TWI260347B - Monoclonal antibody of porcine endogenous retrovirus (PERV) capsid, hybridoma generating the antibody, and use thereof - Google Patents

Abstract

The present invention provides a monoclonal antibody having specificity to porcine endogenous retrovirus (PERV) capsid, a hybridoma generating the antibody, a method for using the monoclonal antibody to test PERV, a method for producing the hybridoma, and a test kit and a pharmaceutical composition containing the monoclonal antibody.

Description

1260347 玖Invention Description: [Technical Field] The present invention relates to a monoclonal antibody against a porcine endogenous retrovirus ('PERV) shell protein, and the use of the antibody, The invention also relates to a fusion tumor producing the monoclonal antibody, and a method of producing the same. [Prior Art] Organ migration is important in human woven science. Because the human body is not easy to obtain, and the feeding of other close relatives, such as the face, makes the hot source become a transplant problem. . Because the pig's eunuch and human organs are the closest in size and function, they are increasingly valued in the consideration of the source of the Yiguan transplant; however, the human and animal common cause, especially the virus, may be infected by the animal organ transplantation process. The transplanted patients have formed a potential crisis that can not be ignored in organ transplant surgery. The newly discovered new pig virus, porcine end-tropic retrovirus (PERV), has gradually received attention and triggered widespread discuss. Porcine endogenous retroviruses are currently divided into three types, namely type A, type B and type c, the main difference being in their surface antigens, which may affect the tissue affinity of the virus. Almost all vertebrate animals have similar primary viruses, but most of them are genetically deficient and mostly non-pathogenic; the vast majority of infectious agents can be bred without specific pathogens (specific pathogens) Free, referred to as SPF) animal individuals to prevent, but naturally exist in the animal genome (Genome) or embryonic infection of the pathogen, although the original host will not cause any disease ' governance but can stretch in the recipient of xenogeneic organs (xen 〇graft recipient) is activated in the body to produce lesions, especially in organ transplant recipients treated with immunosuppressive drugs. The immune function provides effective immune protection. These endogenous retroviruses may be Regeneration in immunosuppressed recipients increases the risk of xenogeneic (pig) organ transplant surgery. For example, Patience et al. and Wilson et al. successfully induced PERV 'release from pig kidney cell line (PK-15 cell line) and primary peripheral blood mononuclear cells, respectively, and confirmed it in vitro. 1260347 human cell lines infected with B cells, T cells and kidney cells (Patience et al., 1997, Nature Med. 3: 282-286; and Wilson et al., 1998, J. Virol 72(4): 3082-3087 In addition, PERV is highly similar to other known reverse prions, such as: feline leukemia virus (FeLV) or murine leukemia virus (MuLV), which are known. The virus has a severe effect on the infected host to induce tumor production or to reduce immune function. (Specke V· et al., 2002, Transpl. Immunol. 9(2-4): 281 -8) At present, most methods for detecting various types of PERV are carried out by means of nucleic acid detection, and related nucleic acid detection techniques have been disclosed in W0. 97/40167, W0 98/53104, W0 00/00829, W0 00/0419, WO 00/11187, USP 6, 100, 034, and USP 6, 261, 806, etc.; however, these methods are only available in the sample under test. PERV nucleic acids are tested, but it is not possible to interpret whether it is an activated PERV virus or a latent inactivated virus. Since the US Food and Drug Administration (FDA) has regulated all biomedical materials from pigs or recipients of xenotransplantation, it is required to follow the detection of pERV, and the best ones are both nucleic acid detection and serum testing. (PHS Guideline 〇n Infectious Disease Issues in Xenotransplantation, Jan. 19, 2001) (Source Animal, Product Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans, Feb. 2001) 'The development of PERV detection technology In addition to the established pERV nucleic acid assay, the researchers attempted to perform PERV assays using serological assays involving the preparation of specific antigens, the development of positive control serum and high-valency antibodies. In the preparation of antigens, Matthews et al. first used the perv whole virus protein released by PERV-infected human kidney epithelial cell line 293(+) cells as an antigen to detect PERV antibodies by Western hybridization analysis (Matthews et al. Human, 1999, Transplantation 67(7): 939-43); then, paradis and Galbraith et al. used recombinant PERV p30-Gag protein as an antigen to detect the presence of antibodies in serum by enzyme-linked immunoassay (Paradis et al. , 1999, Science 285: 1236-1241; and Galbraith et al., 2000, J. Vlrol· Meth〇ds 9〇 (2): 115-124). In the study of positive control serum and antibody preparation, early detection of 1260347 cross-reactive antibody by reverse transcription of other animals was used as positive control serum, such as simian sarcoma-associated virus (SSAV). p29-Gag, gibbon ape leukemia virus p30-Gag, FeLV p27-Gag, FeLV gp71-Env, baboon endogenous retrovirus Gag and MuLV Gag antiserum; There are developments of polyclonal antibodies such as PERV p27-Gag, plO-Gag and pl5-Env (Krach et al., 2000, Xenotransplantation 7(3): 221-9; Tacke et al., 2000, Virology 268: 87-93). However, because the PERV enterprise clearing test method must consider the specific factors of the raw antigen and the local mussel antibody, the difficulty of the S product is not easy to overcome, and it still needs to be highly specific and south thin. The early strain antibody of sensitization was activated by early detection of the PERV virus. SUMMARY OF THE INVENTION In view of the above-mentioned highly specific and highly sensitive porcine endogenous retrovirus (PERV) antibodies, the present invention provides a lion-synthesis cell line which can produce an anti-retroviral retrovirus. Monoclonal antibody to capsid protein. The aforementioned fusion tumor was deposited at the Center for Culture and Conservation of the Institute of Food Industry Development on May 7, 1992. The registration number is BCRC960194. The present invention also provides a monoclonal antibody against the anti-retroviral capsid protein, which can be extended from the following groups: (a) a single antibody secreted by the fusion tumor; and (b) the aforementioned monoclonal antibody An antigen-binding fragment in (a). Lissie's monoclonal antibody system is designed to target a conserved region of the crustacean retrovirus's shell protein, 11] to detect, prevent or treat infections of different types of porcine reverse transcripts. The present invention also relates to a pharmaceutical composition for treating or preventing endogenous retroviral infection in pigs, the composition of which comprises at least the monoclonal antibody produced by the aforementioned fusion tumor and the carrier which is pharmaceutically acceptable. < The invention is directed to the treatment or prevention of porcine endogenous retrovirus infection, wherein the composition comprises at least the aforementioned monoclonal antibody or antigen binding thereof; The other purpose is to provide a set of 1260347 groups for detecting endogenous retrovirus shell proteins in pigs, the composition of which comprises at least the single: Further, the solid support of the package can be: microporous disc, microcapsule (5) ex b, body paper (brane filter paper), glass, Shi Xi wafer 2

