CN116143887A

CN116143887A - Antigen epitope peptide of African swine fever virus p72 protein, monoclonal antibody aiming at antigen epitope peptide and application of monoclonal antibody

Info

Publication number: CN116143887A
Application number: CN202211096917.7A
Authority: CN
Inventors: 郑海学; 石正旺; �田宏; 周改静; 罗俊聪; 张晓阳; 万颖; 茹毅; 杨帆
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2022-09-08
Filing date: 2022-09-08
Publication date: 2023-05-23

Abstract

The invention belongs to the technical field of biology, and particularly relates to an epitope peptide of an African swine fever p72 protein, a monoclonal antibody aiming at the epitope peptide and application thereof. The invention is characterized by treating African swine feverThe toxic p72 gene is cloned into a prokaryotic expression vector pET-30a, a prokaryotic expression vector pET-30a-p72 is constructed, and p72 recombinant protein is obtained through expression and purification. Immunizing a Balb/c mouse with p72 recombinant protein, fusing spleen cells of the mouse with myeloma SP2/0 cells to obtain hybridoma cells, screening by an indirect ELISA method to obtain monoclonal antibodies against the p72 recombinant protein, identifying that epitope peptides of p72 protein corresponding to the antibodies are 20-39 peptide segments of African swine fever virus p72 protein, wherein the amino acid sequence is ²⁰ ILAQDLLNSRISNIKNVNKS ³⁹ 。

Description

Antigen epitope peptide of African swine fever virus p72 protein, monoclonal antibody aiming at antigen epitope peptide and application of monoclonal antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an epitope peptide of an African swine fever p72 protein, a monoclonal antibody aiming at the epitope peptide and application thereof.

Background

African swine fever (African swine fever, ASF) is an acute, febrile, virulent infectious disease caused by African swine fever virus (African swine fever Virus, ASFV) with a mortality rate as high as 100%. The world animal health Organization (OIE) lists it as an animal epidemic that must be reported, and China lists it as a class of animal epidemic. At present, no commercial vaccine is available, and early discovery, early diagnosis, early treatment and improvement of biosafety management level are main measures for preventing and controlling African swine fever at present.

ASFV is a nuclear double-stranded DNA virus, is in an icosahedral structure, has a genome length of 170-193 kkb, has 150-167 open reading frames, can code 150-200 proteins, and comprises an outer envelope protein, a capsid protein, an inner envelope protein, an inner core-shell protein and a genome of the virus from outside to inside. The outer envelope protein is composed of CD2v, p24, p12 and the like, wherein the CD2v protein mediates erythrocyte adsorption and can provide important basis for diagnosis of ASFV. The capsid protein consists essentially of a major protein p72, encoded by B646L. The p72 protein is the main structural protein of ASFV, accounts for about 31-33% of the total mass of the virus, has small variation, has strong antigenicity and immunogenicity, and is an ideal target molecule for diagnosing African swine fever.

Disclosure of Invention

The invention adopts a cell fusion technology, successfully obtains a monoclonal antibody specific to p72 protein through subcloning screening, carries out epitope identification, and discovers that peptide fragments of ASFV p72 protein can be identified ²⁰ ILAQDLLNSRISNIKNVNKS ³⁹ ) The peptide fragment is subjected to conservation analysis to find that different strains are highly conserved, so that valuable information is provided for p72 protein structure and functional analysis, reference is provided for ASFV vaccine design, and important materials are provided for ASFV diagnosis.

The invention adopts the following technical scheme:

cloning the African swine fever virus p72 gene into a prokaryotic expression vector pET-30a to construct a prokaryotic expression vector pET-30a-p72. pET-30a-p72 is transformed into competent cell BL21, and p72 recombinant protein is induced to be expressed and purified to obtain the protein with higher purity. And immunizing a Balb/c mouse with the purified p72 protein, and fusing the spleen cells of the mouse with the myeloma cells SP2/0 of the mouse when the antibody titer reaches the standard, so as to obtain hybridoma cells, and screening positive hybridoma cell strains specific to the p72 protein of the African swine fever virus by using an indirect ELISA method.

The invention injects the hybridoma cells which are expanded and cultured into the abdominal cavity of a Balb/c mouse, collects ascites, and purifies to obtain ASFV p72 protein monoclonal antibody. The ASFV p72 protein monoclonal antibody is identified as an antibody subtype, and the heavy chain of the antibody is analyzed to be IgG2a, and the light chain is analyzed to be Kappa chain.

