CN110452884B - anti-BCoV-N protein monoclonal antibody hybridoma cell line, antibody and application - Google Patents
anti-BCoV-N protein monoclonal antibody hybridoma cell line, antibody and application Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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Abstract
The invention relates to an anti-BCoV-N protein monoclonal antibody hybridoma cell strain which is generated by cell fusion of splenocytes of mice and SP2/0 cells, and the preservation number of the strain is CGMCC No. 17693. The invention also relates to a monoclonal antibody secreted by the anti-BCoV-N protein monoclonal antibody hybridoma cell strain, and a preparation method and application of the monoclonal antibody. The monoclonal antibody prepared by the invention can perform specific reaction with BCoV in Western blot and IFA experiments, and the specific experiment result shows that the monoclonal antibody has no cross reaction with common bovine viruses such as BRV, BVDV, IBRV, BPIV3 and the like, which indicates that the monoclonal antibody has better affinity with active BCoV virus particles, and further proves that the antibodies have important commercial value in the aspects of preparing BCoV antigen diagnostic kits and basic research.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to an anti-BCoV-N protein monoclonal antibody hybridoma cell line, an antibody and application thereof.
Background
Bovine Coronavirus (BCoV) belongs to the order of the nested virus (Nidovirales), the genus Coronaviridae (Coronaviridae), a pleomorphic, enveloped, single-stranded, plus-strand RNA virus. BCoV is an important source of respiratory tract infection of cows, newborn calf diarrhea and adult bovine winter diarrhea, from 1972, BCoV is reported by a plurality of countries and is distributed worldwide, the milk production performance of cows is influenced, the growth, development and production performance of calves after the calves are cured are influenced, BCoV is mainly transmitted by two ways of digestive tract and respiratory tract, infected cows expel toxin at a specific time to infect flocks, and no effective vaccine exists in China at present, so that early diagnosis has important significance for preventing and controlling the disease.
BCoV particles share 5 major structural proteins: nucleocapsid protein N, envelope protein M, fiber protein S, small membrane protein E and hemagglutinin lipase glycoprotein HE, nucleocapsid protein (N) containing 3 structural domains is an alkaline phosphoprotein, plays a role in coronavirus RNA synthesis, and has a hydrophobic region with better immunogenicity of N protein predicted by DNAStar software, and high conservation, and the obtained recombinant protein is soluble expression, and is an active protein obtained by purifying under a nickel column natural condition, has better affinity and is easy to combine with an antibody. However, the biggest disadvantage of using E.coli to express recombinant proteins to prepare monoclonal antibodies is that the prepared monoclonal antibodies cannot recognize viral particles because of the easy occurrence of antigen conformation changes.
Hybridoma technology has been a well established technology since the 70's in the 20 th century. The test period from the preparation of the antigen to the freezing of the hybridoma cells is about 5 months on the premise that the experiment can be carried out smoothly. The experimental period is long, the method is a comprehensive experimental technology, relates to the steps of mouse immunization, cell culture and the like, and is influenced by various experimental factors. In conclusion, the application of hybridoma technology to the production of anti-BCoV-N protein monoclonal antibodies has not been reported.
Disclosure of Invention
The invention aims to provide an anti-BCoV-N protein monoclonal antibody hybridoma cell line, wherein the preservation number of the strain is CGMCC No. 17693.
The second purpose of the invention is to provide the monoclonal antibody secreted by the anti-BCoV-N protein monoclonal antibody hybridoma cell strain.
The third purpose of the invention is to provide a preparation method of the monoclonal antibody.
The fourth purpose of the invention is to provide the application of the monoclonal antibody.
The invention is realized by the following technical scheme:
a monoclonal antibody hybridoma cell strain for resisting BCoV-N protein is generated by cell fusion of splenocytes of mice and SP2/0 cells, and the preservation number of the strain is CGMCC No. 17693.
And II, the monoclonal antibody secreted by the anti-BCoV-N protein monoclonal antibody hybridoma cell strain.
