are that a PCR instrument is connected for 20min at 50 ℃, and the reaction is kept stand for 2min at 4 ℃, and the obtained one-step cloning product is pFastBac TM HTB-SP-E2HZ recombinant plasmid.
TABLE 2 one-step cloning of the ligation System
(3) Recombinant plasmid pFastBac TM Transformation and identification of HTB-SP-E2HZ
Competent cells E.coli DH 5. Alpha. Were removed from-80℃and placed on ice to thaw, and then 20uL of one-step cloning product (pFastBac-E2 HZ) was added, gently mixed, ice-bathed for 30min, and rapidly placed on ice for 3min after heat shock at 42 ℃. 900uL of antibiotic-free LB liquid medium is added into an ultra-clean bench, shaking culture is carried out for 1h at 37 ℃ by a shaking table, centrifugation is carried out for 5min at 5000rpm, 900uL of supernatant is discarded, the residual medium is used for light and heavy suspension bacterial precipitation, the bacterial precipitation is evenly coated on an LB plate containing 50ug/mL of ampicillin, and the plate is reversely cultured for overnight at 37 ℃. And (3) selecting monoclonal bacterial colonies, identifying the monoclonal bacterial colonies as positive by using an E2HZ specific primer PCR, performing amplification culture, sending to a sequencing company for sequencing, and storing the monoclonal bacterial liquid with correct sequence in a refrigerator at the temperature of minus 80 ℃.
(4) Construction of recombinant bacmid
pFastBac to be sequenced correctly TM HTB-SP-E2HZ is transposed to E.coli DH10Bac competent cells, and the competent cells are placed on a shaking table at 180rpm at 37 ℃ for shake culture for 4 hours; 10uL of the culture was diluted 10-fold with 90uL of the medium without antibody, and then uniformly spread on a previously prepared blue-white selective culture plate, and cultured at 37℃for 48 hours. White single colonies are selected and cultured in LB liquid medium containing 50ug/mL kanamycin, 7ug/mL gentamycin and 10ug/mL tetracycline (hereinafter referred to as 'three antibodies') at 180rpm under 37 ℃ in a shaking way, and PCR identification is carried out by using a universal primer M13 after bacterial liquid is turbid. And (5) identifying correct bacterial liquid for expansion culture, and extracting recombinant rod particles. The arrangement method of the blue-white selective culture plate comprises the following steps: adding 50ug/mL kanamycin, 7ug/mL gentamicin and 10ug/mL tetracycline into LB agar, mixing, pouring into LB three-antibody plate, and preserving at 4 ℃ after solidification. 100uL of X-gal (20 mg/mL) and 5uL of IPTG (1 mol/L) were mixed before use and spread evenly on the triple antibody plates.
(5) Extraction of recombinant rod particles
Inoculating positive bacterial liquid into 10mL of LB liquid medium containing the three antibodies in a ratio of 1:1000, and carrying out shaking culture at 37 ℃ and 200rpm for overnight; centrifuging at 4000rpm for 10min, discarding supernatant, and adding 500uL Solution I to the thallus precipitate to resuspend thallus; adding Adding 500uL Solution II, mixing, gently inverting the EP tube for several times, and standing at room temperature for 4min; adding 500uL Solution III, reversing and fully mixing; centrifuging at 4000rpm for 10min, transferring the supernatant to a new EP tube, repeating centrifuging at 4000rpm for 10min, and transferring the supernatant to another new EP tube; adding equal amount of pre-cooled isopropanol at-20deg.C, mixing, standing on ice for 15-30min; centrifuging at 12000rpm at 4deg.C for 10min, discarding supernatant, adding 700uL of precooled 70% ethanol, and performing light suspension precipitation; repeating the previous operation once, discarding the supernatant, and air-drying the precipitate; 60uL of sterile ddH was added 2 After O is dissolved and precipitated by light weight suspension, the concentration and purity of the extracted recombinant rod particles are measured by Nano Drop.
2. Preparation of recombinant baculovirus expressing CSFV 2 type E2 protein
(1) Sf9 cell culture
The culture medium is Sf-900 containing 1% of anti-biological-anti-biological (100X) TM III SFM medium. The day before transfection, sf9 cells were passaged into T25 cell flasks to a cell density of about 70-80% and cultured in an incubator at 27.5 ℃.
(2) Recombinant Bacmid-E2HZ transfected Sf9 cells
Taking Bacmid-E2HZ 1-2 ug, adding 250uL Sf-900 without double antibody TM In a III SFM culture medium, blowing and uniformly mixing, and marking as A liquid; 8uL of Cellfectin II Reagent transfection Reagent is added into another 250uL of Sf-900TM III SFM culture medium without double antibody, and the mixture is uniformly mixed by vortex and marked as B solution; mixing the solution A and the solution B, and incubating for 15-20min at room temperature. During incubation, T25 cells passaged the previous day were used with antibody-free Sf-900 TM III SFM medium was rinsed 2 times to remove the diabodies in the medium. After the incubation of the mixture was completed, it was added dropwise to Sf9 cells which were washed without medium, and 1.5mL of Sf-900 without diabody was added TM SFM medium, 27.5 ℃ incubation for 5h. The transfection mixture was aspirated and 5mL Sf-900 containing 1% of anti-abiotic (100X) was added TM III SFM medium.
(3) Obtaining recombinant baculovirus
After the above-mentioned transfected Sf9 cells were cultured in an incubator at 27.5℃for 72 to 96 hours, obvious swelling of SF9 cells was observed, and cells were collapsed in the late stage of transfection and tended to dissociate from the flask. Directly collecting a culture medium containing viruses, centrifuging at 4000rpm to collect supernatant and cell sediment, wherein the supernatant is the P1 generation recombinant baculovirus, sucking 500 mu L P generation baculovirus to infect 1 bottle of new T75 Sf9 cells, and harvesting the P2 generation recombinant baculovirus after 72 hours; similarly, the P3 generation recombinant baculovirus was harvested. Subpackaging the P1-P3 generation recombinant baculovirus into a 1.5mL centrifuge tube, and storing in a refrigerator at-80 ℃.