= "Can be: radioactive material, enzyme, light substance, phycoerythrin: prime I. The re-purpose of the present invention provides a method for detecting endogenous retrovirus in pigs. v includes the following steps. The single antibody contact of this report, · & (8) epidemic analysis system is a trait-derived retrovirus. The aforementioned test sample may be a lysate or a lysate. The aforementioned immunoassay may be Western hybridization method, immunoglobulin assay or immunofluorescence assay. S S 酵 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合 融合_ cell fusion; and (e) screening for fusion tumors that produce antibodies specific for the anti-viral capsid protein. [Embodiment] The following is a further improvement through the actual payment - the technical features and advantages of the invention. Example 1: Design, Preparation and Purification of Antigens 1·1 Gene Alignment Analysis The present invention was designed with the sequence of PERV (GenBank Database Registration No. AF038601), from pig st cells, purchased from the National Standard Stem Center ( ATCC), numbered atcc CRL-1746, selected to produce PERV p and iral uglyna gene, and planted in pBluescdpt II SK(-) plastid to obtain a expression vector with a sense vector gene, named pST- Gp (Zhang Yuting et al., 2001, Chinese Veterinary Medicine 27 (2): 94-103). The obtained pST-gp plastid DNA was cleaved with restriction enzymes and sputum, and the DM ends were filled with Klenow enzyme, and then ligated with the gamma RV-cut pET-ca (1) vector, and transformed into Escherichia coli. DH5a competent in cells, isolated transform 1260347 promoter in the same direction, named as strain of shellfish DNA, select c{) na direction and Τ7 pST-GAG 〇 == enzyme Μ cut after the available job size is 6, _ The second pair of fragments and the thiol pair are subjected to electrophoresis recovery, wherein the size of 6, the base pair self-joining (self-llgatlGn), and PERV^ pSWNB3;

This plastid has been deposited in the Food Industry Development Research Institute on February 31, 2009. The registration number is (10)9. The other is the PET-bird (1) carrier that is cut by the colleague hi. Performing the conjugation, and then transforming and selecting the ship direction to be the same as the T7 promoter, the expression vector encoding the PERV capsid gene gene sequence is named pst-gag-m, which is matched with Zhongqianguo. It can be deposited at the Food Industry Development Research Institute on February 31, 2009. The registration number is ^Jianzhou. Since porcine endogenous retroviruses are currently mainly divided into three types, namely a type, β type and C type, to prepare a single antibody that can detect all types of PERV virus, in the design of antigen, the present invention _ GCG The towel is translated into Wei software. The ST-printed nucleic acid sequence can be translated into 512 gag amino acids, and the amino acid sequence of PERV gag known in the protein database (SWISS-PR0T) (registration number is AF03860KA type). ), Y17013 (type B), chest 3_ and chest 3_ (c type) into