Thirdly, the preparation method of the monoclonal antibody comprises the following steps:
(1) the constructed pET-32a-BCoV-N recombinant strain is induced to express to generate pET-32a-BCoV-N protein;
(2) the pET-32a-BCoV-N protein is soluble protein and is used as antigen to immunize a mouse;
(3) obtaining splenocytes of the immunized mice, and fusing the splenocytes with SP2/0 cells to generate hybridoma cells;
(4) the hybridoma cells were injected into mice to generate monoclonal antibodies against the BCoV-N protein.
And fourthly, the application of the monoclonal antibody in resisting bovine coronavirus.
Adopt above-mentioned technical scheme's positive effect: the invention uses DNAStar software to predict the main antigen epitope of the N protein of the BCoV-CD strain, prepares the recombinant BCoV-N protein containing the epitope by a prokaryotic expression technology, can perform soluble expression, and obtains the BCoV-N protein with natural activity by nickel column purification.
Drawings
FIG. 1 shows the result of the purification of pET-32a-BCoV-N protein according to the present invention;
FIG. 2 shows the screening results of the positive hybridoma cell lines;
FIG. 3 shows the ascites method of the present invention for producing antibodies;
FIG. 4 shows the result of Western blot identification according to the present invention;
FIG. 5 shows the result of the identification of the ascites monoclonal antibody IFA of the present invention;
FIG. 6 shows the result of specific identification of IFA by the monoclonal antibody of the present invention;
FIG. 7 shows the chromosome number identification (1000X) of the monoclonal antibody of the present invention.
The hybridoma cell 2F9 of the invention is preserved in patent programs in 27.5.2019 in China patent office or the preservation center approved by the International patent organization, the preservation unit is totally called China general microbiological culture Collection center (CGMCC), the preservation unit address is as follows: the No. 3 Xilu Beijing province of Chaoyang, the preservation number is CGMCC No. 17693.
Detailed Description
Sources of the biological material in the present invention:
1. bovine coronavirus (Bovine coronavirus, BCoV) CD strain: the sequence analysis and prokaryotic expression of the bovine coronavirus S gene, high Guobao, Wangming, Liuming and the like, Chinese veterinary medicine for animal husbandry, No. 7 of volume 45 in 2018, and 1740 page 1749.
2. Bovine Rotavirus (Bovine Rotavirus, BRV) DQ strain: the isolation and genotype identification of the new-born bovine rotavirus, Xijinxin, Houxinlin, Donghuxing, etc., the university of eight agricultural cultivations, Vol.22, No. 5, page 56-59, 2010.
3. Bovine parainfluenza virus type 3 (Bovine parainfluenza virus, BPIV3) HJ-1 strain: the separation and identification of bovine parainfluenza virus type 3 and the research of the growth and death rule of infected bovine antibodies, Zhouyilong, Wu Hao, Nippon Gao, and the like, the Chinese veterinary Co-morbidity proceedings, No.1 of volume 27 in 2011, and pages 23-28.
4. Bovine Viral Diarrhea Virus (BVDV) strain HJ-1: the separation and identification of bovine viral diarrhea virus Heilongjiang isolate, Zhangzhouhua, Wudan Dan, Zhouyilong, etc., Chinese veterinary medical report for prevention, No. 10 of 37 vol.2015, page 805-807.
5. IBRV DQ strain: cloning and sequence analysis of gI gene of IBRV DQ isolate, Wang, Zhouyilong, Lizhou army, Hou Xilin, Pravanci, animal medicine progress, 9 th of 2007, pages 23-25.
6. BCoV DQ strain: the isolation and identification of bovine coronavirus, Guanxin, puvavan, Hou Xilin, university of eight agricultural reclamation colleges of Heilongjiang, 2 nd 2007, pages 55-58.
7. S/P20 cells, MDBK (bovine kidney cells), Vero (Vero green monkey kidney cells) and HRT-18 (human rectal cancer cells) were purchased from Shanghai cell Bank, Chinese academy of sciences;
8. BCoVN protein recombinant Escherichia coli (pET-32 a-N): an ELISA detection kit for bovine coronavirus, patent numbers: 201810424384.8, filing date: year 2018, month 5, day 7, publication No.: CN108318686A, published: 24/7/2018.