(4) IFA identification of recombinant proteins
Sf9 cells with good growth state are inoculated into a 96-well cell culture plate, so that the cell density is 70-80%. 8 duplicate wells were set, of which 4 were inoculated with 10ul of P1 generation virus solution, the remaining 4 wells were untreated and placed in a 27.5 ℃ incubator as negative control for culture, and IFA after cytopathic observation identified if Sf9 cells expressed recombinant CSFV 2 type E2 protein. The IFA procedure is as follows: discarding the cell culture medium, washing the cells once with PBS, and drying by suction; adding 80% acetone pre-cooled at-20deg.C into each hole, and fixing at-20deg.C for more than 30 min; discarding cold acetone, washing with PBS for 2 times, and drying; 50uL of mouse anti-6 XHis monoclonal antibody and swine E2 monoclonal antibody hybridoma cell supernatant (monoclonal antibody 6D 10) diluted in a ratio of 1:1000 (PBS) are respectively added into the wells, and incubated for 1h at 37 ℃; discarding the primary antibody, washing with PBS for 2 times, and drying; 50uL of FITC-labeled goat anti-mouse IgG diluted 1:1000 was added to each well and incubated at 37℃for 1h in the absence of light. Discarding the secondary antibody, observing fluorescence under an inverted fluorescence microscope, photographing and preserving. As shown in FIG. 2, sf9 cells infected with recombinant baculovirus were specifically recognized by the mouse anti-6 XHis monoclonal antibody and the swine fever E2 monoclonal antibody 6D10 (cells exhibited significant fluorescence), while uninfected control cells were non-fluorescent, indicating that His-tagged proteins of interest were expressed.
(5) SDS-PAGE and Western blot identification of recombinant proteins
SDS-PAGE and Western blot identification the supernatant and precipitate of the P1, P2 generation recombinant baculovirus obtained in step (3), and the Sf9 cells cultured normally were used as negative control. After the P1 and P2 cell pellet was resuspended in PBS, the supernatant (S) and pellet (P) were sampled and E2HZ protein expression was detected with classical swine fever E2 mab 6D 10. The operation procedure is as follows:
preparing a separating gel and a concentrated gel with the concentration of 12 percent according to the specification; adding 40uL of protein sample to be detected into 10uL of 5 Xprotein Loading Buffer, blowing and mixing uniformly, boiling in water bath for 10min, and centrifuging at 5000rpm for 1min; assembling the electrophoresis tank, filling the electrophoresis liquid into the inner tank and the outer tank, and spotting protein samples according to the sequence (negative control, S1, P1, S2 and P2); run protein electrophoresis following the procedure of 80V 30min,120V 60min.
Coomassie brilliant blue staining: after carefully separating the protein gel from the gel plate, putting the protein gel into a newly prepared coomassie brilliant blue dye solution, shaking and dyeing for 2-4 hours at 37 ℃, putting the gel into a decolorizing solution for decolorizing, and changing the decolorizing solution every 1 hour until the protein gel has no background (the result is shown in fig. 3-A);
immunoblotting: cutting out PVDF film with the size similar to that of the protein glue to be detected, soaking the PVDF film with methanol for activating for 30s, soaking in wet buffer solution for several minutes, and soaking the filter paper and the sponge in the wet buffer solution simultaneously. And (3) layering and discharging bubbles according to the sequence of (-) thick sponge-Bao Lvzhi-glue-PVDF film-Bao Lvzhi-thick sponge (+) and performing constant-current transfer printing for 40min at 200 mA. Closing: the transferred PVDF film was put into TBST containing 5% skimmed milk powder and shake-closed at 37℃for 1h. Washing: the skim milk was discarded and the PVDF membrane was washed 3 times with TBST for 5min each. Incubating primary antibodies: the swine fever E2 monoclonal antibody 6D10 hybridoma cell supernatant was added and incubated overnight at 4℃on a shaker. Washing: the primary antibody was discarded and the PVDF membrane was washed 3 times with TBST for 5min each. Incubating a secondary antibody: HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) was added and incubated on a shaker at 37 ℃ for 1h. Washing: the secondary antibody was discarded and the PVDF membrane was washed 3 times with TBST for 5min each. Color development: FDbio-Pico ECL was added for color development and photographed, and recombinant E2HZ protein was secreted and expressed in the culture supernatant as shown in FIG. 3-B.
Expression and purification of CSFV 2 type E2 protein
(1) Expression of recombinant proteins
Sf9 cells cultured in T75 cell culture flask were transferred to a flask containing 100mL of Sf-900 TM III cell shake flasks of SFM medium were grown in suspension at 27.5℃in an incubator. Cell density was monitored every 24h and reached 2.0X10 s 6 At individual/mL, depending on recombinant baculovirus titer,the P3 generation recombinant baculovirus is inoculated into the Sf9 cells in suspension culture at the ratio of infection ratio (MOI) of 1, the culture is continued to be carried out at the temperature of 27.5 ℃ for 96 hours by shaking culture at 140rpm, the supernatant and cell sediment are obtained by centrifugation at 4000rpm for 5 minutes, and the target protein is in the supernatant.