Line alignment analysis, the results shown in the first figure, the amino acid similarity of different pERV gag reached 94~99%, and the conservation was high in the end region and the terminal region, but the C-terminal region included the main The perv is 7-Gag and pl〇-Gag, so a conserved region of the C-terminus can be selected as an antigen to prepare a monoclonal antibody capable of detecting all types of pERV viruses. Preparation of 1·2 antigen 1·2·1 In vitro induction of PERV capsid fusion protein Purification of PERV capsid fusion protein as antigen for subsequent immunization, transfection of pST-GAG-CB7 plastid DNA into Escherichia coli Representation 9 1260347 NovaBlue (DE3) and BL21 (DE3)-RIL hosts and select transgenic strains of anti-canemycin (kanamycine), select a single colony of transgenic plants and inoculate 2 ml LB medium (containing 25pg /ml Kangmycin), after shaking at 37 ° C overnight culture, take 150 μl of the bacterial solution diluted with fresh LB medium (containing 25 卩 / 1111 Kangmycin) 10 times, shake culture at 37 ° C for 2 hours Thereafter, a chemical inducer of 1-isopropylthio-β-D-galactose 22 (isopropyithi〇gaiactoside, IPTG for short) was added thereto, and cultured at 37 ° C for 2 hours with shaking to induce expression of the per v-shell protein fusion protein. . Finally, the bacterial solution was centrifuged at 6, rpm for 10 minutes at 4 ° C. The suspension was incubated with 15 〇 μl of 50 mM Tris-HC1 buffer, and then an equal volume of 2-fold concentration of sodium dodecyl sulfate was added. Load the dye [S〇dium dodecyl sulfate (SDS) loading dye], shake it and mix it in a 1〇〇C water bath for 5 minutes, then place it on an ice bath to extract the total protein of the bacteria. · 5% sodium sulphate-polyacrylamide gel (SDS-PAGE) for protein electrophoresis analysis' SDS-PAGE protein electrophoresis analysis is carried out according to standard methods (see Molecular Cloning-A) Laboratory Manual; Cold Spring Harbor, 1982-1989), SDS-PAGE The protein size of ST-GAG-CB7 was analyzed by coomassie blue after dyeing at 43. 5 thousand Å. 1· 2· 2 Soluble analysis of PERV capsid fusion protein The inoculum of the induced activity was centrifuged at 6,000 rpm for 1 minute at 4 C to remove the α solution, and then resuspended in a binding buffer. The bacteria were disrupted by an ultrasonic shatterer at 1 Torr and 〇〇〇 rpm at 4. The mixture was centrifuged for 10 minutes, and the soluble protein-containing supernatant was aspirated and placed in a new centrifuge tube; the pellet was dissolved in a urea-containing binding buffer at 10,000 rpm. (: Centrifuge for 1 minute, and take the supernatant containing the insoluble protein to another new centrifuge tube. 10 Add soluble egg and self-soluble protein solution to the 12-fold concentration of sodium dodecyl sulfate. After oscillating and mixing, it was placed in a lOOt water bath for 5 minutes, and then placed on an ice bath. Each 10 μ丨 was subjected to protein electrophoresis analysis with 12·5% SDS-page. The results showed that the PERV shell protein fusion protein was not Soluble protein. / 1· 2· 3 Purification and confirmation of PERV capsid fusion protein Since the PERV capsid fusion protein expressed by Escherichia coli is an insoluble protein, after extracting the insoluble protein by the method of step 1.2.2, The purified PERV capsid fusion protein was obtained by a His-Bind affinity chromatography column (purchased from Novagene), followed by washing, rinsing, etc. The purified protein was taken as 12·5% sodium dodecyl sulfate-poly After electrophoresis analysis of the acrylamide-separating colloid, the protein was disrupted to the pVDF-Plus hybrid membrane using a semi-dry transfer cell (Semi-Dry Transfer Cell, purchased from BIO-RAD) (PVDF-Plus membrane) S-Protein Alkaline Phosphatase Conjugate antibody (S-Tag) was purchased from MSI) with a 5% blocking solution for 1 hour at room temperature. Western Blot Kit; Novagen) was washed 4 times in 1 volume of Phosphate-buffered saline (PBS) after 2 hours at room temperature, and finally color reaction (S-Tag Western Blot Kit; Novagen The results showed that the purified protein could be bound by the antibody of S_protein alkaline phosphatase conjugate, so it can be confirmed that ST_GAG-CB7 is a fusion protein. 1.2.4 Quantitative extraction and purification of PERV capsid fusion protein The protein was placed in a dialysis membrane (Dialysis Tubing-3/4 in. Diameter; GIBCOBRL) and dialyzed overnight at 4 ° C in 1 volume of phosphate buffer, and concentrated by polyethylene glycol (PEG). The right amount of protein is quantified by Bradford (Protein 1260347

Assay Dye; BIO-RAD) for protein quantification. Example 2: Preparation of fusion tumor cell line 2.1 Immunization of mice As shown in the second figure, 6 to 1 week old healthy MLB/c mice were selected for immunization, and the first application was performed. 100 // g purified p-capsid protein fusion protein antigen was mixed with an equal amount of complete adjuvant (Freund, s adjuvant complete, purchased from GIBC0BRL) and injected into the peritoneal cavity of mice; after two weeks, the use was incomplete. The adjuvant (Freund's adjuvant incomplete, purchased from sigma) was mixed with the prepared antigen for a second application; after two weeks, the mouse was tested for immune response' and the anti-PERV cap protein fusion protein was detected. The antibody price was chosen, and the mouse with better response was selected three times without antigen (1〇〇飓/time) without any adjuvant, and sacrificed 4 to 8 days after the first injection to increase the antibody amount. 2· 2 Detection of immune response After fixing the mouse, gently squeeze the mouse tail by hand and find the vein on the back of the tail. Disinfect the cotton at 70 cm from the base of the tail. Use a 2% needle to puncture the needle. Intravenous, the effluent blood was inhaled into a microcentrifuge tube with a micropipette, and after being placed at 37 C for 1 hour, the blood was transferred to a refrigerator at 4 ° C overnight; and centrifuged at 14 rpm for 1 〇 every other day. The supernatant was placed in another new microcentrifuge tube, which was serum; the serum was diluted to different folds with one volume of phosphate buffer for Western Blotting and Enzyme Linked Immunoassay (Enzyme) -Linked Immunosorbent Assay, ELISA) test. 2. 3 production of fusion tumor cell line When it is determined that the mouse has been successfully immunized, the cervical spine is sacrificed, the mouse is cut through the left back, and the spleen is taken out and placed in a culture medium containing 10 ml (Dulbecco's Modified Eagle Medium, DMEM for short). In the dish, the spleen was transferred to another Petri dish containing DMEM medium, and the spleen was cut with tissue scissors to allow most of the spleen cells to flow out 12 1260347; the cell suspension was aspirated into a 50 ml centrifuge tube, and 10 ml was taken. DMEM culture solution was added to the culture solitary tract, and the spleen cells were disrupted and repeated once; it was allowed to stand at room temperature for 5 minutes, and the supernatant was taken to another 50 ml centrifuge tube, and centrifuged at 1, rpm for 5 minutes to remove After the supernatant, 10 ml of DMEM medium was added to suspend the spleen cells, and the cells were counted in a hemocytometer. On the other hand, the myeloma cells (FO cell line, number ATCC CRL-1646) were dispersed, and the cells were counted in a hemocytometer; an appropriate amount of bone tumor cells were mixed with the spleen cells.

