AU2021105168A4 - Monoclonal Antibody against African Swine Fever Virus Helicase D1133L and Hybridoma Cell Strain Secreting the Monoclonal Antibody and Application Thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and relates to a monoclonal antibody against African swine fever virus helicase D1133L and a hybridoma cell strain secreting the monoclonal antibody. The monoclonal antibody is prepared by mouse abdominal cavity culture and in vitro cell culture of hybridoma cell 7D12, and the titer test results show that the titer of ascites antibody produced by mice induced by monoclonal antibody secreted by hybridoma cell strain 7D12 is 107, and the titer of hybridoma cell culture supernatant reaches 1:1280. The monoclonal antibody prepared by the invention has accumulated key biological materials for studying ASFV helicase function and its molecular mechanism, and the indirect immunofluorescence antigen detection method established based on the biological characteristics of the monoclonal antibody provides support for accurate and rapid diagnosis of ASFV, and the monoclonal antibody prepared by the invention can significantly inhibit or block the replication of ASFV and provide possible product support for prevention and treatment of ASFV.

Description

Monoclonal Antibody against African Swine Fever Virus Helicase D1133L and

Hybridoma Cell Strain Secreting the Monoclonal Antibody and Application

Thereof

TECHNICAL FIELD

The invention belongs to the technical field of bioengineering, and particularly

relates to a monoclonal antibody against African swine fever virus helicase D1133L

and a hybridoma cell strain secreting the monoclonal antibody, and also relates to the

application of the hybridoma cell strain and the monoclonal antibody.

BACKGROUND

African swine fever (ASF virus) is a virulent animal infectious disease caused by

African swine fever virus, which mainly infects pigs with a morbidity and mortality as

high as 100%. Other host animals can also carry the virus (soft tick). The clinical

manifestations are high temperature, cyanosis, conjunctival hemorrhage and ascites,

accompanied by respiratory disorders and neurological symptoms, and the incidence

and mortality are all 100%; the main storage host is spleen, and the spleen of diseased

pigs is many times larger than that of normal pigs. When the disease is extensive and

serious, the growth of pigs is blocked and needs to be culled, resulting in serious

economic losses. Like other poxviruses, the immune escape mechanism evolved from

ASFV can help the virus survive under the host immune defense condition and form

persistent infection. At the same time, the diversity of antigen/immune target among

different strains of ASFV makes the virus infect the same host repeatedly.

ASFV belongs to African classical swine fever virus family, and is the only one,

which is a double-stranded linear DNA virus. Under electron microscope, the size of

virus particles was 200nm, with lipid capsule outside and double-stranded DNA core

inside The virus particles are icosahedron, corresponding to 1892-2172 shell particles

(each shell particle has a diameter of 13 nm); there is a hole in the center, which is

hexagonal, and the distance between shell and particle is 7. 4 nm-8.1 nm. The ASFV

genome is a single molecule linear double-stranded DNA with covalently closed ends,

which is 170kb-90kb in size. The whole genome contains 151 ORFs, which can

encode 150-200 kinds of protein, including 5 helicases named D1133L, B962L,

QP509L, Q706L and A589L, all belonging to the helicase superfamily II. The

research proves that QP509L and Q706L belong to the deadbox family of the helicase

superfamily IIDEXH/D-box family among the five helicases of ASFV, and indirectly

proves that D1133L is a member of this family through gene sequence analysis.

Sequence analysis showed that the sequences of five helicases among different ASFV

strains were relatively conservative. D1133L gene is located in the central segment of

ASFV genome, which is about 40kb away from BQ96R. It replicates 12 hours after

virus DNA begins to replicate, and it is a late gene, which can encode and produce

125.847 kDa protein.

ASF infection can often be preliminarily diagnosed by typical clinical symptoms

and epidemic situation. However, ASF is often confused with the clinical symptoms

of classical swine plague. In this case, laboratory diagnosis is very necessary.