(2) Purification of recombinant proteins
1.5mL of nickel column gel particles were pipetted into the purification column void column and the nickel column was equilibrated with 3 bed volumes of 50mM PBS to remove ethanol; adding the balanced nickel column gel particles into a supernatant containing target proteins, and combining overnight at 4 ℃ on a test tube inversion mixer; adding a protein sample combined by a nickel column into a purification column empty column, opening a speed control valve switch to enable liquid to flow out, and collecting a river liquid; washing the hybrid protein with 30mL of 50mM PBS containing 10mM imidazole and 20mM imidazole, respectively, and collecting the washing solution; the target protein was eluted with 5mL of 50mM PBS containing 500mM imidazole, and the eluate was collected. The purity of the protein was checked by SDS-PAGE, and as a result, as shown in FIG. 4, a CSFV type 2E 2 eukaryotic expression protein (hereinafter referred to as SP-E2 HZ) was obtained, having a size of about 45kDa, and the protein concentration was measured by using a Novirzan BCA protein concentration measuring kit.
The result of gene sequencing shows that the cDNA sequence of the coding region of CSFV 2 type E2 protein is shown as SEQ ID NO. 5.
2. Mixed immunization of mice
Whole virus immunization
3 female BALB/C mice of 6 weeks old were subjected to 300uL of CSFV type 2 strain HZ08 (virus titer: 10) 4.5 TCID 50 100 uL) was emulsified by mixing with an equivalent amount of 206VG adjuvant, and the mice were immunized subcutaneously at multiple spots on the nuchal portion, and the second and third immunization with HZ08 field toxin were performed in the same manner after 14 days and 28 days of initial immunization. The orbital vein of the mice 35 days after HZ08 wild virus immunization is sampled, the separated serum is diluted according to 1/500 and is used as a primary antibody, FITC marked goat anti-mouse IgG which is diluted according to 1/1000 is used as a fluorescent secondary antibody, IFA is used for measuring the specific antibody of CSFV 2 type E2 protein in the serum of the mice, and negative control is set. As shown in FIG. 5, specific antibodies to CSFV type 2E 2 protein were raised in mice.
(II) protein immunization
Immunization of SP-E2HZ protein was performed on mice 42 days after full virus immunization:
purified CSFV 2 type E2 protein SP-E2HZ was mixed with an equivalent amount of 206VG adjuvant for emulsification, and the mice were subjected to multipoint subcutaneous immunization at the nuchal portion of the mice at a dose of 50-100 ug/dose, and protein di-and tri-immunization was performed in the same manner 14 days and 28 days after the initial immunization. One mouse was selected for impact immunization by intraperitoneal injection of 50ug of CSFV 2 type E2 protein 35 days after protein immunization. After 3 days, mice spleen cells were prepared for fusion.
3. Preparation of hybridoma cell lines
Preparation of spleen cells
Collecting the eyeball of the mice subjected to the immunization treatment, taking blood and then removing the neck for killing; immersing the cadaver in 75% alcohol for 2min for disinfection, taking out, draining the alcohol, fixing on a clean foam plate in a supine mode, and putting into a biosafety cabinet; sequentially shearing the skin, the muscle layer and the peritoneum from the position close to the right of the midline of the abdomen by using sterile scissors forceps, and fully exposing the abdominal cavity; taking sterile forceps and scissors, cutting off peritoneum, and finding and separating spleen; placing spleen in a sterile culture dish containing RPMI 1640, and separating connective tissue and fat on the surface of the spleen; rinsing the spleen 3 times with a new RPMI 1640 medium; placing a sterile 200-mesh copper net in a sterile culture dish with the diameter of 100mm, infiltrating the RPMI 1640 on the net surface, placing the separated spleen on the net surface, grinding the spleen by using a sterile round bottom test tube, continuously flushing the spleen by using the RPMI 1640 until the spleen is crushed, and filtering the copper net; washing the copper mesh clean by using RPMI 1640; spleen cell suspension in a petri dish was collected and centrifuged at 300Xg for 5min at room temperature in a sterile round bottom centrifuge tube and the cell pellet resuspended in 10mL RPMI 1640 for use.
(II) preparation of myeloma cells
Mouse myeloma cells SP2/0 in log phase in 2T 75 flasks (commercial product, CRL-1581 TM ) About 1/2 to 1/5 of the number of spleen cells, the medium was discarded, and the adherent cells were resuspended in 10mL each of RPMI1640 after one wash with RPMI 1640. Collecting SP2/0 suspension in another sterile round bottom centrifuge tube, centrifuging at 300Xg for 5min at room temperature, discarding supernatant,cell pellet was resuspended with 10ml rpm 1640.
(III) cell fusion
The collected spleen cells were mixed with myeloma cells SP2/0, centrifuged at 300Xg for 5min at room temperature, the supernatant was discarded, and the RPMI1640 was repeatedly washed 2 times, and the supernatant was discarded. The centrifuge tube was tapped on a paper towel to mix the two cells evenly to a slurry. Suspending the centrifuge tube in a sterile water bath at 37 ℃, sucking 1mL of 50% PEG1450 preheated at 37 ℃ to drop in 1min near the bottom of the tube, shaking while dropping to fully mix the cells, standing for 30s, and stopping gradient dilution: namely, different volumes of RPMI1640 (1 mL, 2mL, 3mL, 4mL, 5mL and 6mL respectively) preheated at 37 ℃ in advance are added step by step, each volume is added in 1min, shaking is carried out while adding, and standing is carried out for 30s between each liquid adding; centrifugation at 300Xg for 5min at room temperature, discarding supernatant, resuspending cell pellet with 150mL HAT screening medium (containing RPMI1640, 20% fetal bovine serum, 1% anti-Antimycotic and 1% HAT) and spreading uniformly into 10 96 well cell culture plates. After 3-4 days, culture was continued with HT screening medium in half-changed medium (containing RPMI1640, 20% fetal calf serum, 1% anti-animal and 1% HT).
4. Screening for identification of monoclonal hybridoma cell lines
Screening of positive hybridoma cell lines
When the hybridoma cell division aggregation after successful fusion reaches about 1/3 of the hole bottom area (8-10 days), hybridoma supernatants are taken for IFA screening.