After combining, the cell number ratio was 1:3, and after centrifugation at 1,000 rpm for 10 minutes, the supernatant was removed; after being placed in a 37 t: water bath for 1 minute, 1 ml of 37 ° C preheating was added. % PEG1500 for cell fusion; then slowly add 10 ml DMEM medium, centrifuge at 600 rpm for 3 minutes, remove the supernatant; add 50 ml of 20% fetal bovine serum (FBS) and one HY culture (containing bran) with Penicillin/Streptomycin/Amphotericin B (10%, 〇〇〇U/ml, 10 mg/ml, 0·025 mg/ml) L-glutamine, bovine Insulin, Oxaloacetate and sodium pyruvate were used to suspend the cells, first placed in an incubator at 37 ° C 7% C 〇 2 After four hours, the fused cells were removed and 110 ml of HY medium containing 1% HAT (10 mM sodium hypoxanthine, 40 μΜ aminopeterin, and L6 mM thymidine) was added. After thorough mixing, dispense into 96-well culture dish (200 μΐ/well), place at 37 ° C, 7% C〇2 Incubator for 10-15 days. Example 3: Screening of fusion tumor cell strain 3.1 Enzyme-linked immunoassay test After one week of cell fusion, about 40 to 50 cells of fusion tumor cells were observed, and the culture supernatant was taken for enzyme-linked immunoassay for fusion tumor Screening of cell strains; since the immune antigen of the above experiment was prepared by Escherichia coli (BL2i(dE3)-RIL), 13 1260347 must prepare an egg self-quality scale group of the host cell gut g. The prepared broad-shell protein fusion protein antigen and E. coli eggs were dissolved in -coating buffer (coatlng Buffer; 15 _ % listen, % secret

In Na·, _.6), place it in a 96-well immunoenzyme reaction tray (〇5 μ_11), move to the refrigerator and let it stand overnight; wash the three volumes in one volume, each time about -min; A 5% skim milk powder solution was used for one hour in the listening incubator and then washed three times with one volume of PBS.

The culture supernatant of the fusion tumor cells was taken out, and the 96-well immunoenzyme reaction plate (μΐ/well) which had adhered to the pERV capsid fusion protein antigen or Escherichia coli protein was separately injected, and the serum previously isolated from the heart was doubled. The volume was diluted by 1, and the 〇〇〇 was used as a positive control. After 2% of the effect in the 3rc incubator, 'washed three times with one volume of pbs, and added a second antibody, a hydrogen peroxide, goat anti-mouse serum antibody (g〇at anti with this ❻igG peroxidase conjugate, purchased from Chemicon), placed in a 37 ° C incubator for 1 hour, washed three times with one volume of PBS, and then added to the loo μ immunization reaction negative (ΤΜΒ / Ε solution ' 3, 3 ', 5, 5 ' - tetramethybenzidine , purchased from Chemicon), allowed to stand at room temperature for 30 minutes in the dark, and then added 50 μM of 〇. 25 M HCl to terminate the reaction. The absorption spectrum at a wavelength of 450 ηιτι was measured by a spectrophotometer. Comparing the difference between the PERV capsid fusion protein antigen and the E. coli protein, several fusion tumor cells which recognize only the PERV capsid fusion protein without recognizing the E. coli protein were obtained. 3.2 Western hybridization assay As the PERV capsid fusion protein antigen of step 1.2 of the example has a His-Tag protein N-terminus, it is necessary to prepare a protein of His-Tag-containing Escherichia coli as a control group to avoid a false positive reaction. Add His-Tag protein and PERV capsid fusion protein to an equal volume of twice the concentration of sodium dodecyl sulfate (SDS) loading dye, shake and mix, and then in 14 1260347 100 C water bath After 5 minutes, it was placed on ice and analyzed by protein electrophoresis using SDS-polyacrylamide gel (SDS-PAGE) with 12.5% 5% sodium sulphate-polyacrylamide. . Using a semi-dry protein breaker to break into the PVDF-P1US hybrid membrane, containing 5%. 1% Tween 20 5% skim milk powder (dissolved in one volume of pbs) at room temperature for 1 hour after 'one volume PBS After washing 4 times, the culture supernatant of the selected fusion tumor cells was added for 2 hours at room temperature, washed 4 times with one volume of pbs, and then added with a second antibody-assay dish of acetic acid goat anti-mouse serum. The antibody was incubated at room temperature for 1 hour, washed twice with PBS, and finally added to the enzyme reaction solution (BCIP/NBT membrane phosphatase substrate system, purchased from KPL) for 2 minutes at room temperature. The reaction solution was stopped once with a stop reaction solution (1 mM Tri# imMEDTA), and the results were interpreted. As shown in the third figure, the line indicates the protein size mark (pr〇s丨e (7)丨〇r, ie ei η marks), from top to bottom, 118 '76, 5〇, 38, Shen, 19 and 13 kilo 呑 ki (kilodalton, kD for short); line 2 is the total protein of PERV shell protein fusion protein induced by iptg; line 3 is the PERV shell protein fusion purified by the colloidal column Protein ·, line 4 is the total protein of the cell which expresses His Tag protein by IprG; line 5 is the U protein shell of Escherichia coli. As a result, the antibody secreted by the fused cell was only recognized as PERV, which was 43.5 kD in size and did not react with the His_Tag protein. 3. 3 The single-stranded tumor of the fusion tumor was confirmed to be a fusion tumor cell strain capable of secreting PERV capsid antibody by enzyme-linked immunoassay and western hybridization assay, and the cells in the % well culture plate were broken up with a micropipette. Then, the fresh culture solution is diluted with _ times and (10) times, and then dispensed in another new 96-well culture plate for several days, and then the above-mentioned enzyme-linked immunoassay test and Western hybridization analysis test are repeated, and only The cell population of a cell population was screened out for 15 1260347 'this is a single-body fusion tumor cell line. 3. 4 Proliferation culture of the fusion tumor cell line The obtained single-body fusion tumor cells were transferred from a 96-well culture plate to a 24-well culture plate, and transferred to a 6-well culture plate in about three days, and finally transferred to a 25T culture flask. The cells can be stored frozen in about one week. In the process of subculture, the culture supernatant is collected first, and then