Therefore, the diagnosis needs further laboratory tests by negative staining with electron microscope, serological methods, histopathology, PCR detection, RFLP and animal inoculation. Studies have confirmed the feasibility of these methods, however, these detection methods are more or less limited in application, which makes it difficult to detect pathogens as early as possible and is not conducive to large-scale epidemiological investigation; at present, there is no immunoassay kit for serological detection at home and abroad. Molecular biological methods have high specificity and accurate results, but they have high requirements for experimental conditions and operators, so they are mostly used in laboratory research, so it is difficult to apply in the clinical stage. Enzyme immunoassay technology organically combines the high efficiency of enzymatic reaction with the high specificity of immune reaction. It has the advantages of high sensitivity, strong specificity, low requirements for instruments and equipment, low cost, simple and quick operation, no radioactive pollution, high degree of automation, long preservation time of reagents, etc. It is suitable for large-scale on-site detection work and may become a diagnostic method with great popularization value. With the increasing losses caused by ASFV to pig industry year by year, it is inevitable to carry out extensive ASFV monitoring, and there is no ASF detection standard suitable for the whole world. Under the background of no commercial vaccine of ASFV, the development of IFA and ELISA diagnostic kits for detecting African swine fever is of great significance to the epidemic monitoring, epidemiological investigation and improvement of immunization strategies.

SUMMARY

According to the invention, the African swine fever virus helicase D1133L is

necessary for the replication of the African swine fever virus. According to the

invention, the D1133L gene of the ASFV is knocked out by adopting the crispercas9

gene editing technology, and IFA results show that the ASFV cannot replicate and

cannot survive after the D1133L gene is knocked out. The Q -RT -PCR results showed

that interfering D1133L gene significantly inhibited the replication of ASFV after

interfering D1133L gene with Si-RNA interference sequence.

In addition, the overexpression of D113L protein in the invention is detected by

Luciferase and Western blot, and the results show that D1133L can inhibit host

natural immune nuclear factor NF-KB.

The amino acid sequence of the helicase D1133L is shown in SEQ ID NO: 1.

On the basis of the above findings, the present invention provides a hybridoma

cell strain secreting monoclonal antibodies against African swine fever virus helicase

D1133L, specifically, the purified protein of African swine fever virus helicase

D1133L expressed by Escherichia coli is used as immunogen to immunize BALB/c

mice, and its spleen lymphocytes are fused with SP2/0 myeloma cells (5:1). A

hybridoma cell line stably secreting monoclonal antibody against D1133L protein was

obtained by screening, named as hybridoma cell line 7D12, which was preserved in

China Center for Type Culture Collection with the number of CCTCC NO:

C2020180.

According to the invention, the hybridoma cell strain 7D12 is inoculated into the

abdominal cavity of a mouse, and when the abdominal cavity of the mouse is extremely inflated, ascites is extracted, and upper oil and sediment are removed to obtain a mouse ascites antibody, which is a monoclonal antibody against African swine fever virus helicase D1133L.

According to the invention, hybridoma cell strain 7D12 is inoculated into

DMEM culture medium containing 20% FBS and 1% antibiotics for culturing, cells

are separated, and supernatant of cell culture medium is collected to obtain

monoclonal antibody, which is monoclonal antibody against African swine fever virus

helicase D1133L. It was found that the subtype of the monoclonal antibody was IgGI.

The antibody titer determination results show that the titer of ascites antibody

produced by mice induced by the hybridoma cell strain of the invention is 107, and

the titer of hybridoma cell culture supernatant reaches 1:1280.

Another object of the present invention is to apply the hybridoma cell strain

7D12 or the monoclonal antibody mentioned above in the preparation of reagents for

diagnosing or detecting African classical swine fever virus antigen infection.

Yet another object of the present invention is to apply the above monoclonal

antibody in the preparation of ASFV inhibitors.

The invention also provides a kit containing the monoclonal antibody.

The invention has the beneficial effects that:

According to the research of Q-RT-PCR and IFA, the monoclonal antibody

against African swine fever virus helicase D1133L can inhibit ASFV replication in a

dose-dependent manner, so that the monoclonal antibody can be used for preparing

ASFV inhibitors.

In addition, Western blot detection results show that the monoclonal antibody of

the invention can be specifically combined with the ASFV antigen. IFA results show

that the supernatant of hybridoma cell culture can react with PAM cells infected by

ASFV, and fluorescence signals can be seen under fluorescence microscope,

indicating that the McAb obtained by the invention can react specifically with ASFV.