Positive screening system: antigen cell plates used for screening were infected with 200TCID 50 PK-15 cells of CSFV HZ08 strain; hybridoma cell culture supernatant to be detected, secondary antibody of FITC labeled goat anti-mouse IgG (1/1000 diluted use), positive control of laboratory self-made CSFV E2 monoclonal antibody 6D10 (see article PMID:33493566 for specific data) and negative control of 1640 screening medium.
(II) screening for identifying hybridoma cell lines
And taking the hybridoma cell strain supernatant identified as positive for IFA identification screening.
Identification screening system: antigen cell plates used for screening were each stained with 200TCID 50 CSFPK-15 cells of the V HZ08 strain and vaccine C strain; the primary antibody was hybridoma cell culture supernatant initially identified as positive well, and the secondary antibody, positive control and negative control were as described in (one). According to the result of the identification IFA, hybridoma cell holes with identification ability (specifically identifying HZ08 strain and not identifying C strain) are selected for amplification and monoclonalization. The results of the differential screening are shown in FIG. 6.
(III) subcloning
Mix target positive well cells and count, take about 100 cells to 15mL 1640 medium (containing RPMI1640, 20% fetal bovine serum, 1% anti-animal) mix, inoculate into a 96 well cell culture plate, observe and record cell clone number per well. After about 8-10 days, the culture supernatant was taken for IFA detection, positive cell wells were recorded, and thus 2-3 rounds of screening were performed until a single cloned positive hybridoma cell line was obtained.
Through screening and subcloning, 2 monoclonal cell strains which stably secrete CSFV 2 type E2 protein specific antibodies are finally obtained, and are respectively named as 6A12 and 11B8, and the secreted antibodies are called as monoclonal antibodies 6A12 and 11B8 in the invention.
5. Identification of monoclonal antibodies 6A12 and 11B8
1. Subtype determination of monoclonal antibodies
The monoclonal antibodies 6A12 and 11B8 obtained above were identified according to the instructions of the protein antibody subtype identification kit. The principle of the subtype identification of the antibody is to utilize an enzyme-linked immunosorbent assay (ELISA) to determine the binding condition of the antibody to be detected and antibodies with different subtype specificities (provided in an identification kit), identify the antibody specifically bound with the antibody to be detected, and obtain the subtype of the antibody to be detected according to the specificity of the bound antibody. The identification method comprises the following steps: the subclass of the monoclonal antibody to be tested was judged according to the detection of absorbance by a microplate reader according to the kit instructions, and the results are shown in FIG. 7, wherein the monoclonal antibodies 6A12 and 11B8 are IgG1 subclasses of kappa light chains.
IFA identification of reactivity of the monoclonal antibodies 6A12 and 11B8 with CSFV strains
The PK-15 cells are inoculated into a 96-hole cell culture plate one day in advance, a CSFV HZ08 strain infection hole, a CSFV C strain infection hole and a normal cell hole are respectively arranged,HZO8 and C strains with 200TCID 50 Each hole. Discarding cell culture supernatant after 48h, washing with PBS for 2 times, adding 100 uL-20deg.C pre-cooled 80% acetone into each well, and fixing at-20deg.C for more than 30 min; washing with PBS for 2 times, adding supernatants of monoclonal antibodies 6A12 and 11B8 hybridoma cell lines to be detected, setting positive control (CSFV E2 monoclonal antibody 6D 10) and negative control (1640 culture medium), and incubating at 37 ℃ for 1h; PBS is washed for 2 times, the secondary antibody is FITC marked goat anti-mouse IgG (used by 1/1000 dilution), and the secondary antibody is incubated for 1h at 37 ℃; the secondary antibodies were discarded, fluorescence was observed under an inverted fluorescence microscope and stored by photographing, as shown in fig. 8-a, monoclonal antibodies 6a12 and 11B8 could detect CSFV HZ08 strain on PK-15 cells, but not C strain, indicating that monoclonal antibodies 6a12 and 11B8 have the potential to discriminate between CSFV 2 type strains and vaccine C strain.
3. Reactivity of the antibodies 6A12 and 11B8 with CSFV E2 by immunoblotting
Mixing prokaryotic expression proteins of CE2 and HZE2 with 5×protein loading buffer solution according to the volume of 4:1, respectively, and performing SDS-PAGE electrophoresis (electrophoresis program is 80V 30min,120V 45min) after 10min in boiling water; transferring the separated proteins onto nitrocellulose membrane, blocking with 5% skimmed milk powder, adding culture supernatants of hybridoma cells 6A12 and 11B8, incubating overnight at 4deg.C, and rinsing with Tris salt buffer (TBST) containing 0.05% Tween-20 for 3 times; goat anti-mouse IgG was added at 1:5000 dilution, incubated at 37℃for 1h, and rinsed 3 times with TBST; color development was performed according to FDbio-Pico ECL luminophor instructions. As shown in FIG. 8-B, the monoclonal antibodies 6A12 and 11B8 bind only to HZE protein, a specific band appears at 45kDa, but not to CE2, demonstrating that monoclonal antibodies 6A12 and 11B8 are CSFV type 2E 2-specific antibodies, and also that monoclonal antibodies 6A12 and 11B8 recognize a CSFV E2 linear epitope.
4. Epitope identification
(1) Construction of CSFV 2 type E2 protein truncated fragment expression vector
In order to define the antigen epitope recognized by monoclonal antibodies 6A12 and 11B8, a series of recombinant plasmids containing CSFV 2 type E2 truncated fragments are constructed, and different truncated E2 proteins are expressed in a prokaryotic expression system.