The invention also determines that the antigen epitope peptide aimed at by screening positive hybridoma cells is an African swine fever virus p72 protein 20-39 peptide segment by truncated expression of the African swine fever virus p72 protein, and the amino acid sequence is ²⁰ ILAQDLLNSRISNIKNVNKS ³⁹ 。

The beneficial effects of the invention are as follows:

the invention obtains 1 monoclonal antibody which can specifically aim at ASFV p72 protein, and can well identify ASFV p72 protein; subtype identification shows that the heavy chain of the monoclonal antibody is IgG2a, and the light chain is Kappa chain; through truncated expression of the p72 protein, the monoclonal antibody of the p72 protein can specifically identify 20 aa-39 aa peptide segments of ASFVp72 protein, and the amino acid sequence is finally determined as follows ²⁰ ILAQDLLNSRISNIKNVNKS ³⁹ . By comparing ASFV of different types, the peptide segments of p72 protein 20 aa-39 aa are found to be highly conserved, and the peptide segments are the basis for designing, diagnosing and identifying ASF vaccines.

Drawings

FIG. 1 is a diagram showing the analysis of prokaryotic expression SDS-PAGE of ASFV p72 protein of example 2, the induced group expresses p72 protein with a size consistent with that expected, and the uninduced group does not express the protein. The arrow in the figure shows the target protein.

FIG. 2 is a diagram of SDS-PAGE analysis of the ASFV p72 protein of example 2, showing that the size of the purified ASFV p72 protein is consistent with the expectations and the purity is higher. The arrow in the figure shows the target protein.

FIG. 3 is a SDS-PAGE analysis of ASFV p72 protein monoclonal antibody after ascites purification in example 5, heavy and light chains are properly sized and the antibody purity is higher.

FIG. 4 shows the detection of the subtype of ASFV p72 monoclonal antibody of example 6, which shows the heavy chain of IgG2a and the light chain of Kappa chain.

FIG. 5 is an epitope identification chart of ASFV p72 monoclonal antibody of example 7, illustrating that the minimum epitope of the monoclonal antibody is ²⁰ ILAQDLLNSRISNIKNVNKS ³⁹ 。

FIG. 6 is a result of an ASFV p72 monoclonal antibody epitope conservation analysis in example 8, which shows that the monoclonal antibody epitope is conserved among different strains.

Detailed Description

The following detailed description of the present invention is provided to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of the present invention.

EXAMPLE 1 construction of recombinant prokaryotic expression vector pET-30a-p72

ASFV gene is searched through NCBI, ASFV p72 (genbank number: CBW 46748.1) gene sequence is synthesized and constructed to prokaryotic expression vector pET-30a #BamHI andXhoi), recombinant plasmid pET-30a-p72 is obtained. The ASFV p72 gene sequence is shown in SEQ ID NO. 1, the amino acid sequence encoded by the ASFV p72 gene is shown in SEQ ID NO. 2, and the expected protein size is 72kDa.

EXAMPLE 2 prokaryotic expression and purification of recombinant p72 protein

Recombinant plasmid pET-30a-p72 was transformed into competent cell BL21, and the transformed bacterial liquid was uniformly spread on LB solid medium containing kanamycin (100. Mu.g/ml), and cultured in inversion at 37℃overnight. The monoclonal colony was picked up and inoculated into LB liquid medium containing kanamycin (100. Mu.g/ml), and cultured at 37℃for 12 hours at 220r/min, as seed bacterial liquid. Inoculating the seed bacterial liquid into two bottles of fresh LB liquid culture liquid containing kanamycin (100 mug/ml) according to the proportion of 1:100, and culturing at 37 ℃ and 220r/min until OD _600nm When the value is 0.6-0.8, IPTG with final concentration of 0.5mmol/L is added into one bottle as an induction group, the other bottle is used as a control group, and the culture is continued for 4-6 h at 37 ℃ and 220r/min without treatment. The cells were collected by centrifugation at 12000r/min for 2 minutes in 500. Mu.l each of the induction group and the control group, resuspended in 80. Mu.l of 8M urea, and then allowed to stand at room temperature for 20 minutes, and then added with 5 XSDS-PAGE Loading Buffer, boiled at 100℃for 8 minutes, and analyzed for protein expression by SDS-PAGE.

As shown in fig. 1, the induced group expressed p72 protein, the size was as expected, and the control group (i.e., the uninduced group) did not express p72 protein.

As shown in fig. 2, the purified ASFV p72 protein was of a size consistent with the expectations and of higher purity.