Example 1
Example 1 illustrates purification and concentration determination of recombinant BCoV N protein:
carrying out induction expression on the constructed pET-32a-BCoV-N recombinant bacteria, centrifuging at 12000r/min for 5min, collecting bacteria, carrying out ultrasonic disruption on the bacteria liquid, separating supernatant, purifying N protein by using Ni-NTA His-Bind resin, and carrying out concentration determination on the purified pET-32a-BCoV-N protein according to the method of the BCA kit instruction.
pET-32a-BCoV-N protein is purified according to Ni-NTA His-Bind resin under natural condition, imidazole elution buffer solutions with different concentrations are adopted to elute target protein, and SDS-PAGE gel analysis shows that the elution efficiency is the largest when the imidazole concentration is 20mM, and the recovery efficiency is the highest when elution is carried out for 2-4 times, which is shown in figure 1, M: low relative molecular mass protein Marker; 1: supernatant of the bacterial liquid; 2-9: the elution times of 20mM imidazole are from 1 to 9 times.
Example 2
Example 2 illustrates BALB/c murine immunization:
the purified recombinant pET-32a-BCoV-N protein is used as an antigen, and a foot pad immunization method is applied to immunize female BALB/c mice with age of 6-8 weeks, wherein the process is as follows: adjusting the concentration of pET-32a-BCoV-N protein to 200 mug/mL, mixing and emulsifying with equivalent Freund complete adjuvant, taking 100 mug of emulsion to immunize a foot pad, immunizing 50 mug of the two rear foot pads respectively, immunizing a mouse for two times after one week, taking 200 mug/mL pET-32a-BCoV-N protein to mix and emulsify with equivalent Freund incomplete adjuvant, taking 100 mug of emulsion to immunize the mouse with the foot pad, immunizing the two rear foot pads respectively for 50 mug, immunizing the same with the four-way immunization method as the two-way immunization, detecting the serum titer of the mouse by established indirect ELISA until the serum titer is more than 8000 times, directly injecting 100 mug of pET-32a-BCoV-N protein into the abdominal cavity of the mouse, strengthening immunization and carrying out cell fusion within one week.
Example 3
Example 3 illustrates hybridoma cell line establishment:
1. preparation of SP2/0 cells:
about 2 weeks before the fusion, the frozen SP2/0 cells were thawed and cultured in a medium containing 20% fetal calf serum at 37 ℃ with 5% CO2Culturing in a cell culture box, carrying out subculture of SP2/0 cells for 12h before fusion, gently blowing down the cells with RPMI-1640 during fusion, centrifuging for 5min at 1000r/min, repeating for 2 times, and counting the cells for later use.
2. Preparation of a breeding layer:
taking 1 healthy BALB/c mouse, cutting off the neck, completely soaking and disinfecting in ethanol, fixing a pin on an anatomical plate, lifting the skin of the abdomen of the mouse by using forceps, cutting off the skin by using scissors, completely tearing the skin of the mouse by using the forceps to expose a large area, wiping and disinfecting the skin by using alcohol cotton, sucking RPMI1640 culture solution containing 20% fetal calf serum and 50 XHAT by using a 5mL disposable sterile injector, injecting the RPMI1640 culture solution into the abdominal cavity of the mouse (taking care of avoiding pricking the intestinal tract), keeping the position of the pin still, slightly kneading and massaging the abdomen of the mouse by using a hemostatic forceps with an alcohol cotton ball, slowly extracting the liquid in the abdominal cavity, injecting the liquid into a sterile culture dish, repeating the steps for 3-5 times to suck out macrophages in the abdominal cavity of the mouse as much as possible, preparing 50mL RPMI1640 culture solution containing 20% fetal calf serum and 1 XHAT together, the aspirated macrophages were mixed well and applied to a multi-channel pipette at 100. mu.L/wellThe amount of the extract is added into a 96-well cell culture plate, 4-5 cell culture plates can be fully paved, and then the cell culture plate is placed at 37 ℃ and 5% CO2Observing the growth state of the cells after about 18-24 h after the culture in the incubator, and if the cells are not polluted and adhere tightly, using the cells as feeder layer cells.