After the cells were photographed with 0.05/Q Trypsin/EDTA, fresh DMEM medium containing 2% fbs was added and centrifuged at 1, rpm for 5 minutes. After removing the supernatant, fresh DMEM containing 2% FBS was added. The culture medium suspends the cells, and after counting the cells by the hemocytometer, an appropriate amount of the cells is continuously cultured, and in the process of subculture, the HY culture solution containing 20% FBS can be replaced with the DMEM culture solution containing 20% FBS. . 3. 5 Preservation and storage of the fusion cell line The cells in the culture flask were photographed with 〇·05% Trypsin/EDTA, and then added to the DMEM medium containing 20% FBS, centrifuged at 10,000 rpm for 5 minutes, and removed. To clear the solution, add the fresh DMEM medium containing 20% FBS to suspend the cells; after counting by the hemocytometer, 'centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and then add the appropriate amount of cell cryopreservation solution (including 10% DMS0). DMEM culture medium) suspend the cells so that the cell concentration becomes 2 X 10/ml 'Imly mix the cell suspension with iml and add the cells to the cell cold; in the east small tube' and seal the bottle mouth, first place at -20 ° C 30 After a minute, transfer to -8 (TC placed overnight, then transferred to a liquid nitrogen barrel for storage. The present invention screened a fusion tumor cell line that produces PERV shell protein monoclonal antibody, named PERV GAG-5A11, already in the Republic of China Deposited and researched at the Center for Culture and Preservation of the Food Industry Development Research Institute on May 7, 1992. The registration number is BCRC960194. Example 4: Production and purification of monoclonal antibodies 4. Typing of individual antibodies (typing) With ImmunoPure® Monoclonal Antibody Isotyping Kit II ( From the PIERCE), the typing of the monoclonal antibody was carried out. 50 μL of the coating antibody working solution (coating antibody working 16 1260347 solution) was added to the 96-well immunoenzyme reaction plate and placed at 4 ° C overnight. After removing the supernatant, the solution was added. 125 μΐ-folding buffer buffer was placed at 37 ° C for 1 hour; the supernatant was removed, and after washing four times with 125 μM wash buffer, 50 μM of monoclonal antibody (fused tumor cell line PERV) was added. GAG-5Α11 culture supernatant) or positive control group (mouse monoclonal antibody IgG1 ic chain) and placed at 37 ° C for 1 hour; the supernatant was removed and washed four times with 125 μM wash buffer, then Add 50 μΐ of subclass-specific rabbit anti-mouse immunoglobulins or normal rabbit serum, and let it stand at 37 ° C for 1 hour; remove the supernatant to 125 After washing the μΐ washing buffer four times, add 50 μL of alkaline phosphatase conjugated goat anti-rabbit IgG working solution and place at 37 °C. After 1 hour of action; after removing the supernatant, wash it four times with 125 μL of washing buffer; then add 1 μm of a concentration of pNpp (p-nitrophenyl phosphate) solution and protect it from light at room temperature. The absorption spectrum at a wavelength of 405 nm was measured by a spectroscopic spectrometer for about 30 minutes. As shown in the fourth figure, the prepared monoclonal antibody (PERV GAG-5A11) was identified as IgG2b after typing (typing), and its light chain was κ chain. 4. 2 Ascites preparation Eight weeks old healthy BALB/c mice were selected and injected into the peritoneal cavity of mice with 5〇〇μ1 incomplete adjuvant. After every other week, 3χ106 fresh fusion tumor cells PERV GAG-5A11 Inject into the abdominal cavity of the mouse; after 1 day, the ascites was aspirated, centrifuged at 3, rpm for 10 minutes, and the supernatant was aspirated into a new tube; placed at 56 ° C for 45 minutes, at 3,000 rpm. Centrifuge at 1 rpm for a minute, pipette the supernatant into a new tube, and store at 2 °C °C, which contains high concentrations of monoclonal antibodies. 4. 3 Purification of monoclonal antibody The culture supernatant of the fusion tumor cell line PERV GAG-5A11 was used at 4 rpm. Deviate. In 10 knives 1 , after taking the last month's liquid and 〇·45//in transition film, add a volume of 17 1260347 starting buffer (8 7 31 ^ 1) 1 ^ 61 ', containing 〇. 〇5~11']: [3-[1〇10118.6, 〇.15 M NaCl, 0.02% NaN; 3) diluted; the ascites treated as described above is diluted with twenty volumes of starting buffer, and then 0 · 45//m filtration membrane filtration; the culture supernatant or ascites of the above-mentioned treated fusion tumor cell line PERV GAG-5A11 was passed through a packed HiTrap Protein-A affinity chromatography layer (purchased from Pharmacia). Wash with the starting buffer, then flush out the IgG antibody with Elect buffer (containing 0.05 Μ glycine-HCl ρΗ2·3, 0·15 M NaCl), and add ΐ/4 volume to the buffer ( Neutralizing buffer containing 0.5 M phosphate buffer, ρΗ7· 7 ) to neutralize the pH, the purified IgG antibody can be obtained, and then placed in a dialysis membrane and dialyzed overnight in a volume of PBS at 4 ° C, and then taken out and centrifuged. The tube was subjected to vacuum drying and stored at 4 °C. 4·4 Purity analysis of monoclonal antibodies The purified IgG antibody was used to detect the purity of the antibody by protein electrophoresis. Add _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Sodium-polyacrylamide guanidine separation colloid (SDS-PAGE) for protein electrophoresis analysis; SDS-PAGE stained with coomassie blue (Coomassie blue), as shown in the fifth figure,