The result of the specificity test of the monoclonal antibody shows that the

monoclonal antibody of the invention has good specificity and only reacts with

ASFV, but does not react with FMDV, SVV and CSFV. Therefore, that monoclonal

antibody can be used for preparing reagent for diagnosing or detecting African

classical swine fever virus antigen infection and for detecting ASFV.

Generally speaking, the monoclonal antibody prepared by the invention has

accumulated key biological materials for studying the function of ASFV helicase and

its molecular mechanism. The indirect immunofluorescence antigen detection method

established based on the biological characteristics of the monoclonal antibody

provides support for accurate and rapid diagnosis of ASFV. The monoclonal antibody

prepared by the invention can significantly inhibit or block the replication of ASFV,

thus providing possible product support for prevention and treatment of ASFV.

The titer determination result of the antibody of the invention shows that the titer

of ascites antibody produced by mice induced by the hybridoma cell strain of the

invention is 107, and the titer of hybridoma cell culture supernatant reaches 1:1280.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is the result of Luciferase and Western blot detection of inhibition of

nuclear factor NF-KB by D1133L. Fig. 1A is a graph of Luciferase detection results,

in which abscissa indicates different proteins, vec indicates control, D1133L indicates

D1133L protein, and ordinate indicates transformation level of NF-KB; Fig. lB shows

the results of Western blot.

In Figure 2, A is a schematic diagram of crispercas9 knocking out D1133L, and

B is an indirect immunofluorescence test verification result diagram.

Figure 3 is a graph of inhibition of ASFV replication after Si-RNA interference

with D1133L by Q-RT-PCR.

Figure 4 is a detection chart of expression form of D1133L protein in

Escherichia coli. In the figure, 1 is supernatant, 2 is precipitate and 3 is Marker.

Figure 5 is a graph showing the isolation and purification results of D1133L

protein.

Figure 6 is a graph showing the identification results of monoclonal antibody

subtypes of the present invention.

Figure 7 is a Western blot analysis of monoclonal antibody reactivity of lysate

after ASFV infection with PAM cells; M: molecular weight of pre-dyed protein; 1:

PAM cell lysate, 2: ASFV infected PAM cell lysate.

Figure 8 is the result of indirect immunofluorescence detection of reactivity

between monoclonal antibodies and clinical isolates of ASFV. Al is the 594nm color

diagram of red fluorescent labeled secondary antibody after the supernatant of 7D12

hybridoma cell culture is mixed with PAM cells infected by ASFV, A2 and B2 are the

DAPI staining diagram of PAM cells. B Iis the staining pattern of 7D12 hybridoma

cell culture supernatant mixed with normal PAM cells.

Figure 9 is a graph showing the detection result of the inhibition of ASFV

replication by the monoclonal antibody of the present invention. In which:

Figure 9: A is a graph of Q-RT-PCR results; Fig. 9B shows the dose-effect

diagram of supernatant from Mock, 10tM, 20[M, 40tM and 80 m hybridoma cells

7D12 from left to right. The first line is 594nm color diagram of red fluorescence

labeled secondary antibody; the second line is the 488nm color diagram of green

fluorescent labeled secondary antibody, and the third line is the DAPI staining

diagram of PAM cells; the fourth row is the Merge diagram.

Preservation information:

Date: September 20, 2020;

Name of preservation unit: China Center for Type Culture Collection;

Preservation number: CCTCC NO: C2020180;

Address: Wuhan University, Wuhan, China;

Classification and name: Hybridoma cell line 7D12.

DESCRIPTION OF THE INVENTION

The technical scheme of the present invention will be described in detail with

specific examples below.

Sources of virus species, cells, experimental animals and biochemical reagents

used in this example:

(1) The recombinant PET-28a-D1133L and SP2/0 cells were preserved by the

epidemiology team of foot-and-mouth disease and new disease in Lanzhou veterinary

institute, Chinese academy of agricultural sciences (reference for preparation method:

yang min. isolation, identification and biological characteristics of malignant bone

tumor side group (SP) cells[D].2011.Yun-Gang Z,Ju-Lun Y,Li W.Construction of

prokaryotic expression plasmid PET-28a (+)-Ha-Ras and expression and

purification of p21ras[J].The Journal of Practical Medicine,2007.)