3. Popliteal lymph node cells and spleen cell preparation:
removing eyeballs of a BALB/c mouse which is immunized for 3d, collecting whole blood in a 1.5m L centrifugal tube, standing overnight at 4 ℃, obtaining positive serum as separated serum, killing the mouse due to dislocation of cervical vertebrae after the eyeballs are picked, soaking and disinfecting the mouse in 75% alcohol for 5min, placing the mouse in a super clean bench of a biological safety cabinet which is sterilized by ultraviolet for 30min, fixing the mouse on a foam board, and performing the following operations in the biological safety cabinet: lifting the skin of the abdomen of the mouse by using forceps sterilized by high temperature and high pressure with one hand, carefully shearing an inverted triangle opening by taking an ophthalmic scissors sterilized by high temperature and high pressure with the other hand, blunt-tearing the skin of the abdomen of the mouse to completely expose the peritoneum, carefully shearing the abdomen of the mouse, aseptically taking out the whole spleen of the mouse, placing the whole spleen of the mouse into an aseptic flat dish containing a serum-free RPMI1640 basic culture medium, taking a 1mL syringe to suck the culture medium, gently puncturing and slowly washing and blowing the spleen, removing the spleen after the color of the spleen is changed from deep red to light red, gently tearing the skin on the inner side of the thigh by a scissors, taking out the popliteal lymph node, placing the popliteal lymph node into the culture dish, cutting the skin on the left side of the abdomen and chest of the mouse by a scissors, taking out the spleen, placing the popliteal lymph node into a sterilized 200-mesh copper net, gently rolling the popliteal lymph node by using a 5mL rubber plug syringe, then removing the copper net and residual tissue fragments, after the popliteal lymph node cells and the spleen cells were mixed well, the mixture was centrifuged at 1000r/min for 5min, and the centrifugation was repeated 2 times while counting the cells.
4. Cell fusion:
SP2/0 cells were mixed with splenocytes at a ratio of 1: 8 (SP2/0 cells and popliteal lymphocytes in a ratio of 1: 5), centrifuging for 5min at 1000r/min, discarding the supernatant, centrifuging the mixed cells again at 1000r/min for 5min, sucking the supernatant with a rubber-tipped dropper, flicking the cells at the bottom of the centrifuge tube with fingers, scattering, mixing, and adding into a 500mL beakerImmersing a centrifugal tube into warm water with 400mL of sterile distilled water with the water temperature of 38 ℃, standing in a water bath for 2-3 min, sucking PEG 1450750 muL preheated at 37 ℃, slowly adding along the wall of the centrifugal tube for 1min, gently blowing up cells for several times, continuously standing in the water bath for 30s, sucking 1640 basic culture medium by a rubber head dropper, slowly adding along the wall of the centrifugal tube for 1min, adding 3mL within 1min, then slowly adding 30mL1640 basic culture medium within 4min, centrifuging for 4min, discarding supernatant, slowly purging the cells by using 30mL basic culture medium, centrifuging for 4min, discarding supernatant, adding 50mL 20% serum culture medium and 1mL HAT into a culture dish, uniformly mixing, slowly blowing up the uniformly mixed cells, transferring into the culture dish, slowly adding the cells into a 96 pore plate containing feeder layer cells, wherein each pore is 125 muL, and finally, standing at 37 deg.C for 5% CO2Cultured in an incubator.
PEG4000 and PEG1450 are commonly used for cell fusion, the PEG4000 has larger molecular weight, the cell loses water faster, the fusion is more violent, the action time is shorter, but the damage to the cell is larger; PEG1450 has smaller molecular weight, slower cell dehydration process, milder fusion process, longer action time, easy control of operation process and less damage to cells, so PEG145 is selected as a cell fusion agent in the invention.