Line 1 indicates ProSieve color protein markers, from top to bottom, 118, 76, 50, 38, 26, 19, and 13 kD, respectively; line 2 is ascites; Lu line 3 is ascites via HiTrap Protein-A. Affinity chromatographic analysis of purified IgG antibodies, the size of which is heavy chain 50 kD and light chain 25 kD° Example 5: titer analysis of monoclonal antibodies 5. 1 immunization with enzyme Analytical method for the analysis of the strength of a single antibody, taking a known concentration of antigen, using enzyme-linked immunoassay to detect the price of individual antibodies. The purified PERV capsid fusion protein is dissolved in double coating buffer (15 _

Na£〇3, 35 mM NaHC〇3, ρΗ9·6), placed in a 96-well immunoenzyme reaction tray (0.25 18 1260347 pg/well), and transferred to a 4t refrigerator for overnight storage; washed three times with one volume of pBS, Each time about -minute; add 5% of the skim milk powder solution, in the 3η: incubator, after washing three times with - volume PBS, then add the first antibody (the antibody to be tested PERV GAG-5A11, to - Double volume PBS diluted different times), placed in a stepwise incubator for ^ hours, three times the volume of PBS, three times; add a second antibody-catalase goat anti-mouse serum antibody, placed in 3rt After the incubation box is used for 2 hours, it is washed three times in one volume; after that, the receptor of the immune enzyme reaction (ΤΜΒ/Ε solution) of 1〇〇μ1 is recognized, and it is allowed to stand at room temperature for 3 minutes, and then added to %. ^·25 M HCl, the reaction was terminated, and the absorption spectrum at a wavelength of 45 Å was measured by a spectrophotometer. As a result, as shown in the sixth graph, the antibody titer of PERVGAG-5AU ascites was 49, and that of the pure PERV GAG-5A11 IgG antibody was 48. 5. 2 Sensitivity analysis of monoclonal antibodies (enzyme-linked immunoassay) Take different concentrations of antigen (1, 2^, 2, g, 2-Vg, 2-Vg, 2\g, 2-Vg), - Detection of the sensitivity of individual antibodies by enzyme-linked immunoassay. The purified pERv capsid fusion protein antigen was dissolved in double coating buffer (15 mM Na 〇 3, 35 mM NaHa) 3, ρ Η 9· 6) ' and then different concentrations of antigen were placed in a 96-well immunoenzyme reaction disk. , and moved to a refrigerator at 4 ° C overnight; rinse three times with one volume of PBS for about one minute each time; then add 5% skim milk powder solution, double the time in the incubator at 37 ° c, double After washing the volume of PBS three times, the first antibody (single antibody pERV GAG-5A11, 0·56 mg/ml; diluted 5 times in PBS with one volume) was added, and placed in a 3rc incubator for 1 hour. The solution was washed three times with one volume of PBS, and the subsequent steps were the same as those described in Example 5.1. As shown in the seventh panel, the monoclonal antibody PERVGAG-5A11 was detected by enzyme-linked immunoassay, and its sensitivity was as high as 2'Lg of antigen. 5·3 Sensitivity analysis of monoclonal antibodies (Western hybridization assay) Different concentrations of antigen were taken and the sensitivity of individual antibodies was detected by Western blot analysis. The purified PERV protein fusion protein antigen was dissolved in monotonic sodium dodecyl sulfate to fill 19 1260347 dye, and then different concentrations of antigen were taken for protein electrophoresis analysis; and the semi-dry protein breaker was used to break to PVDF- Plus hybrid membrane, containing 1% of 5% non-fat dry milk powder (dissolved in one volume of PBS) for 1 hour at room temperature, washed 4 times with one volume of PBS, adding the first antibody (Single antibody, 〇·56mg/ml; diluted 5000 times with one-fold PBS), 2 hours at room temperature, 4 times with one volume of PBS; then add a second antibody, the subsequent steps are the same as the steps of the examples. 3·2 stated. As shown in the eighth figure, line 1 indicates the protein size marker (ProSieve c〇1〇r pr〇tein markers), which are 76, 50, 38, 26, 19, 13 and 10 kD from top to bottom; 3, 4, 5, 6, 7, 8 and 9 are purified PERV capsid fusion proteins of 2, 21⁄4, 21⁄4, 2, g, 2\g, 2^g, 2>g and 2>g, respectively; The monoclonal antibody PERVGAG-5A11 was detected by Western blotting analysis and its sensitivity was up to 2 antigens. Example 6: Specificity analysis of monoclonal antibodies In terms of specificity analysis of monoclonal antibodies, since PERV is a mammalian c-type retrovirus, it is desirable that the prepared PERV monoclonal antibody does not reverse transcription with other humans. The virus produces a cross-reactivity. In this example, a human-linked immunodeficiency virus (Murex HIV-1, 2, 0) and a human sputum leukemia virus (Murex HTLV Ι + ΙΙ) enzyme-linked immunoassay kit (purchased from Murex Biotech) were used. Test; using a 96-well immunoenzyme reaction plate to which HIV-1, 2, 0 or HTLV-I+II antigen is attached, add 50 μM of sampie diluent, and then add test sample mouth (single antibody) PERV GAG-5A11) or control group (positive control is Anti-HIV serum or Anti-HTLV serum; negative control is normal human serum), placed at 37 ° C for 30 minutes, Rinse with washing solution five times; add 50 μΐ of conjugate, put it at 37t: after 30 minutes, rinse with washing solution five times; add 1〇〇μ1 of the receiving solution, put it at 37T: after 30 minutes, then add After 50 μΐ, 18 M H2SO4 terminates the reaction, and finally the spectroscopic spectrum 20 1260347

