KR100369533B1 - Monoclonal antibody specific for vp6 of porcine rotavirus and hybridoma cell line producing same - Google Patents

Abstract

The present invention provides a monoclonal antibody capable of specifically recognizing and binding to the VP6 protein of porcine rotavirus, a fusion cell line secreting the antibody, a method for producing the monoclonal antibody using the fusion cell line, and the monoclonal antibody. The present invention relates to a method for detecting swine rotavirus by using the present invention. The monoclonal antibodies of the present invention can accurately and quickly diagnose whether swine rotavirus is infected in a swine secretion sample test, thereby confirming the propagation path of rotavirus and prematurely. It can be used as a good means of basic biology research to study the physiological activity, differentiation and growth regulation of mammalian cells because it can enhance the prevention effect by the discovery and have specific reactivity against rotavirus of all mammals except pigs. Can be.

Description

MONOCLONAL ANTIBODY SPECIFIC FOR VP6 OF PORCINE ROTAVIRUS AND HYBRIDOMA CELL LINE PRODUCING SAME}

The present invention provides a monoclonal antibody that can specifically recognize VP6, a structural protein of porcine rotavirus, a fusion cell line secreting the same, a method for producing a monoclonal antibody using the fusion cell line, and the monoclonal The present invention relates to a method for detecting swine rotavirus using an antibody.

Rotavirus is Leo virus (Reovirus), ohreubi virus (Orbivirus), Pitot Leo virus (Phytoreovirus), Fiji belongs to the virus (Reoviridae) Leo birida together with (Fijivirus), divided in groups A by serological classification to F that Rotavirus, which belongs to group A, is the most widespread virus and infects humans, monkeys, horses, pigs, dogs, cats, rabbits, mice, cows, sheep, etc., and causes diarrhea. Pigs, mice, etc. as host animals, other information is not yet known. Rotavirus causes severe diarrhea when infected with animals, human rotavirus causes severe diarrhea in newborns and infants, swine rotavirus causes diarrhea in pigs of all ages, especially in piglets within 5 weeks of birth. The mortality rate is 100%, and the 5 week old pig also has a mortality rate of over 90%. The disease was allowed to grow in monkey kidney cells in 1963 by Mahlerbe et al., But the rotaviruses of group B and group C have yet to be established. When the disease is caused by rotavirus, firstly, the mortality rate of piglets is very high, secondly, there is no appropriate treatment measures, and thirdly, prevention measures against viruses are difficult, and fourth, the control effect by the vaccines currently developed. There is a problem that is low.

In other words, rotavirus is a common etiology that causes enteritis in many animals including humans. The size of the particles is 70 nm in diameter, and the genome consists of double-stranded ribonucleic acid. Is composed of triple structure with VP1, VP2, VP3, VP4, VP6 and VP7 as main structural proteins. Specifically, VP1 and VP3 protect ribonucleic acid, VP2 is the main component of the core protein surrounding the nucleic acid, and VP6 and VP7 constitute the inner and outer outer proteins, respectively. VP4 promotes viral infection and is located outside of the VP7 protein. The most effective prophylactic method of reducing diarrheal disease in pigs by swine rotavirus is to establish anti-defective measures through early diagnosis of swine rotavirus infection. As a diagnostic method for a virus, a virus-aware antibody is produced and a method of measuring viral infection using the antibody is generally used. In this case, it is more specific to use a monoclonal antibody than a polyclonal antibody.

However, since the core protein is located inside the virus particle, the antibody against it cannot recognize the living virus particle, and the antibody against the envelope protein can recognize the virus particle. Among the coat proteins VP6, VP7, and VP4, the VP7 protein is composed of different epitopes due to different sequences of the rotavirus subtypes, and the VP4 protein is present only when the rotavirus has a complete structure. The use of antibodies against them as proteins is extremely limited. On the other hand, the VP6 protein is the most frequently expressed envelope protein during the production or propagation of rotavirus, and the most effective method is to use an antibody against the virus particles having a common antigen structure in rotavirus belonging to group A. Specifically, the VP6 protein is a protein having a molecular weight of about 45 kDa, which has a nucleotide sequence that does not change frequently in the rotavirus subtype, and is highly homologous to the VP6 protein of rotavirus that infects animals other than pigs. Therefore, monoclonal antibodies against VP6 protein can be very useful for the detection of rotavirus.