5. Culturing the fusion cells:
after the fusion, 20% serum medium containing HAT can be added as appropriate depending on the number and state of fused cells at 5d, 8d and 11 d. And (3) carrying out ELISA detection and screening of positive hybridoma cell strains secreting the antibody when hybridoma cells in a 96-well cell culture plate grow to about 1/4 at the bottom after fusion at 7-10d, further carrying out subcloning and screening of the positive hybridoma cell strains stably secreting the antibody, wherein after 4 times of subcloning, all cloned cells are distributed in a single colony and the positive detection rate is about 100%, thus obtaining 5 positive hybridoma cell strains relatively stably secreting the specific monoclonal antibody, and respectively naming the 5 obtained monoclonal antibodies as 2F9, 2E3, 7A1, dC4 and cB 9.
6. Cloning of positive hybridoma cell lines:
cloning positive hybridoma cells by adopting a limiting dilution method, preparing feeder layer cells before cloning, slightly blowing up the hybridoma cells to be cloned in original holes to form cell suspension by the same method, ten times diluting, the cells were counted, diluted with 1 × HT and 20% serum medium, and added to 96-well plates containing feeder layer cells, adding 1.5 clones/well, observing with microscope after cell pile appears after 7 days, marking clone number, simultaneously, changing a proper amount of liquid, screening positive cell strains again by using an indirect ELISA method, screening the positive cell strains with good specificity, high titer and good growth state, and (4) cloning the positive cells for multiple times in the same way until all cell stacks are detected to be positive after cloning, stopping cloning, and transferring the positive cell strains to a 6-well plate for amplification culture.
7. Freezing and storing the hybridoma cells:
selecting cells in logarithmic growth phase, discarding supernatant, slightly blowing up the cells by using a basic culture medium to form cell suspension, counting the cells of the suspension, transferring the suspension into a 15mL centrifuge tube, centrifuging the suspension for 5min at the speed of 1000r/min, discarding supernatant, and mixing serum and DMSO according to the ratio of 9: 1, slowly adding into a centrifuge tube, and slightly blowing to form cell suspension to make the cell concentration reach 1 × 106Adding into a freezing tube, wherein each tube is 0.8mL, marking, sealing, placing the freezing tube at 4 ℃, 1h, -20 ℃, 1h, -80 ℃ overnight, and placing in liquid nitrogen for long-term storage the next day.
8. And (3) recovering the cells:
rapidly taking out the freezing tube from liquid nitrogen, placing in 42 deg.C warm water, rapidly shaking to rapidly melt the freezing solution, sterilizing 75% ethanol surface, centrifuging for 5min at 1000r/min, discarding supernatant, adding 1mL basal medium, transferring the lightly suspended cells to 5mL 20% serum culture medium cell bottle, 37 deg.C, 5% CO2Cultured in an incubator.
Example 4
Example 4 illustrates monoclonal antibody preparation:
1. the ascites method produces a large amount of antibodies:
postpartum female BALB/c miceSeveral mice were injected with 0.5mL of sterilized liquid paraffin into each abdominal cavity, gently rubbing the abdominal parts for 2min, and one week later, positive hybridoma cells (each 5X 10/one) were injected into the abdominal cavity of the mice5) After several days, the abdominal cavity of the mouse is obviously enlarged, and after 14 days, until the abdominal cavity of the mouse is swollen and slow to move, a 20mL syringe needle is taken to puncture the abdominal cavity slightly, ascites is collected and placed at 37 ℃ for 1h, 10000 r/min is taken out, centrifugation is carried out for 10min, and the supernatant is taken out and stored at-80 ℃.
2F9, 2E3, 7A1, dC4 and cB9 hybridoma cell lines were selected, BALB/c-line white mice sensitized with Freund's complete adjuvant were intraperitoneally inoculated, and the abdomen of mice around 14d was extremely enlarged and listened, at which time ascites were collected, as shown in FIG. 3, A: inoculating 13d mice, and obviously enlarging the abdomen, which is 3-5 times of that of the control mice; B. c: ascites fluid was collected.