(2) ASFV strain was isolated by the epidemiological team of foot-and-mouth

disease and new diseases of Lanzhou Veterinary Institute of Chinese Academy of

Agricultural Sciences and stored in the National African Swine Fever Regional

Laboratory (Lanzhou, China).

(3) Eight-week-old female BALB/c mice were purchased from the Experimental

Animal Center of Lanzhou Veterinary Institute, Chinese Academy of Agricultural

Sciences.

(4) Standard fetal bovine serum and modified culture medium RPMI 1640 were

purchased from Gibco Company. Complete Freund's adjuvant, incomplete Freund's

adjuvant, selective media HAT and HT, PEG(MW4000) for cell fusion, reagent kit

for monoclonal antibody subtype identification and horseradish peroxidase labeled

sheep anti-mouse IgG(HRP-IgG) were purchased from Sigma Company. HRP

labeled Alexa goat anti-mouse IgG secondary antibody (wavelength 594 is red

fluorescence) and HRP labeled Alexa goat anti-rabbit IgG secondary antibody

(wavelength 488, green fluorescence) were purchased from Proteintech Company.

Example 1 helicase D1133L inhibits host nuclear factor NF-KB.

)x106 293t cells were spread in a 6-well plate and placed in 5% C02 at 37C for

2h. the overexpression plasmid D1133L (which was prepared by inserting D1133L

gene between BamHI and EcoRI of pCDNA3.1 plasmid) was transfected for 24h,

then stimulated with TNF-a for 12h, and NF-KB was detected by Luciferase and

Western blot.

As shown in fig. 1A, compared with the control vec, D133L protein inhibited

the transformation level of nf K b; Fig. lB shows that at protein level, with the

increase of D1133L dosage, the expression of NF-KB gradually decreases. Generally

speaking, D1133L inhibited nuclear factor NF-KB in a dose-dependent manner.

Example 2: Necessity of African swine fever virus helicase D1133L for

replication of African swine fever virus

2.1 The knockdown helicase D1133L gene ASFV cannot replicate

With the endogenous knock-out of D1133L gene in crispercas9, according to the

framework shown in fig. 2A, according to the principle of homologous

recombination, ASFV without Dl133L gene was formed.

1x106 PAM cells were spread on a 6-well plate, placed in 5% C02 at 37C for2

hours, transfected with 1 g D1133L knockout plasmid, and then inoculated with

0.1M of ASFV. After passing to the 9th generation, the ASFV strain and WT-ASFV

without D1133L gene were verified by indirect immunofluorescence test.

As shown in fig. IB, the results of indirect immunofluorescence test showed that

there was almost no fluorescence in ASFV group with D1133L gene deletion, which

indicated that ASFV could not replicate and survive after D1133L gene deletion.

2.2 Si-RNA interference D133L significantly inhibited ASFV replication

Search D1133L sequence by NCBI, and design si RNA interference sequence

by snap gene analysis software (Si-RNA-negetive control fam:forward primer 5'

UUCUCCGAACGMGUCACGUTT-3'andR: 5'-ACGMGACACGUUCGGAGAATT-3';

Si- RNA-1067 fam: forward primer 5'-CCAUCAAGGGUCUCCAAUUTT-3'and R: 5'

-AAUMGGAGACCCUMGAMGGTT-3'), which were synthesized by Gene Pharma

company in Shanghai.

1x106 PAM cells were spread in a 6-well plate, and placed in 5% C02 at 37C

for 2 hours. After transfecting 1 g of Si-RNA with Poly Plus, they were inoculated

with O.1M ASFV. After 12 hours, the samples were collected and detected by

Q-RT-PCR. As shown in fig. 2. the replication of ASFV is significantly inhibited

after Si-RNA interferes with D1133L gene.

The above results indicated that the helicase D1133L gene is necessary and

indispensable for the replication of ASFV.

Example 3: Preparation of Monoclonal Antibodies

3.1 Purification and detection of recombinant protein

The genetically engineered bacteria (recombinant PET-28a-D1133L) expressing

D1133L protein after induction were cultured, and the cells were collected, crushed, and supernatant and precipitate were collected respectively. the electrophoresis detection results are shown in fig. 4, and D1133L protein band appears in the collected precipitate, indicating that D1133L protein is expressed as inclusion body.