Infectious gastroenteritis viruses (Saif and wesley in 1992) and swine pandemic diarrhea virus (Pansaert et al., 1981) are viruses composed of the same ribonucleic acid, which are swine-infected viruses such as swine rotavirus that cause severe diarrhea. Therefore, it is necessary to develop a vaccine according to an infectious agent by diagnosing the exact cause of viruses causing these diarrhea, but no antibody has yet been developed to distinguish between these viruses and rotaviruses.

In order to establish an accurate diagnosis system for swine rotavirus that causes severe diarrhea in pigs, the present inventors immunized mice (Balb / c) with swine rotavirus particles, and then separated spleen cells of mice. A fusion cell line producing a monoclonal antibody against the protein of porcine rotavirus by fusion with myeloma of the mouse, and culturing the fusion cell line and murine monoclonal antibody having high specificity for porcine rotavirus from the culture The present invention was completed by confirming that the monoclonal antibody has the characteristics of distinguishing rotavirus from the swine infectious gastroenteritis virus and the swine epidemic diarrhea virus.

An object of the present invention is to provide a monoclonal antibody that specifically recognizes swine rotavirus VP6 protein, a fusion cell line capable of producing the same, and a method for mass production of the monoclonal antibody by culturing the fusion cell line.

Another object of the present invention is to provide a method for detecting porcine rotavirus using the monoclonal antibody.

1 is a result of confirming the specificity for porcine rotavirus of the monoclonal antibody of the present invention by SDS-polyacrylamide gel electrophoresis.

Figure 2 shows the results confirmed by immunoblotting the specificity for the swine rotavirus of the monoclonal antibody of the present invention.

Figure 3 shows the results of verifying the specificity for the swine rotavirus of the monoclonal antibody of the present invention by the enzyme immunoassay method.

In order to achieve the above object, in the present invention, a monoclonal antibody produced by the mouse-derived fusion cell line NS1-RIO (KCTC 0654BP) and specifically binding to the VP6 protein of porcine rotavirus and the fusion cell line NS1-producing the same RIO (KCTC 0654BP) is available.

In addition, the present invention comprises the method of producing a monoclonal antibody against swine rotavirus, comprising injecting the fusion cell line intraperitoneally of the mouse and then collecting ascites fluid and isolating the monoclonal antibody against swine rotavirus therefrom; Provided is a method for detecting swine rotavirus, characterized in that using the monoclonal antibody.

The antibody of the present invention can be prepared as follows. That is, MA104 cells (porcine pancreatic cell line, obtained from Chungbuk National University) were cultured in a minimal medium containing fetal bovine serum, and then infected with porcine rotavirus to MA104 cells, which resulted in 90 When more than% is isolated, stop the culture and centrifuge the culture supernatant to isolate swine rotavirus. The isolated porcine rotavirus particles were suspended in phosphate buffer and mixed with the same amount of complete Freund adjuvant solution to immunize mice, and the fused cell lines were fused by fusing the immunized splenocytes and myeloma cells. Next, the fusion cell lines that specifically react only with the swine rotavirus antigen are selected by enzyme immunoassay.

Immunization of mice is performed according to conventional methods. In other words, porcine rotavirus as an antigen was first administered intraperitoneally, intravenously, subcutaneously, secondly after 14 to 21 days, and then another antigen was administered. Be sure to As a common auxiliary antigen, complete Freund adjuvant and incomplete auxiliary antigen can be used as appropriate. Splenocytes are obtained 3 to 4 days after the final administration of the immune antigen and used as immune cells. Cell fusion blast cells, for example SP2 / 0 · Ag14 myeloma cells, are used for cell fusion.