2. Determination of monoclonal antibody titer:
the ascites is diluted from 1 ten thousand times to 2 times to 512 ten thousand times, added into an enzyme label plate, and applied with an indirect ELISA method established by taking pET-32a-BCoV-N protein as antigen, negative ascites is taken as a control, immune mouse serum is taken as a positive control, the ratio of the OD value of the parallel diluted monoclonal antibody to the OD value of the negative control is more than the maximum dilution multiple of 2.1, and the maximum dilution multiple is the titer of the antibody.
According to the established ELISA method, cell culture supernatant of 5 positive hybridoma cell lines and antibody titer of prepared mouse ascites are determined, the cell culture supernatant is diluted by 2 times, the prepared ascites is diluted by 10 times, and the measured antibody titer result shows that the titer of the culture cell supernatant and the ascites of 2F9 are the highest and respectively reach 1: 32000 and 1: 109The specific results are shown in table 1:
TABLE 1 hybridoma cell culture supernatants and ascites antibody titers prepared
Test example 1
Test example 1 illustrates the immunological activity and specificity of the monoclonal antibodies:
1. western blot identification:
preparing full monolayer HRT-18 cells, inoculating BCoVDQ strain virus to six-well plate, placing at 37 deg.C and 5% CO2Incubating for 1h in a cell constant-temperature incubator, discarding the supernatant, continuously culturing to 2mL of DMEM containing 0.25% of pancreatin while setting an untreated group as a blank control, pouring out the culture solution when cytopathic effect (CPE) reaches 50%, washing twice with PBS, adding 150 mu L of cell lysate, cracking on ice for 30min, centrifuging at 12000rpm at 4 ℃ for 5min, collecting the supernatant, sucking out 80 mu L of the supernatant to 1.5mL of a centrifuge tube, adding 20 mu L of 5 multiplied protein loading buffer solution, boiling in boiling water for 5min, preserving at-20 ℃ for later use, performing SDS-PAGE electrophoresis, transferring the product to a PVDF membrane, taking the prepared hybridoma ascites monoclonal antibody as a primary antibody, and performing Western blot identification.
Western blot results showed that BCoV-infected HRT-18 cells reacted specifically with 7A1, 2E3, cB9, dC4 and 2F9, producing immunoblots at 55kDa, whereas the control group did not show immunoblots, as shown in FIG. 4, indicating that the monoclonal antibodies produced were able to react specifically with BCoV CD strain.
2. Indirect Immunofluorescence (IFA) assay:
HRT-18 cells infected with BCoV DQ 3d and uninfected cells are used as controls, prepared hybridoma cell ascites monoclonal antibody is used as a primary antibody, DyLight594 labeled goat anti-mouse IgG is used as a secondary antibody, IFA test is carried out, meanwhile, common bovine viruses BRV, BVDV, IBRV, BPIV3, BRSV and the like are selected for specific test, positive serum or monoclonal antibody of corresponding virus is used as a control, the secondary antibody is FITC labeled anti-bovine IgG, BRV infects MA104 cells, and other viruses infect MDBK cells.
The monoclonal antibodies 7A1, 2E3, cB9, dC4 and 2F9 all reacted with BCoV-infected HRT-18 cells to generate red fluorescence, while the uninfected control group did not show red fluorescence, and the negative control group only included one group of photographs, and all other antibodies were similar to the photographs and did not have red fluorescence, as shown in FIG. 5.
7A1, 2E3, cB9, dC4 and 2F9 as primary antibodies were all unable to react with BVDV, BPIV3 and IBDV infected MDBK cells, and BRV infected MA104 cells to generate immunofluorescence, while the corresponding positive sera were able to generate immunofluorescence, as shown in FIG. 6.
Test example 2
Test example 2 illustrates the identification of monoclonal antibody subclasses:
detecting the antibody subclass by using an antibody subclass detection kit, wherein the antibody subclass comprises IgG1, IgG2a, IgG2b, IgG 3, IgG A and IgG M according to SBaclonotypingTMSystem/HRP antibody subclass identification kit instruction (can be in the cell screening process for identification).