AbAnit long 450 coffee absorption spectrum. As a result, as shown in the ninth figure, the monoclonal antibody PERV does not react with the human immunodeficiency virus and the human T cell from the blood virus. Example 7 ··Evaluation of the ability of single antibody to detect PERV 7.1 Detection of pERy virus by Western blot analysis The antigen used in the anterior column is a recombinant fusion protein purified from E. coli, so Whether the individual antibodies can recognize the white 2 to infectious brix virus particles produced by the pig cells requires further analysis of the stone green. Here, the following cell lines were raised: the porcine kidney cell strain 5 and the human kidney epithelial cell line 293 (1) which has been infected with P-P, have been confirmed by the RT-pcR method to release PERV 'no two cells as positive In the control group, other human kidney epithelial cell line 293 was used as a negative control group; the culture supernatant was collected in large quantities, and centrifuged for 1 hour at _=4C to collect virus particles; in addition, the cells were collected and added to the cells. The stroke solution was centrifuged at 4 Torr for 30 minutes, centrifuged at 1 Torr, _ rpm at 4 t: 1 〇 for 4 liters, and the supernatant was aspirated as a cell extract; then an equal volume of twice the concentration of 12 sulphuric acid was added. Sodium filling agent, after mixing i, in loot: water bath for 5 minutes, then placed on ice 'for protein electrophoresis analysis; Xiang semi-dry protein breaker collapsed to PVDF Plus miscellaneous membrane' to contain 〇1 % Tween 2〇 5% skim milk powder (dissolved in one volume of PBS) for 1 hour at room temperature, washed 4 times with one volume of pBS; add the first antibody (per antibody PERVGAG-5A11, 0.56mg/ Ml; diluted 5000 times with one volume of PBS), 2 small at room temperature At the time, the cells were washed 4 times with one-fold volume of PBS; then a second antibody-alkaline phosphate goat anti-mouse serum antibody was added, and after 1 hour at room temperature, it was washed 4 times with 1 volume of PBS, and finally added. The enzyme reaction was carried out by a BCIP/NBT membrane phosphatase substrate system (purchased from KPL) at room temperature for 2 minutes, and then washed once with the termination reaction solution (UMTris and 1 mM EDTA), and the results were interpreted. As shown in the tenth figure, 'line 1 indicates the protein size (pr〇Sievec〇i〇r pr〇tein 21 1260347 markers), from top to bottom, U8, 76, 5〇, 38, 26, 19, 13 And 10 kD; line 2 is the culture supernatant of porcine kidney cell line PK15; line 3 is the culture supernatant of PERV-infected human kidney epithelial cell line 293(+); line 4 is human kidney epithelial cell line 293 The supernatant was cultured; line 5 was the cell extract of PK15; line 6 was the cell extract of 293(+); line 7 was the cell extract of 293. To evaluate the ability of monoclonal antibodies to detect PERV virions from cell lines, we found that the monoclonal antibody PERV GAG-5A11 we produced can bind to the protein of PERV virions released by PK15 and 293(+). It is 65 kD or 63 kD (for GAG precursors) and 26 kD, and does not react with 293. 7.2 Detection of PERV virus by cell immunostaining In the steps of this example, the following cell lines were cultured, including porcine kidney cell line ρκΐ5 and human kidney epithelial cell line 2 9 3 (+) and human kidney that had been PERV-supplemented. The epithelial cell line 293 was cultured in a 6-well culture plate overnight; after removing the culture supernatant, the cells were washed twice with one-fold volume of PBS; the cells were fixed with 95% alcohol for 1 minute, and then washed twice with one-fold volume of PBS. 5 minutes each time; drip a drop of blocking serum diluted with one volume of ρβ$ for 45 minutes at room temperature, and then wash it four times with one-fold volume pbs containing 5%·05% Tween 20; One antibody (per antibody PERVGAG-5A11, diluted 2 times in PBS of one volume; negative control group was directly added to one volume of PBS), placed in a 37 ° C incubator for 1 hour, containing 〇·Tween 20-fold volume PBS wash four times; then add a second antibody (magnetic biochemically second step antibody), after 30 minutes at 37 ° C incubator, with 0. 05% Tween 20 one volume pBS is washed four times; then ABC reagent is added ( ABC Reagent, UntiTectTM Mouse ABC kit, purchased from