Cell fusion between immunized spleen cells and myeloma cells is carried out by known methods such as, for example, Kohler G & Milstein C., Nature , 256 , 495 or modified methods thereof. Immunized spleen cells and SP2 / 0 · Ag14 myeloma cells are mixed at a ratio of 20: 1 to 5: 1, preferably 10: 1, and centrifuged to fuse. The fused cells can be isolated by culturing in a conventional selection medium such as HAT medium to destroy unfused cells and selectively growing the fused cells.

The fusion cells thus obtained can be treated with known restriction protocols (Current Protocols in Immunology) to obtain individual clones that produce the desired antibodies, resulting in the production of monoclonal antibodies. The fusion cell line selected above was named 'NS1-R1O' and was deposited in the Biotechnology Research Institute Gene Bank on August 19, 1999 as Accession No. KCTC 0654BP.

Clones of fusion cells producing the desired antibody can be searched by enzyme immunoassay.

Mass production of the monoclonal antibody of the present invention can be carried out as follows. Inject the fusion cell line into 5 x 10 ⁶ cells / colum in the abdominal cavity of mice injected with 0.5 ml of pristane (2,6,10,14-tetramethyl pentadecane) in advance and harvest ascites approximately 10 days later. Centrifugation yielded a plurality of supernatants, passed through a glass tube (1 × 10 cm) filled with Protein-G bwad (Sigma co.), And glycine-etchiel (0.1 M Glycine-HCl. PH 2.7) solution. It can be obtained by eluting the antibody.

In the monoclonal antibody against swine rotavirus secreted by the cell line established in the present invention, the subtype of the antibody is immunoglobulin G ₃ type (IgG ₃ ), and it is confirmed by Western blotting to specifically recognize the VP6 protein of rotavirus. As a result of confirming the specificity of the antibody by enzyme-immunoassay, the pig rotavirus does not react with swine infectious gastroenteritis virus and swine epidemic diarrhea virus, which are composed of ribonucleic acid such as rotavirus that causes diarrheal disease in pig High specificity for.

By using the monoclonal antibody of the present invention, specific detection of swine rotavirus from pig secretions, feces, and blood can accurately and quickly diagnose whether swine rotavirus is infected. It can also be used as a good means of basic biology research to study the physiological activity, differentiation and growth regulation of mammalian cells.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Fusion Cell Lines Producing Monoclonal Antibodies to Porcine Rotavirus

(Step 1) cultivation and propagation of porcine rotavirus

When MA104 cells (porcine pancreatic cell line, obtained from Chungbuk National University) are inoculated into a T75 flask containing a minimum medium containing 10% fetal bovine serum, and the bottom of the flask is filled with 70 to 90% of cells. Incubated until. The cultured cells were washed twice with physiological saline, inoculated with 1 ml of pig rotavirus solution (100 cfu) and infected for 1 hour, followed by the addition of 20 ml of medium (50 µg / l trypsin). Incubated for hours.

(Step 2) Isolation of Porcine Rotavirus

After centrifuging the rotavirus medium of step 1 containing 110,000 x g for 1 hour, the virus precipitate was dissolved in 200 µl of saline. Slowly pour this solution onto the phosphate buffer containing 60% sugar, then centrifuge at 130,000 x g for 2 hours 30 minutes to elute the virus bands, then suspend in phosphate buffer and again. Centrifugation at 130,000 x g for 2 hours 30 minutes yielded purified swine rotavirus precipitate.

(Step 3) Immunization of Mice

In order to obtain the immunized mice required for the development of the fusion cell line, the phosphate buffer containing porcine rotavirus particles (20 ug) isolated in step 2 and the same amount of complete Freund adjuvant until emulsified The mixture was mixed well and then injected intraperitoneally in 6-8 week old Balb / c mice. Two weeks later, the same phosphate buffer containing rotavirus particles in the same amount as the first injection was mixed with Freund's incomplete auxiliary antigen. Subsequently, serum was separated from blood collected from the blood vessels of the tail of the mouse after 4 to 5 days, and the antibody titer was confirmed. Then, 3 to 4 days before the cell fusion, 10 ug of 0.85% saline containing 10 μg of pig rotavirus particles Was injected intravenously and intraperitoneally.