The procedure was as follows: the target fragment amplified by PCR is connected to pET30a vector to transform DH5 alpha competent cells. Amplifying and culturing positive monoclonal colony with correct sequence, extracting plasmid, converting to Rossetta competent cells, PCR identifying and screening positive monoclonal colony, amplifying and culturing to OD 600 =0.6, and expression of truncated E2 protein was induced at 16 ℃ after addition of an amount of IPTG (final concentration of 1 mM/mL). The truncated E2 protein is further identified by Western blot with monoclonal antibodies 6A12 and 11B 8. As shown in FIG. 9-A, the monoclonal antibody 6A12 reacts with recombinant truncated proteins aa1-332 and aa1-72, but does not react with aa1-56 and aa199-332, which indicates that the epitope recognized by the monoclonal antibody 6A12 is located within the aa1-72 region of CSFV 2-type E2 protein; the monoclonal antibody 11B8 reacted with recombinant proteins aa1-332, aa199-332, aa1-279, but not aa204-332, aa1-269, indicating that the epitope region recognized by monoclonal antibody 11B8 was located within the aa199-279 interval (FIG. 9-B).
(2) Construction of CSFV 2 type E2 specific amino acid mutant protein expression vector
To further determine the key amino acid positions for the identification of CSFV 2 type E2 and vaccine strain C E2 by antibodies 6a12 and 11B8, a series of point mutations were introduced into CSFV 2 type E2 by sequence alignment (fig. 10) based on the different amino acid positions of the different CSFV 2 strains and vaccine strain C2.
The specific procedure is as follows: the macrocycle of pET30a-E2HZ plasmid is amplified by designing a primer introducing directed amino acid single-point mutation, linearization plasmid macrocycle self-connection is carried out after the action of homologous recombinase, and positive monoclonal colony expansion culture with correct sequencing is carried out, so that the extracted plasmid is transformed into Rossetta competent cells. Screening positive monoclonal colony, and enlarging culture to OD 600 =0.6, and expression of E2 mutant protein was induced at 16 ℃ after addition of a certain amount of IPTG (final concentration of 1 mM/mL).
The obtained single-point mutation plasmids of amino acids are pHZE2-3, pHZE2-16, pHZE2-20, pHZE2-24, pHZE2-36, pHZE2-40, pHZE2-45, pHZE2-47, pHZE2-49, pHZE2-56, pHZE2-72, pHZE2-200, pHZE2-212, pHZE2-213, pHZE2-235, pHZE2-253, pHZE2-268 and pHZE2-270, namely, aa1-72 of CSFV 2 type E2 and aa199-279 of CSFV type 2 type E2 are replaced by amino acids at positions corresponding to vaccine strain C2 one by one. S3A, N16D, P L, E G, G5432D, D40N, R45K, I S, T49V, I K, I47S, T V, I T, R K, Q200V, K212D, E213G, V235I, E S, L268S, G270E.
E2 mutant and monoclonal antibodies 6A12 and 11B8 are subjected to further reactivity identification through Western blot. The results showed that the reactivity with mab 6a12 was lost after the mutation P20L, E, 24, G, D N, respectively, of CSFV 2 type E2 (fig. 11-a), the reactivity with mab 11B8 was lost after the mutation E213G of CSFV 2 type E2 (fig. 11-B), indicating that the key amino acid sites for mab 6a12 to identify CSFV 2 type epidemic strain E2 and vaccine C strain E2 were proline at position 20, glutamic acid at position 24, and aspartic acid at position 40, and that for mab 11B8 to identify CSFV 2 type epidemic strain E2 and vaccine C strain E2 were glutamic acid at position 213.
The gene sequencing result shows that the heavy chain variable region amino acid cDNA sequence of the monoclonal antibody 6A12 is shown as SEQ ID NO.6, and the light chain variable region amino acid cDNA sequence is shown as SEQ ID NO. 7.
6. Establishment of blocking ELISA
1. Determination of antigen coating concentration and detection of monoclonal antibody dilution ratio
The reaction titers of the monoclonal antibodies 6A12 and 11B8 were determined by indirect ELISA using purified SP-E2HZ as coating antigen. Adopting a square titration method to optimize the reaction conditions: the SP-E2HZ protein coating concentration is 1ug/mL, 2ug/mL and 4ug/mL respectively, the 6A12 dilution ratio is 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and the 11B8 dilution ratio is 1:1000, 1:2000, 1:4000. Adding the detection monoclonal antibody, reacting for 1h at 37 ℃, washing with PBST for 3 times, and drying by beating; adding HRP-marked goat anti-mouse IgG secondary antibody diluted by 1:10000, reacting for 1h at 37 ℃, washing by PBST for 3 times, and beating to dryness; the color development was stopped with TMB for 10min,2M sulfuric acid, OD 450nm And (5) reading. As shown in FIG. 12, the OD increased with increasing dilution of the monoclonal antibody 450nm The reading drops and OD is selected 450nm The lowest antigen coating concentration and the maximum mab dilution ratio at readings approaching 1.0. Therefore, the SP-E2HZ protein coating concentration was subsequently selected to be 1ug/mL, with a 6A12 dilution ratio of 1:8000 and an 11B8 dilution ratio of 1:1000 for blocking establishment of the ELISA method.
2. Blocking ELISA preliminary establishment
And detecting the dilution ratio of the monoclonal antibody according to the determined coating concentration of the SP-E2HZ antigen. Preliminary attempts were made to establish a blocking ELISA.