The genetically engineered strain (recombinant PET-28a-D1133L) expressing

D1133L protein after induction was cultured at 37C and 37°C200rpm for 5 hours,

and then the strain liquid was harvested, centrifuged at 5000 g for 10 minutes, and

then added with 10 mL PBS. The bacterial solution was resuspended, and the

samples were placed in ice bath for ultrasonic cracking (30% power) for 3 s with an

interval of 15 s and ultrasonic cracking for 40min. Then centrifuge at 10,000 g for 20

min, discard the supernatant, and keep the cell precipitate. The recombinant protein

was purified by GE's nickel column HIS purification system, and the operation

process was as follows:

The genetically engineered bacteria (recombinant PET-28a D1133L) expressing

D1133L protein after induction were cultured, and the cells were collected, crushed,

and supernatant and precipitate were collected respectively. the electrophoresis

detection results are shown in fig. 4, and D1133L protein band appears in the

collected precipitate, indicating that DI133L protein is expressed as inclusion body.

The genetically engineered strain (recombinant PET-28a D1133L) expressing

Dl133L protein after induction was cultured at 37C and 37C, 200 rpm for 5 hours,

and then the strain liquid was harvested, centrifuged at 5000 g for 10 minutes, and

then added with 10 mL of PBS. The bacterial solution was resuspended, and the

samples were placed in ice bath for ultrasonic cracking (30% power) for 3 s with an interval of 15 s and ultrasonic cracking for 40 min. Then centrifuge at 10,000 g for min, discard the supernatant, and keep the cell precipitate. The recombinant protein was purified by GE's nickel column HIS purification system, and the operation process was as follows:

1) Re-suspend the cell pellet with buffer B, and shake it carefully at room

temperature for 60min to make it clear and transparent, so as to avoid foaming.

2) Centrifuge 10000 g for 30min, discard the precipitate, and transfer the

supernatant to another centrifuge tube.

3) 1 mL of 50% ninta resin was suspended and added to 4 mL of cell lysate,

which was mixed gently and combined at room temperature for 60min.

4) After carefully adding the mixture of lysate and ninta resin suspension into the

empty column with closed lower end, remove the closed cover at the lower end of

the column, collect the effluent and save it for SDS-PAGE analysis.

5) Rinse the foreign protein twice with 4 mL buffer C, and save the rinsed

components for SDS-PAGE analysis.

6) Elute the target protein with 0.5 mL buffer D for 4 times, and then with 0.5

mL buffer E for 4 times, and collect the fractions for SDS-PAGE analysis.

As shown in fig. 5, the purified D1133L protein has higher purity and higher

protein yield.

3.2. Immunize mice

Ten-week-old female BALB/c mice were immunized three times with purified

D1133L protein as immunogen. The immunization program is as follows: first

immunization, each mouse was mixed with 50 g D1133L protein and the same

amount of FCA to make emulsifier, and injected subcutaneously and

intraperitoneally in the neck and back; the second exemption was carried out 2

weeks after the first exemption, and FCA was replaced by FICA, with the same dose

and method as above; three exemptions were given 2 weeks after the second

exemption, with the same dose and method as the second exemption. One week

before cell fusion, the method of strengthening immunity was intraperitoneal

injection of 100 g purified D1133L protein.

3.3 Cell Fusion

The results of cell fusion were better 3-5 days after strengthening immunization

in immunized mice. The steps of cell fusion are as follows: the peritoneal

macrophages of BALB/c negative mice are taken according to the conventional

method and spread on a 96-well cell culture plate for later use. The positive mice

were killed by neck cutting, and their spleen cells were taken aseptically. The spleen

cells and SP2/0 myeloma cells were fused with PEG4000 according to the ratio of

:1, and the fused cells were spread on the prepared feeder cells (Kohler G ,Milstein

C. Continuous culture of fused cells secreting antibody of predefined specificity. [J].

Nature ,1975 ,256(5517):495-497).