(Step 4) Cell Fusion and Culture

Three to four days after the last immunization with the antigen, anesthetized mice with ether, and the spleen located on the left side of the body were taken out, and finely ground with a tissue homogenizer, a Hans' buffered saline solution (HBSS). ) To make a suspension, and the splenocytes obtained at this time were put into a 15 ml centrifuge tube and centrifuged. This procedure was repeated twice to wash the splenocytes sufficiently with HBSS.

On the other hand, SP2 / 0.Ag14 cell line as a parent cell for cell fusion was cultured in RPMI medium containing 10% fetal calf serum (FBS) while maintaining a maximum density of about 5 x 10 ⁵ / ml. The parental cells, SP2 / 0.Ag14, were also washed by centrifugation twice with HBSS buffer.

Then, the cells were resuspended in HBSS buffer so that the splenocytes and the parental cells were each 10 ml, and the cells in each suspension were counted to obtain 1 x 10 ⁸ splenocytes and 1 x 10 ⁷ SP2 / 0.Ag14 myeloma cells. The suspension was mixed in 50 ml centrifuge tubes and then centrifuged again to precipitate. The precipitate in the centrifuge tube was dispersed by tapping lightly with a finger and allowed to stand at 37 ° C. for 1 minute and then 45% (w / v) polyethylene glycol (PE) -5% (w / v) contained in HBSS buffer. v) 1 ml of the mixed solution of DMSO was slowly added for 1 minute and shaken slightly for 1 minute. Thereafter, culture medium (RPMI) was added and the suspension was centrifuged again, and the cell pellet was resuspended in HAT medium at 1-2 × 10 ⁵ / ml, and the 96 well micro titer plate was used. 0.2ml aliquots were placed on a microtiter plate and incubated in a CO ₂ humidity incubator whose temperature was adjusted to 37 ° C.

(Step 5) Selection of Fusion Cells Producing Monoclonal Antibodies

Selection of the fusion cells was carried out by enzyme immunoassay (Enzyme immunoassay) using a microplate coated with porcine rotavirus. 50 μl (2 μg / ml) of porcine infectious rotavirus antigen was added to the microplate and attached to the plate surface. The unreacted antigen was removed by washing with phosphate buffer-Tween 20 (PBST) solution. It was. 50 µl of the fusion cell culture was added to each well and allowed to react for 1 hour, followed by washing sufficiently with phosphate buffer-Tween 20 solution to remove unreacted culture. Goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP, Sigma co.) Was added thereto and reacted at room temperature for 1 hour, followed by washing with PBST solution sufficiently. Subsequently, the substrate solution (OPD) of peroxidase was added and reacted, and the degree of reaction was measured at 492 nm using an ELISA reader.

Subsequently, a group of fusion cell lines that produce antibodies having a specifically high binding capacity with the swine rotavirus antigen is selected first, followed by a selection of monofusion cell lines that produce monoclonal antibodies by limiting dilution of monoclonal antibodies. Clones were obtained. The clones showing the highest titers among these clones were selected and cryopreserved, and the double immunodiffusion method confirmed that the subtype of the antibody secreted by the clone was immunoglobulin G ₃ type (IgG ₃ ). This fusion cell line was named 'NS1-R10' and was deposited on August 19, 1999 as Accession No. KCTC 0654BP to the Bank of Biotechnology.

Example 2 Mass Production of Monoclonal Antibodies Specific to Porcine Rotavirus

In order to mass produce monoclonal antibodies against swine rotavirus from the fusion cell line established in Example 1 above, 0.5 ml of pristane was injected intraperitoneally per valgus mice. One week later, 5 x 10 ⁶ cells were injected per mouse into the fusion cell line obtained in Example 1, and ascites fluid was collected from the inflated abdominal cavity. Since the ascites solution contained fusion cells grown at high concentrations, the ascites solution was centrifuged at 10,000 xg to precipitate the fusion cells, which were then passed through a Protein-G glass tube (1 × 10 cm) and the antibody was used as a glycine etchiel solution. Was eluted and dialyzed in 50-fold phosphate buffer for 16 hours and then centrifuged to store the supernatant at -20 ° C.