The results show that the light chains of the 5 monoclonal antibodies are all Kappa chains, the heavy chains of 2E3 and 2F9 are IgG2a, and the heavy chains of cB9, dC4 and 7A1 are IgG1, and the specific results are shown in Table 2:
TABLE 2 identification of the subclass of BCoVN protein monoclonal antibodies
Test example 3
Experimental example 3 illustrates the identification of chromosome number of hybridoma cells:
the chromosome counting adopts a colchicine method, and comprises the following steps:
(1) taking out the hybridoma cells and the SP2/0 cells from the culture box;
(2) adding 0.05mL of colchicine with concentration of 20 μ g/mL into each bottle of cells to make the final concentration reach 0.1 μ g/mL, placing into a cell culture box for culturing for 2.5h to stop cell chromosome division at metaphase;
(3) the cells were gently blown down and added to a centrifuge tube, centrifuged at 1000r/min for 5min and the supernatant discarded. 2mL of KCl solution with the concentration of 0.075mol/mL is sucked by a pipette and then resuspended and precipitated, and the precipitate is put into a water bath kettle with the temperature of 37 ℃ for 30 min;
(4) adding 1mL of cell fixing solution (the volume ratio of methanol to glacial acetic acid is 3:1), pre-fixing for 2-3 min, centrifuging for 5min at 1000r/min, and removing supernatant. Then adding 1mL of cell fixing solution, fixing at room temperature for 2-3 min, and centrifuging at 1000r/min for 5 min. Repeating the fixing step once;
(5) the cell sediment is made into suspension by using a fixing solution and used for smear, and about 200 to 500 mu L of the suspension is generally added. The volume of the added fixed liquid is determined according to the amount of the cell sediment, and the fixed liquid can be used for selecting suspensions with multiple concentrations with the best effect;
(6) dropping 1-2 drops of cell suspension on the glass slide, repeatedly wiping the glass slide for several times before use, drying the glass slide, and precooling for 10 min. Dyeing is carried out by using Giemsa dyeing liquid, and the dyeing time is 15-20 min generally. Finally, washing with distilled water for 1-2 times, and airing at room temperature;
(7) 10 cells of each of the hybridoma cell and the SP2/0 cell are respectively selected to ensure that chromosomes are well dispersed, complete in shape, free of overlapping and loss, and observed under a microscope. Chromosomes were counted and the average of the number of chromosomes was found.
Chromosome counting of the heteropositive hybridoma cells by a colchicine method shows that chromosomes of all hybridoma cell lines are uniformly distributed, each chromosome is clear, and the number of chromosomes of the hybridoma cell lines is close to the sum of the number of chromosomes of myeloma cells (55-65) and mouse spleen cells (40) and is between 95 and 105, as shown in figure 7.
Test example 4
Experimental example 4 demonstrates the identification of the stability of antibody secretion by hybridoma cells:
hybridoma cell lines 7A1, 2E3, cB9, dC4 and 2F9 are continuously passaged for 3 months, the frozen hybridoma cells are recovered after 3 months, the growth state of the cells is adjusted, when the state is good, cell supernatants are collected, and the titer of the hybridoma cell supernatants is respectively detected by using an indirect ELISA method. The antibody titer is compared to determine the stability of the antibody secreted by the hybridoma cell strain, the result is shown in table 3, and the result shows that the titer of the antibody secreted before and after cryopreservation has no significant change, which indicates that the hybridoma cell strain has good stability.