Diluting serum (to-be-detected serum) collected for 28 days after virus challenge of CSFV 2 strain QZ14 and serum (to-be-detected serum) collected for 28 days after immunization of vaccine C strain and one CSFV standard negative serum according to a ratio of 1:10, incubating for 1h at 37 ℃, washing with PBST for 3 times, and drying by beating; 50uL of monoclonal antibody 6A12 diluted in a ratio of 1:8000 or monoclonal antibody 11B8 diluted in a ratio of 1:1000 are added into each hole, incubation is carried out for 1h at 37 ℃, PBST is washed 3 times, and the mixture is dried by beating; 50uL of goat anti-mouse IgG secondary antibody marked by HRP diluted by 1:10000 is added into each hole, incubated for 1h at 37 ℃, washed by PBST for 3 times, and patted dry; each well was developed by adding 100. Mu.LTMB for 10min, and the development was stopped by adding 2M sulfuric acid, and OD was measured 450nm And reading the value. As shown in fig. 13, when 6a12 is used as the monoclonal antibody, the blocking rate of the CSFV 2 epidemic strain QZ14 infected porcine serum antibody is significantly different from that of the C strain immunized porcine serum antibody, so that the subsequent experiment selects 6a12 as the monoclonal antibody to establish a blocking ELISA system.
3. Serum dilution ratio optimization
And detecting the influence of serum dilution factors on the blocking rate according to the determined SP-E2HZ antigen coating concentration and the detection monoclonal antibody 6A12 dilution ratio.
Diluting serum (serum to be detected) collected after 28 days of virus challenge of CSFV 2 strain QZ14 and one CSFV standard negative serum respectively according to 1:5, 1:10, 1:20 and 1:40, incubating for 1h at 37 ℃, washing with PBST for 3 times, and drying; adding 50uL of monoclonal antibody 6A12 diluted in a ratio of 1:8000 into each hole, incubating for 1h at 37 ℃, washing by PBST for 3 times, and beating to dryness; 50uL of goat anti-mouse IgG secondary antibody marked by HRP diluted by 1:10000 is added into each hole, incubated for 1h at 37 ℃, washed by PBST for 3 times, and patted dry; 100. Mu.L TMB was added to each well and the color development was stopped by adding 2M sulfuric acid for 10min, and OD was measured 450nm And reading the value. The blocking rate was calculated as follows: blocking rate (%) = (1-serum OD to be tested) 450nm ) Negative serum OD450nm . The results showed that the blocking rate decreased with increasing serum dilution, so that the serum dilution ratio of 1/5 was selected for the subsequent experiments (Table 3).
TABLE 3 blocking Rate of different dilutions of CSFV type 2 strain QZ14 challenge serum against monoclonal antibody 6A12
4. Sensitivity test
The sensitivity of blocking ELISA was tested based on the determined SP-E2HZ antigen coating concentration, the test mab 6A12 dilution ratio and the serum dilution ratio.
Pig serum (7, 14, 21, 28) (serum to be tested) harvested on different days after virus challenge of CSFV 2 strain QZ14 and one CSFV standard negative serum are diluted 1:5, 50 uL/hole, incubated for 1h at 37 ℃, washed 3 times with PBST, and dried by beating; adding 50uL of monoclonal antibody 6A12 diluted in a ratio of 1:8000 into each hole, incubating for 1h at 37 ℃, washing by PBST for 3 times, and beating to dryness; 50uL of goat anti-mouse IgG secondary antibody marked by HRP diluted by 1:10000 is added into each hole, incubated for 1h at 37 ℃, washed by PBST for 3 times, and patted dry; 100. Mu.L TMB was added to each well and the color development was stopped by adding 2M sulfuric acid for 10min, and OD was measured 450nm And reading the value. The results showed that the serum antibody blocking rate of QZ14 challenge was as high as 67.8% for 21 days (table 4). The blocking ELISA system was detected at day 21 after the artificial infection of pigs with CSFV type 2 strain QZ 14.
TABLE 4 blocking Rate of monoclonal antibody 6A12 by serum at different time points of challenge with CSFV 2-type strain QZ14
5. Specificity experiments
11 parts of pig serum (4 parts of pig serum of 21 days of virus challenge of CSFV type 2 epidemic strain, 3 parts of pig serum of 28 days of vaccine C strain immunization, 2 parts of BVDV type 1 infected pig serum, 2 parts of BVDV type 2 infected pig serum) and 1 part of CSFV Negative pig serum (Negative serum) are taken and detected by adopting the blocking ELISA system established above. The operation procedure is as follows:
(1) Indirect ELISA
The coating concentration of the antigen corresponding to the serum (CSFV epidemic strain E2, CSFV vaccine strain C2 or BVDV E2) is 5ug/mL, the serum is diluted according to 1:1000, 50 uL/hole, incubated for 1h at 37 ℃, washed 3 times by PBST, and dried by beating; 50uL 1 was added per well: 10000-diluted HRP-labeled rabbit anti-pig IgG secondary antibody is incubated for 1h at 37 ℃, PBST is washed 3 times, and the secondary antibody is dried by beating; 100. Mu.L TMB was added to each well and the color development was stopped by adding 2M sulfuric acid for 10min, and OD was measured 450nm And reading the value. The results showed that the results of indirect ELISA of 11 sera with the corresponding coating antigen showed that these sera were positive sera for the corresponding virus (FIG. 14-A), but could not distinguish between CSFV 2-type epidemic infection serum antibodies, vaccine C-strain immune serum antibodies and BVDV infection serum antibodies.
(2) Blocking ELISA
Serum positive for the indirect ELISA was tested using the blocking ELISA system established above. The results showed that the blocking rate of serum antibodies was greater than 47% for 21 days of challenge with 4 circulating CSFV 2 strains, and less than 13% for 28 days of challenge with 4 BVDV strains with 3 vaccine C strains (fig. 14-B).