Oncogene), after 30 minutes at 37 ° C, washed four times with PBS containing one part of we·05% Tween 2〇, and added NovaRED substrate (Vect〇r N〇vaRED kit, available from Vector Laboratories). After 3 seconds at room temperature, rinse twice with deionized water; finally add HE dye, and apply for 3 〇 seconds at room temperature to perform nucleus 22 1260347 柒 color, after washing once with de-ball water, then stand upside down Microscope observation and photography. As shown in Figure 11, the results showed that the prepared monoclonal antibody pERVGAG-5An could bind to the PERV virions released by PK15 and 293(+) without cross-reactivity with 293, and in the negative control group. There is no cross reaction. Example 8: Detection of PERV antigen infection in humans using monoclonal antibodies The detection of pERV antigen was performed using the serum of a pig farm worker, and human kidney epithelial cell line 293 (+) infected with pERV was used as a positive control group. First, the serum of the pig farm staff was added to an equal volume of twice the concentration of sodium dodecyl sulfate to fill the dye, and the mixture was shaken, and then placed in a 100 C water bath for 5 minutes, and then placed on an ice bath for protein electrophoresis analysis; The pvDF-pius hybridization membrane was disrupted using a semi-dry protein disrupter, and the subsequent steps were as described in Example 7.1 of the Examples. As shown in Figure 11, line 1 indicates protein size markers, H8, 76, 50, 38, 26, 19, 13 and 1〇kD from top to bottom, respectively; line 2 is human renal epithelial cells infected with PERV. The culture supernatant of strain 293 (+); line 3 to line 1 is the serum of the farm staff. PERV GAG-5A11, a PERV coat protein antibody, was used for PERV antigen detection in pig farm staff. As a result, no PERV antigen was detected in the serum test of 3 farm staff, while the positive control group 293 (+) 63 kD (for GAG precursors) and 26 kD PERV viral proteins can be detected. The above-described embodiments are intended to be illustrative of the present invention and are not intended to limit the invention to the specific forms disclosed. The scope of the present invention is defined by reference to all modifications and similar variations without departing from the spirit and scope of the invention. [Simple description of the schema] The first panel shows the results of the amino acid sequence alignment analysis of different types of porcine endogenous retrovirus shell protein gene (perv gAG). The figure shows the preparation process of the PERV capsid monoclonal antibody of the present invention. The second panel shows the ability of the antibody of the fusion tumor cell of the present invention (PERV GAG-5A11) to recognize the pERV capsid protein. 23 1260347 The fourth panel shows the results of the typing analysis of the monoclonal antibody PERV GAG_5AU of the present invention. The fifth panel shows the results of electrophoretic analysis of the purified monoclonal antibody PERV GAG_5AH of the present invention. The sixth graph shows the results of the valence analysis of the purified monoclonal antibody PERV GAG_5AU of the present invention. The seventh graph shows the results of analyzing the sensitivity of the monoclonal antibody pERy GAG-5A11 of the present invention without using an enzyme-linked immunoassay. .

The eighth graph shows the results of analysis of the sensitivity of the monoclonal antibody p-layer GAG-5A11 of the present invention without Western hybridization analysis. The ninth panel shows the results of the specificity analysis of the monoclonal antibody PERV GAG-5A11. The tenth figure (four) (five) field hybridization analysis method for the detection of individual antibodies should be 5An identification 苐11 map shows the results of PERV virus. The twelfth image shows the results of the detection. The expression of pERV GAG_5A11 was not detected by cell immunostaining. The identification of PERV antigen was observed by using a single plant minus pm 5A11.

twenty four

Claims (1)

Pick up, apply for patent scope: 1. - A kind of fusion tumor, which can produce anti-secret anti-transcription squamous protein material antibody, the fusion tumor was deposited in the Food Industry Development Research Institute on May 7, 1992. Research Center, the registration number is BCRC960194. 2. According to the fusion tumor described in the middle of the paste, the surface cells are produced by fusion with the spleen cells of the animal producing the anti-endogenous retrovirus shell protein antibody. 3. According to the scope of the patent application! The fusion tumor of the above, wherein the aforementioned monoclonal antibody system is designed for a common conserved region of a different shell protein of the endogenous retrovirus. 4. A monoclonal antibody against a porcine reversible virulence capsid protein, which may be selected from the group consisting of: (4) a monoclonal antibody produced by a fusion tumor of the third aspect of the patent application; and b) an antigen-binding fragment of the aforementioned monoclonal antibody (a). 5. According to the cap-specific sylvestris, the capsid-specific strain system is designed for the conserved regions of different types of _-derived retroviruses. 6. If you apply for a single plant domain as outlined in 专 4, it can detect, steal or treat infections of different types of pig endogenous retroviruses. - 7·-species test _, sexual retrovirus set, including at least the patent application of the individual antibody described in item *. 8. A kit for detecting endogenous retroviruses in pigs, which comprises at least a monoclonal antibody produced by the fusion tumor described in the scope of claim ♦. 9. If the kit described in Section 7 or Item 8 of the patent application is applied, it may further comprise a solid phase support, a signal generator and a coloring substance. 10. The kit of claim 9, wherein the solid support may be: microporous disc, latex beads, microspheres, membrane paper (membrane ηι^qing), glass , wafers, metals or ceramics. 11. If the kit described in claim 9 is applied, the aforementioned substance may be: radioactive substance, enzyme, phosphorescent substance, phycoerythrin, biotin or fluorescent substance. 25 1260347 12. Test the endogenous reversal of pigs (4) Test the sample with ί;;: method, including at least the following steps (b) by immunoassay; whether; 13. If the scope of patent application is 12 The age of endogenous reverse transcriptional disease exists in Qin. Liquid (lysate). The method of jin, the sample to be tested is the serum or the fine running solution. 14. The method described in claim 12, the aforementioned immunoassay method can be parent method, agglutination reaction, enzyme-linked immunity. Law or immune law. The method for preparing a fusion tumor according to the above-mentioned patent scope of the invention, comprising: a purified human porcine endogenous retrovirus shell protein c-terminal conserved region; " Body eggs acquire spleen cells of the aforementioned animals, fuse with myeloma cells; and screen for fusion tumors that produce antibodies specific for porcine endogenous retrovirus shell proteins. Once 26