TABLE 3 identification results of stability of antibody secreted by positive hybridoma cells
Test example 5
Test example 5 illustrates epitope analysis for monoclonal antibodies:
the epitope of the monoclonal antibody was analyzed by a superimposed ELISA method using monoclonal antibodies 2F9, 2E3, dC4, 7a1 and cB9 as the first incubation antibodies, and 2F9, 2E3, dC4, 7a1 and cB9 as the second incubation antibodies, respectively, for 3 replicates per experiment, as follows:
(1) diluting BCoV-N protein by using a carbonate buffer solution with the pH of 9.6 to coat an ELISA plate, wherein 100 mu L/hole (the antigen concentration is 2.5 mu g/mL) is coated for 1h at 37 ℃;
(2) washing with PBST for 3 times, adding 200 μ L1% gelatin into each well for sealing, and sealing at 37 deg.C for 0.5 h;
(3) washing the plate 3 times with PBST, diluting a monoclonal antibody hybridoma cell culture supernatant (2F9, 2E3, dC4, 7A1 and cB9) with PBST according to the proportion of 1/10, 100 mu L/well, and incubating at 37 ℃ for 1.5 h;
(4) adding another monoclonal antibody supernatant (2F9, 2E3, dC4, 7A1 and cB9) (1/10 dilution), acting for 1.5h at 37 ℃, and washing 3 times by PBST;
(5) washing the plate for 3 times, diluting the goat anti-mouse IgG labeled with HRP according to 1/5000, incubating at 100 mu L/hole for 1h at 37 ℃;
(6) washing the plate for 3 times, adding substrate color development solution TMB, 100 μ L/hole, and developing at room temperature in dark for 10 min;
(7) the reaction was finally stopped by adding 2mol/L sulfuric acid, 50. mu.L/well and the OD450nm value was determined, which was verified when P/N > 2.0.
(8) As a result, AI ═ A1.2-A1/A2X 100%
A1 and A2 are OD values of the single monoclonal antibodies, A1.2 is OD value of the superposed monoclonal antibodies, and the judgment standard is as follows: AI > 50% indicates that the recognition antigenic sites are different, AI < 50% are site-similar or identical.
The epitope of the monoclonal antibody was analyzed by the overlay ELISA method, and the specific results are shown in tables 4 and 5:
TABLE 4 OD of monoclonal antibody overlay ELISA450Value of
TABLE 5 AI values of monoclonal antibody overlay ELISA
The results show that 2F9 superimposed with 2E3 and dC4, respectively, both had AI values > 50% and recognized epitopes different from one another, while 2E3 and dC4 recognized epitopes not different.
In summary, the following steps: the 5 anti-BCoV-N protein hybridoma cell strains successfully constructed by the research can effectively identify BCoV particles with infection activity and have strong specificity, wherein the best effect is achieved by using the 2F9 hybridoma cell strain, and a material basis is laid for preparing a BCoV antigen detection kit in the next step.
Claims (2)
1. An anti-BCoV-N protein monoclonal antibody hybridoma cell strain is characterized in that: is generated by cell fusion of mouse spleen cells and SP2/0 cells, and the preservation number of the hybridoma cell strain is CGMCC No. 17693.
2. The monoclonal antibody secreted by the anti-BCoV-N protein monoclonal antibody hybridoma cell strain of claim 1.
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| US5672350A (en) * | 1989-08-22 | 1997-09-30 | Veterinary Infectious Disease Organization | Recombinant bovine coronavirus E2 and E3 polypeptides and vaccines |
| CN108318686A (en) * | 2018-05-07 | 2018-07-24 | 黑龙江八农垦大学 | A kind of bovine coronavirus ELISA detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5672350A (en) * | 1989-08-22 | 1997-09-30 | Veterinary Infectious Disease Organization | Recombinant bovine coronavirus E2 and E3 polypeptides and vaccines |
| CN108318686A (en) * | 2018-05-07 | 2018-07-24 | 黑龙江八农垦大学 | A kind of bovine coronavirus ELISA detection kit |
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Effective date of registration: 20231228 Address after: 201210 No. 39, Lane 100, Banxia Road, Zhoupu Town, Pudong New Area, Shanghai Patentee after: Shanghai Maibang Biotechnology Co.,Ltd. Address before: No.5 Xinfeng Road, high tech Development Zone, Daqing City, Heilongjiang Province, 163000 Patentee before: HEILONGJIANG BAYI AGRICULTURAL University |