3.4 Screening and subcloning of cell lines

Coat polystyrene plate with purified D1133L protein at 1 g/well at 37C for 3

h; PBST (0.01 mol/L, pH 7. 3, PBS+0. 05% Tween-20) was washed for 3 times,

min/time; Add PBST /BSA (0.01 mol/L, pH 7.3, PBST +2% BSA), 200 [L/well,

and seal at 37C for 2h or at 4C overnight; PBST was washed 3 times, 5min/time,

patted dry, and stored at 20°C for later use. The best dilution ratio of antigen was

determined by square array test, that is, ASFV D1133L protein was coated on

96-well plate, and square array titration test was carried out to obtain the

concentration of antigen and the dilution ratio of positive serum of prokaryotic

expression protein D1133L immunized mice. Meanwhile, the serum of negative

mice was set as negative control. According to the reaction results, the best coating

concentration of antigen was selected. The supernatant of hybridoma cell culture

was detected by conventional indirect ELISA. The ELISA plate was placed on the

ELISA microplate reader, and S/P > 2. 1 was used as the positive criterion. Clonal

pores of hybridoma cells resistant to D1133L protein were selected.

According to the above screening method, the positive hybridoma cells were

subcloned. Sub-cloning adopts limited dilution method. Primitive pore cells are

diluted with HT medium and then subdivided into 96-pore cell plates, and one

primordial pore cell is divided into one plate. After subcloning, observe the number

and status of cells in each well. Take the well which secretes antibody stably and is a

single clone as far as possible after subcloning for the second subcloning. After three

times of subcloning, the positive rate of the subclone board which was originally

monoclonal should reach 100%. A hybridoma cell strain capable of stably secreting monoclonal antibodies specific to D1133L protein was obtained, named as hybridoma cell strain 7D12, which was preserved in China Center for Type Culture

Collection, and its microbial preservation number was CCTCC NO: C2020180.

3.5 Mass preparation of monoclonal antibodies

Ascites antibody: 10-week-old healthy BALB/c mice were intraperitoneally

injected with liquid paraffin (0.5 Ml/mouse). After 1 week, 105 hybridoma cells

were injected into the abdominal cavity. After 7-10 days, ascites was extracted once

every 2 days when the mouse abdomen was extremely distended. The extracted

ascites was centrifuged at 1,000 g for 10 minutes to remove the upper oil and

sediment, and the supernatant was stored separately at 20°C or70°C forlateruse.

Hybridoma cell culture supernatant: Hybridoma cell line was cultured with 20%

FBS+1% double antibody (penicillin and streptomycin) +DMEM for 7D12. After 24

hours, the cells were separated and the supernatant of cell culture medium was

collected for later use.

Example 4 detection of biological characteristics of monoclonal antibodies

4.1 Determination of antibody titer

Ascites antibody:

The purified Dl133L protein was diluted with the coating solution, and then

added to the ELISA reaction plate at 1 g/well, and left overnight at 4C. The next

day, pour out the liquid 3min the hole and wash it for 3 times, each time for 3

minutes. Add 200 pL sealing solution to each well, stand at 37C for lh, and wash for 3 times, each time for 3min. The ascites antibody prepared in Example 3 was serially diluted 10 times with PBS on another plate, and 100 L/well was added to the closed ELISA plate. Each sample was duplicated in parallel, with PBS as negative control and ASFV positive serum as positive control. Incubate in 37°C incubator for 1 h, wash for 3 times, each time for 3min. Then add horseradish peroxidase-labeled goat anti-mouse antibody (1: 5000), 100 [L/well, incubate at

37°C for 1 h, and wash for 5 times, each time for 3min. Add 100 L/well of freshly

prepared substrate solution, place it in dark place at room temperature for 15min,

then add 50 L/well of stopping solution, and determine the OD450, taking s/p > 2.1

as the positive criterion.

Hybridoma cell culture supernatant:

Same as the above method, coated with D1133L protein, the supernatant of

hybridoma cell culture containing monoclonal antibody prepared in example 3 was

diluted ten times by 9 gradients, and other conditions were the same as ascites

detection.

The maximum dilution of positive reaction is the titer of ascites antibody and the

titer of culture supernatant of positive hybridoma cells.

The detection results are shown in Table 1 and Table 2. The results show that the

titer of ascites antibody produced by induced mice is 107, and the titer of hybridoma

cell culture supernatant reaches 1280.