7. Kit for blocking ELISA
1. The invention further prepares a blocking ELISA kit for detecting type 2 CSFV infection serum antibody based on the monoclonal antibody 6A12 obtained by preparation, and the components are as follows:
(a) CSFV type 2E 2 eukaryotic expression protein SP-E2HZ and pre-coated ELISA plate (100 ng/well);
(b) A sample diluent;
(c) A negative control;
(d) Hybridoma cell line 6a12 culture supernatant (monoclonal antibody 6a12 secreted);
(e) HRP-labeled goat anti-mouse IgG;
(f) Washing liquid: 0.5% PBST (10 mM PBS+0.5% Tween 20);
(g) Color development liquid: TMB substrate (solution A, solution B);
(h) Stop solution: 2M H 2 SO 4
2. The using method of the blocking ELISA kit comprises the following steps:
(1) All kit components must be returned to room temperature 18-25 ℃ before use. The reagents should be mixed by gentle vortexing.
(2) Diluting serum with sample diluent according to a ratio of 1:5, adding 50uL of each well, setting a compound well and a negative control, incubating for 1h at 37 ℃ or incubating overnight at 4 ℃, washing with 0.5% PBST for 3 times, each time for 5min, and drying by beating;
(3) Diluting the monoclonal antibody 6A12 with a sample diluent 1:8000, adding 50uL of each well, incubating for 1h at 37 ℃, washing with 0.5% PBST 3 times for 5min each time, and drying by beating;
(4) HRP-labeled goat anti-mouse IgG was diluted 1:10000 with sample dilution, 50ul was added per well, incubated for 1h at 37 ℃, washed 3 times with 0.5% pbst for 5min each time, and patted dry;
(5) 100uL of TMB developing solution (equal volume of A solution and B solution are mixed) is added, and the mixture is placed for 10min under the condition of light shielding and room temperature. And (5) timing after the 1 st hole is added.
(6) At 10min, 50uL of stop solution (2M H2SO 4) was added to each well to stop the reaction. The order of adding the stop solution is required to be consistent with the order of adding the color development solution.
(7) The absorbance value of the sample was measured at 450 nm.
(8) The blocking rate of the samples was calculated as follows:
blocking rate (%) = (1-serum OD to be tested) 450nm ) Negative serum OD 450nm 。
(9) And (3) result judgment:
if the blocking rate of the tested sample is greater than or equal to 36%, the sample can be judged as positive, namely the serum contains CSFV 2 type strain antibody;
if the blocking rate of the sample to be tested is less than or equal to 29%, the sample can be judged as negative, namely the serum does not contain CSFV 2-type antibody;
if the blocking rate of the detected sample is 29-36%, the detected sample is judged to be suspicious.
The content of the sequence table is as follows:
SEQ ID NO.1
aagcttgtcgagaagtactagaggatcataatcagccat
SEQ ID NO.2
cccttgtgccccggtcaccagcagcagccatattatac
SEQ ID NO.3
ctgctggtgaccggggcacaagggcggctgtcctgtaaggaagactacag
SEQ ID NO.4
tctagtacttctcgacaagcttttagtgatggtgatggtgatgggtcacgtccagatcaaaccagtat
SEQ ID NO.5
RLSCKEDYRYAISSTNEIGPLGAEGLTTTWKEYNHGLQLDDGTVRAICTAGSFKVIAL
NVVSRRYLASLHKRALPTLVTFELLFDGTSPAIEEMGDDFGFGLCPFDTTPVVKGKYN
TTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFKREKPFPHRVGCVTTVVEK
EDLFYCKWGGNWTCVKGNPVTYMGGQVKQCRWCGFDFKEPDGLPHYPIGKCILAN
ETGYRVVDSTDCNRDGVVISTEGEHECLIGNTTVKVHALDGRLGPMPCRPKEIVSSA
GPVRKTSCTFNYTKTLRNKYYEPRDSYFQQYMLKGEYQYWFDLDVTSEQ ID NO.6
caggtccaactgcagcagtcaggacctgagctggtgaagcctggggcttcagtgaggatatcctgcaaggcttctggctacaccttca
caaactactatatacactgggtgaagcagaggcctggacagggacttgagtggattggatggatttatcctggaaatgttaatactaagt
acaatgagaaattcaagggcaaggccacactgactgcagacaaatcctccagcacagcctacatgcagctcagcagcctgacctctg
aggactctgcggtctatttctgtgcaacctttgatggttactactcttggtttgcttactggggccaagggaccacggtcaccgtctcctcaSEQ ID NO.7
gacattgagctcacccagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatacagggccagcaaaagtgtcagt
acatctggctatagttatatgcactggaaccaacagaaaccaggacagccacccagactcctcatctatcttgtatccaacctagaatctg
gggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaaccta
ttactgtcagcacattagggagcttacacgttcggaggggggaccaagctggaaataaaacgg
Claims (7)
1. a recombinant baculovirus expressed CSFV 2 type E2 protein is characterized in that a cDNA sequence of a coding region of the protein is shown as SEQ ID NO. 5.
2. The method for preparing CSFV 2 type E2 protein according to claim 1, comprising the steps of:
(1) Using cDNA of CSFV 2 strain HZ08 as template, using specific primer shown in SEQ ID NO.3 and SEQ ID NO.4 to make amplification so as to obtain E2 protein coding region cDNA sequence;
(2) Cloning of the E2 protein coding region sequence into the modified pFastBac with secretion Signal Peptide (SP) TM On HTB vector, recombinant plasmid pFastBac is obtained TM HTB-SP-E2HZ; transposing the recombinant plasmid to E.coli DH10Bac competent cells to obtain recombinant Bacmid-SP-E2HZ;
(3) Transfecting the recombinant Bacmid-SP-E2HZ into Sf9 cells to obtain recombinant baculovirus; the recombinant baculovirus expressed CSFV type 2E 2 protein, abbreviated as SP-E2HZ protein, was harvested.
3. The method according to claim 2, wherein BamHI and HindIII are selected as the target gene insertion sites in the step (2), and pFastBac is amplified using the primers shown in SEQ ID NO.1 and SEQ ID NO.2 TM HTB vectors, after linearization, carry secretion signal peptides.