Example 3: Specificity Assay of Monoclonal Antibodies Specific to Porcine Rotavirus by Western Blotting

(Step 1) SDS-polyacrylamide gel electrophoresis

After infecting MA104 cells with porcine rotavirus and diluting with phosphate buffer-Tween 20 (PBST) solution (0.05% Tween 20), the protein concentration was measured and electrophoresed on 10% SDS-polyacrylamide gel. 1 is shown. Uninfected MA104 cells were used as a comparative group. In Figure 1, row M is the standard molecular protein, row 1 is uninfected MA104 cells, and row 2 is rotavirus infected MA104 cells.

(Step 2) Western Blotting Assay

The protein separated in the gel in step 1 was transferred to the nitrocellulose membrane by electric method. Nitrocellulose membranes were treated with 3% bovine albumin solution for 12-14 hours to reduce the nonspecific response of the proteins transferred to the membrane. In order to identify monoclonal antibodies that specifically respond to porcine rotavirus, NS1-R10 monoclonal antibodies of the present invention were added and reacted for 2 hours at room temperature. This nitrocellulose membrane was washed three times with phosphate buffer containing 0.5% Tween followed by goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP, obtained from Sigma co.). And phosphate buffer containing 0.5% Tween and 0.018% (v / v) 4-chloro-1-naphthol and 0.045% (v / v) H ₂ O ₂ were dissolved in phosphate buffer and methyl alcohol. Color reaction was carried out using a substrate dissolved in a mixed solution.

The results are shown in FIG. Here, column M is a standard molecular protein, column 1 is an uninfected MA104 cell, and column 2 is a rotavirus infected MA104 cell, wherein the monoclonal antibody of the present invention is 45 kDa in the protein of a cell infected with porcine rotavirus. It was confirmed that the compound specifically reacts with the molecular weight of the VP6 protein.

Example 4: Specificity Assay for Swine Rotavirus of Monoclonal Antibodies by Enzyme Immunoassay

Porcine rotavirus, swine infectious gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) that infect pigs and cause diarrhea in microplates 50 μl (2 μg) per well / Ml) was added to the plate surface and unreacted antigens were washed away. 50 μl of the fusion cell culture obtained in Example 1 was added to each well, followed by reaction for 1 hour, followed by sufficient washing with phosphate buffer-Tween 20 (PBST) solution. Anti-mouse IgG horseradish peroxidase was added thereto and reacted at room temperature for 1 hour, followed by sufficient washing with PBST solution. Subsequently, the substrate solution of peroxidase was reacted, and the degree of reaction was measured at 492 nm with an enzyme immunoassay.

The results are shown in FIG. 3, and as shown here, the monoclonal antibody of the present invention can be confirmed to be an antibody that specifically reacts only with swine rotavirus VP6 protein.

Thus, using the monoclonal antibody that specifically recognizes the rotavirus VP6 protein of the present invention and the fusion cell line producing the same, it is possible to accurately and quickly diagnose the infection of swine rotavirus in the test of the secretion of swine from rotavirus. It is possible to enhance the prevention effect by confirming the propagation path and early detection, and the antibody of the present invention has a specific reactivity against rotavirus in mammals other than pigs to study the physiological activity, differentiation and growth regulation of mammalian cells. It can be used as a good means of basic biology research.

Claims (5)

Monoclonal antibody produced by mouse-derived fusion cell line NS1-R1O (KCTC 0654BP) and specifically binding to VP6 protein of porcine rotavirus. The method of claim 1, Monoclonal antibody with subclass type of IgG ₃ . Fusion cell line NS1-R1O (KCTC 0654BP), which produces monoclonal antibodies against the VP6 protein of porcine rotavirus. Injecting the fusion cell line of claim 3 into the intraperitoneal of a mouse, and then taking ascites fluid to produce a monoclonal antibody against the swine rotavirus VP6 protein, comprising separating the monoclonal antibody against the VP6 protein of swine rotavirus. How to. The method for detecting swine rotaviruses, wherein the monoclonal antibody of claim 1 is used.