Table 1 Titer of ascites monoclonal antibody

Dilution 101 104 101 106 107 108 Negative multiple control

OD4 5 0 1.551 1.468 1.363 0.808 0.509 0.373 0.193

Table 2 Titer of hybridoma cell culture supernatant

Dilution 10 20 40 80 160 320 640 1280 2560 Negative multiple control

D1133L 0.6 0.44 0.499 0.398 0.363 0.357 0.321 0.293 0.16 0.134

4.2 Subclass identification of monoclonal antibodies

Subclasses were identified by using the mouse monoclonal antibody typing gold

label test paper kit of IsoStrip Company, and the specific steps were operated

according to the instructions of the kit. The main steps are as follows:

A: The antibody was diluted with 1% B- -11 serum albumin (BSA) or

phosphate-buffered saline (PBS) (pH=7.2-7.6). The dilution ratio of hybridoma cell

culture supernatant was 1: 10-1: 100, and the total volume after dilution was 150 [L.

B: Open the latex head on the glass tube, add 150 L diluted sample into the tube

and mix well with the substrate, let it stand at room temperature for 30s, insert the

gold-labeled test paper into the glass tube, take it out after the sample is completely

absorbed, and let it stand at room temperature for 1min. Observation results.

The subtype identification results are shown in fig. 6. The results show that the

subtype of monoclonal antibody prepared in example 3 is IgG1.

Example 5 Application of Monoclonal Antibodies

5.1 Detection of ASFV antigen by indirect immunofluorescence

1. Identification of immunological activity of monoclonal antibody (McAbs)

Western blot was used to analyze the immunological activity of monoclonal

antibodies. The Western blot procedure was as follows: SDS-PAGE electrophoresis

was performed on ASFV cell lysate and pre-dyed protein Marker (gel concentration

was 10%), the electrophoresis product was transferred to PVDF membrane, and the

protein band and pre-dyed protein Marker band of each lane were cut out (stored for

later use). Then, the PVDF membrane strip containing the whole virus cleavage

product was washed in deionized water for 10min, and sealed with 5% skim milk at

room temperature for 1h. The sealed PVDF membrane strip reacted with ascites

induced by 1:2000 times diluted monoclonal antibody at 37C for 1 h, PBST (0.01

mol/L, pH 7. 2; 0. 05% Tween-20 was washed three times, then reacted with HRP

goat anti-mouse IgG (1: 4000) at 37C for 1h, PBST was washed three times, and

then developed with 3,3' diaminobenzidine (DAB) substrate solution.

The Western blot results are shown in fig. 7. the results show that the

monoclonal antibody prepared in example 3 can specifically bind to ASFV antigen.

2. Establishment and application of indirect immunofluorescence (IFA) detection

method

(1) Detection of ASFV antigen

PAM cells infected by ASFV(0.1M) strain and normal PAM were cultured for 2

days, then the supernatant was poured out, lightly washed with PBS for 3 times,

fixed with 4% paraformaldehyde for 15min, washed with PBS for 3 times, permeabilized with 0. 1% triton x 100 for 15min, then washed with PBS for 3 times, and then sealed with 1% BSA for 1 hour. after washing with PBS for 3 times, the supernatant of hybridoma cell culture prepared in example 3 was dropped. After washing with PBS for 3 times, the secondary antibody was HRP-labeled Alexa goat anti-mouse IgG secondary antibody (red fluorescence at wavelength 594nm) or

HRP-labeled Alexa goat anti-rabbit IgG secondary antibody (green fluorescence at

wavelength 488nm) (1:500) and acted at 37C for 1 h; After washing with PBS for 3

times, it was stained with 5 g/mL DAPI for 10min, washed with PBS for 3 times,

and observed under fluorescence microscope.

3. Specific test of monoclonal antibody

Swine foot-and-mouth disease virus (FMDV), seneca virus (SVV), classical

swine fever (CSFV) and Escherichia coli were used

Positive serum, the antibody of hybridoma cell culture supernatant prepared in

Example 3 was detected by ASFV ELISA antibody detection kit to test its

specificity.