4. A monoclonal antibody for identifying hog cholera virus type 2 infection is characterized in that the heavy chain variable region amino acid cDNA sequence of the monoclonal antibody is shown as SEQ ID NO.6, and the light chain variable region amino acid cDNA sequence is shown as SEQ ID NO. 7.
5. The method for producing a monoclonal antibody according to claim 4, comprising the steps of:
(1) The SP-E2HZ protein of claim 1 is purified and then administered to mice immunized with CSFV 2 strain HZ08 whole virus to achieve mixed immunization;
(2) Preparing a hybridoma cell strain by utilizing a mouse myeloma cell SP2/0 and spleen cells of a mouse after mixed immunization; obtaining a monoclonal cell strain capable of stably secreting a specific antibody through screening and subcloning;
(3) Subtype determination, IFA identification, immunoblotting identification and epitope identification are carried out on the obtained monoclonal antibody, and the monoclonal antibody capable of blocking an ELISA system is screened.
6. A blocking ELISA kit for detecting type 2 CSFV infection serum antibodies comprising the following components:
SP-E2HZ protein and a pre-coated ELISA plate thereof, 100 ng/hole;
a sample diluent;
a negative control;
a culture broth comprising the monoclonal antibody of claim 4;
HRP-labeled goat anti-mouse IgG;
washing liquid: 0.5% PBST with 10mM PBS and 0.5% Tween 20;
color development liquid: TMB substrate comprising solution A and solution B;
stop solution: 2M H 2 SO 4 。
7. The method of using the kit of claim 6, comprising the steps of:
(1) Returning all the components to the room temperature of 18-25 ℃, and uniformly mixing the reagents by vortex;
(2) Diluting a serum sample to be detected by using a sample diluent according to a ratio of 1:5, adding 50uL of the sample diluent into each hole of a culture plate, and setting a compound hole and a negative control; after incubation at 37℃for 1h or at 4℃overnight, 0.5% PBST was washed 3 times, 5min each time, and patted dry;
(3) Diluting the monoclonal antibody with a sample diluent at a ratio of 1:8000, adding 50uL of the monoclonal antibody into each hole, and incubating for 1h at 37 ℃; washing with 0.5% PBST for 3 times and 5min each time, and drying;
(4) Diluting HRP-labeled goat anti-mouse IgG with sample diluent at 1:10000, adding 50uL per well, and incubating at 37 ℃ for 1h; washing with 0.5% PBST for 3 times and 5min each time, and drying;
(5) Adding 100uL of TMB developing solution which is prepared at present, and mixing the solution A and the solution B in equal volume; placing for 10min at room temperature in dark place, and starting timing after the 1 st hole is added;
(6) Adding 50uL of stop solution into each reaction hole at 10min to stop the reaction, wherein the sequence of adding the stop solution is consistent with that of adding the color development solution;
(7) Measuring the absorbance value of the sample at 450 nm;
(8) The blocking rate of the serum sample to be tested is calculated according to the following formula:
blocking rate (%) = (1-serum OD to be tested) 450nm ) Negative serum OD 450nm ；
(9) And (3) result judgment:
if the blocking rate of the detected sample is more than or equal to 36%, judging that the detected sample is positive, namely that the serum contains CSFV 2-type strain antibodies;
if the blocking rate of the tested sample is less than or equal to 29%, the tested sample is judged to be negative, namely the serum does not contain CSFV 2 type strain antibody;
if the blocking rate of the detected sample is 29-36%, the detected sample is judged to be suspicious.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294147A (en) * | 2008-05-30 | 2008-10-29 | 中国农业科学院哈尔滨兽医研究所 | Monoclone antibody of swine fever virus resistant wild strain E2 protein, preparation method and application thereof |
| KR101652962B1 (en) * | 2016-04-15 | 2016-09-02 | 대한민국 | A Kit Using Antibodies for Differentiating Recombinant CSFV Vaccinated Swine and Wild Type CSFV Infected Swine, and Differentiating Method Using Thereof |
| CN107557344A (en) * | 2017-08-23 | 2018-01-09 | 广州伯尼兹生物科技有限公司 | One plant of expression CSFV E 2 protein monoclonal antibody hybridoma cell line CSFV 3H3G6 and antibody and kit |
| CN113354740A (en) * | 2021-02-22 | 2021-09-07 | 浙江大学 | Classical swine fever virus self-assembly protein nanoparticle, preparation method and application |
| CN114874995A (en) * | 2022-03-27 | 2022-08-09 | 浙江大学 | Classical swine fever virus type 2E rns Monoclonal antibody hybridoma cell strain of protein and application |
-
2023
- 2023-04-17 CN CN202310408644.3A patent/CN116554282A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294147A (en) * | 2008-05-30 | 2008-10-29 | 中国农业科学院哈尔滨兽医研究所 | Monoclone antibody of swine fever virus resistant wild strain E2 protein, preparation method and application thereof |
| KR101652962B1 (en) * | 2016-04-15 | 2016-09-02 | 대한민국 | A Kit Using Antibodies for Differentiating Recombinant CSFV Vaccinated Swine and Wild Type CSFV Infected Swine, and Differentiating Method Using Thereof |
| CN107557344A (en) * | 2017-08-23 | 2018-01-09 | 广州伯尼兹生物科技有限公司 | One plant of expression CSFV E 2 protein monoclonal antibody hybridoma cell line CSFV 3H3G6 and antibody and kit |
| CN113354740A (en) * | 2021-02-22 | 2021-09-07 | 浙江大学 | Classical swine fever virus self-assembly protein nanoparticle, preparation method and application |
| CN114874995A (en) * | 2022-03-27 | 2022-08-09 | 浙江大学 | Classical swine fever virus type 2E rns Monoclonal antibody hybridoma cell strain of protein and application |
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