ELISA results show that the monoclonal antibody prepared in example 3 only

reacts with ASFV positive serum, but does not react with FMDV, SVV, CSFV

positive serum. The test results are shown in table 3, in which "-" indicates no

reaction and "+"indicates reaction.

Table 3 Specific identification of monoclonal antibodies

Virus FMDV SVV CSFV E.coil ASFV result

+ 5.2 Monoclonal antibodies inhibit the replication of ASFV

PAM cells infected by ASFV strain and normal PAM were cultured for 2 days,

and Mock blank control was set. 10OM, 20jM, 40tM and 80Mm7D12 cell

supernatants were dripped into the ASFV infected group respectively, and then

washed lightly with PBS for 3 times after 24h, fixed with 4% paraformaldehyde for

min, washed with PBS for 3 times, permeabilized with 0. 1% triton X100 for

min, washed with PBS for 3 times, then blocked with 1% BSA for 1h, washed

with PBS for 3 times, and then cultured with hybridoma cells prepared in Example

3. After washing with PBS for 3 times, the secondary antibodies were HRP-labeled

Alexa goat anti-mouse IgG secondary antibody (red fluorescence at 594nm) and

HRP-labeled Alexa goat anti-rabbit IgG secondary antibody (green fluorescence at

488nm) (1:500), which were exposed to room temperature for 5 hours. After

washing with PBS for 3 times, it was stained with 5 g/mL DAPI for 10min, washed

with PBS for 3 times, and observed under fluorescence microscope.

The Q-RT-PCR results showed that the supernatant of hybridoma cell culture

prepared in example 3 interfered with D1133L of ASFV, and then the replication of

ASFV was significantly inhibited, as shown in fig. 9A. The results of IFA are shown

in fig. 9B. with the increase of the supernatant of hybridoma cell culture, the

fluorescence signal gradually decreases, indicating that ASFV replication is inhibited; The fluorescence signal of DAPI decreased gradually, which indicated that the amount of PAM cells decreased with the increase of supernatant of hybridoma cell culture. The results showed that the supernatant of 7D12 hybridoma cell culture could inhibit the replication of ASFV.

In addition, the results of IFA are the same as those of IFA, as shown in fig. 9C.

Any modification, equivalent substitution, improvement, etc. made within the

spirit and principle of the present invention shall be included in the protection scope

of the present invention. There are many other embodiments of the present

invention. Without departing from the spirit and essence of the present invention,

those skilled in the art can make various corresponding changes and modifications

according to the present invention, but these corresponding changes and

modifications should belong to the protection scope of the appended claims.

Claims (10)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A hybridoma cell strain secreting monoclonal antibody against African swine

fever virus helicase D1133L is characterized in that the hybridoma cell strain is 7D12,

which is preserved in China Center for Type Culture Collection with the preservation

number of CCTCC NO: C2020180.

2. A monoclonal antibody against African swine fever virus helicase D1133L

secreted by the hybridoma cell line as claimed in claim 1.

3. The monoclonal antibody according to claim 2 is characterized the amino acid

sequence of the helicase D1133L is shown in SEQ ID NO: 1.

4. The monoclonal antibody according to any one of claims 2 to 3 is

characterized in that the subtype of the monoclonal antibody is IgG1.

5. The application of the hybridoma cell strain according to claim 1 in preparing

reagents for diagnosing or detecting African swine fever virus infection.

6. The application of the monoclonal antibody according to any one of claims 2

to 4 in preparing reagents for diagnosing or detecting African classical swine fever

virus antigen infection.

7. The application of the monoclonal antibody according to any one of claims 2

to 4 in preparation of ASFV inhibitor.

8. A kit is characterized by comprising the monoclonal antibody according to

any one of claims 2 to 4.

9. The preparation method of the monoclonal antibody according to claim 2 is

characterized in that the hybridoma cell strain according to claim 1 is inoculated into the abdominal cavity of a mouse, ascites is extracted when the abdominal cavity of the mouse swells, impurities are removed, and the supernatant is preserved.

10. The method for preparing monoclonal antibody according to claim 2 is

characterized in that the hybridoma cell strain according to claim 1 is inoculated into

DMEM medium containing 20% FBS and 1% antibiotics for culture, cells are

separated, and the cell culture medium is collected and cleaned.

FIGURES 1/6

A Figure 1