After confirming successful expression, the induced bacterial cells were collected by centrifugation at 12000r/min for 2 minutes, sonicated with an appropriate amount of 0.01M PBS (pH 7.4), centrifuged at 12000r/min for 5 minutes at 4℃and the supernatant was aspirated, the pellet was resuspended in an equal volume of supernatant solution PBS, 5 XSDS-PAGE Loading Buffer was added, boiled at 100℃for 8 minutes, and protein solubility was analyzed by SDS-PAGE, resulting in the finding that the p72 recombinant protein exists as inclusion bodies.

EXAMPLE 3 animal immunization

Purified p72 recombinant protein was quantified using BCA protein quantification kit, and then diluted to 0.15mg/ml with PBS. Mixing and emulsifying the diluted p72 recombinant protein with Freund's complete adjuvant in equal volume, and injecting 6 female Balb/c mice with 6-8 weeks age at subcutaneous split point at back, wherein 200 μl/mouse is obtained. Two weeks later, the protein was emulsified with incomplete Freund's adjuvant and boosted, and the same immunization dose and volume were used. Four weeks later, a third immunization was performed, and the method was the same as that of the second immunization. 7 days after the third immunization, the tail part of the mouse is cut off, about 100 mu l of tail blood is collected, serum is separated, the serum antibody titer of the mouse is detected by indirect ELISA, and the mouse with the highest antibody titer is selected for cell fusion.

EXAMPLE 4 preparation of ASFV p72 protein monoclonal antibody

(1) Fusion and selection of hybridoma cells

Resuscitation and culture of SP2/0 cells:

SP2/0 cells were resuscitated 10 days or so prior to fusion. The method comprises the following specific steps: sp2/0 cells were removed from the liquid nitrogen tank and rapidly placed in a 37℃water bath with vigorous shaking to allow rapid thawing of the cells. The thawed cells were transferred to a 15ml centrifuge tube containing 10ml of RPM1-1640 medium, centrifuged at 1000 RPM for 5min, the supernatant was discarded, and 10ml of 10% FBS-containing RPM1-1640 medium was added for aeration and mixing, and transferred to a cell culture flask for adherent culture.

Preparation of feeder cells:

the day before cell fusion, female Balb/c mice with the age of 6-8 weeks are taken, eyeballs are taken for blood collection, blood is collected, neck breakage is killed, and the mice are soaked in 75% alcohol for 5min. Mice were fixed on dissecting plates with the abdomen facing upwards. The abdominal cavity was cut off with scissors at the lower abdomen, 5ml HAT medium was gently injected into the abdominal cavity, and HAT medium was aspirated after several flicks. Repeating the steps once, mixing the extracted cells, adding into 96-well culture plate, 100 μl/well, and placing at 37deg.C with 5% CO ₂ Culturing in a carbon dioxide incubator for 24 hours.

Preparation of spleen cells:

the day before fusion, PBS containing 100. Mu.gp 72 recombinant protein was intraperitoneally injected into the mice with the highest antibody titer. Mice were sacrificed by cervical removal and immersed in 75% alcohol for 5min. Mice were fixed on dissecting plates with their abdomen facing up, the abdominal cavity was cut open, the spleen removed and placed in a 60mm diameter petri dish containing 3ml of complete RPM1-1640 medium, and spleen surface fat and tissue stripped. Spleens were transferred to a cell filtration screen, 3ml of RPM1-1640 were injected into the spleens at different locations using a syringe, the spleens were sheared with scissors, then the spleens were squeezed with the syringe's plug until only fibrous tissue remained on the surface of the screen, the tissue passing through the screen was collected in an underlying sterile petri dish, and the spleen cell suspension was transferred to a 15ml centrifuge tube. Centrifugation at room temperature, centrifugation at 1000r/min for 5min, discarding supernatant, suspending cells with 5ml RPM1-1640 medium, trypan blue viable cell count.

Cell fusion:

the well-grown SP2/0 cells are taken, the culture solution is discarded, the culture solution is washed twice by using the culture medium with the RPM of 1-1640, and then 15ml of the culture medium with the RPM of 1-1640 is blown and evenly mixed, and the mixture is transferred into a 50ml centrifuge tube. The SP2/0 cell number and the spleen cell number are adjusted to be between 1:10 and 1:3 by using an RPM1-1640 culture medium, the mixture is gently transferred into a 50mL centrifuge tube, the mixture is centrifuged for 5min at room temperature of 1000r/min, the supernatant is discarded, the cells are collected, and the bottom of the centrifuge tube is flicked, so that cell aggregates are uniformly mixed. Taking PEG preheated at 37 ℃, sucking by a 1ml suction tube, slowly adding the PEG dropwise into a centrifuge tube, counting the time for reading, adding 1ml PEG for 1min, rotating the centrifuge tube while adding, covering the centrifuge tube after adding, and standing for 1-2 min. The pre-warmed serum-free RPM1-1640 medium 25mL at 37℃was aspirated and added to the centrifuge tube slowly followed by rapidly within 5min to reduce the toxic effects of PEG on cells. Centrifuging at room temperature of 1000r/min for 5min, discarding supernatant, re-suspending cells with 15ml HAT medium, transferring into a culture dish, adding into 96-well culture plate with feeder cells, placing 100 μl/well into 37 deg.C, 5% CO ₂ Culturing in a cell culture incubator.

Screening of positive hybridoma cells:

observing and recording the growth condition of the fused cells, culturing for 3-4 days, allowing the clone of the cells to be seen in the wells, performing half-quantity liquid exchange by using HAT culture medium on the 5 th day, detecting the supernatant of the fused cells by adopting an indirect ELISA method when the cells in the wells grow to 1/3 of the bottom of the wells, and screening positive wells.

The indirect ELISA method is specifically implemented as follows:

the purified p72 recombinant protein was diluted to 1. Mu.g/ml with coating buffer, 100. Mu.l per well, and coated overnight at 2-8 ℃. The plates were blocked with 5% BSA blocking solution, 150. Mu.l/well, for 2 hours at 37℃after washing 4 times with PBST wash. The blocking solution was discarded, and the mixture was dried on absorbent paper at 37℃for 2 hours. Mu.l of the cell supernatant was pipetted into an ELISA plate coated with p72 protein, incubated at 37℃for 30min, washed 4 times with PBST wash, and patted dry. mu.L of goat anti-mouse IgG-HRP diluted 1:15000 with 5% BSA was added, incubated at 37℃for 30min, washed 4 times with PBST wash, and patted dry. Adding 50 μl of substrate solution, incubating at 37deg.C for 15min, adding stop solution, and reading OD on an ELISA _450nm Values. Selecting OD _450nm After the positive cell wells with the highest values were expanded, 3 subclones were performed, each of which was screened by the indirect ELISA method described above. And selecting a hybridoma cell strain which stably secretes the p72 antibody for expansion culture and freezing for later use.

EXAMPLE 5 preparation and purification of ASFV p72 protein monoclonal antibody ascites

Preparation of ascites:

female Balb/c mice of 8 weeks old were each intraperitoneally injected with 0.5ml of sterilized liquid paraffin under aseptic conditions. After two weeks, positive hybridoma cells selected in example 4 of the expansion culture were collected and the cell number was adjusted to 2×10 ⁶ Per ml, each mouse was intraperitoneally injected with 0.5ml of the cell suspension. Mice were observed daily and generally died for about 10 days. Before the death of the mice, the ascites is collected by a syringe, the mice are centrifuged for 10 min at the temperature of 4 ℃ and the speed of 12000r/min, and the intermediate transparent liquid is the monoclonal antibody. Packaging ascites, and storing at-80deg.C.

Purification of ascites:

the abdominal cavity liquid was diluted with a binding solution (20 mM phosphate buffer, 150mM NaCl, pH 7.4) at a ratio of 1:10, and then purified by adding a gravity column preloaded with 1ml of Protein G agarose gel; washing and balancing the filler with 5ml deionized water and 10ml Binding buffer before purification, adding diluted ascites, and collecting flow-through liquid for analysis; the eluate was used for elution, and the eluate was collected 10 times in 1ml tube, and an appropriate amount of 1M tris-HCL was added to each tube for neutralization. After purification, 40. Mu.l of each tube was added with 10. Mu.l of 5 Xloading buffer and boiled in a metal bath at 100℃for 8min; SDS-PAGE electrophoresis analysis of the size and purity of the target protein; as can be seen from FIG. 3, the heavy and light chain sizes of the target protein are as expected, and are ASFV p72 protein monoclonal antibodies. The purer eluted samples were then pooled and stored at-80℃for further use.

EXAMPLE 6 subtype identification of ASFV p72 protein monoclonal antibody

The ASFV p72 protein monoclonal antibody is subjected to subtype identification according to the operation instructions of the Proteinech monoclonal antibody subtype identification kit, and the result shows that the heavy chain of the antibody is IgG2a, and the light chain is Kappa chain, as shown in figure 4.

EXAMPLE 7 identification of an epitope recognized by an ASFV p72 protein monoclonal antibody

Preliminary identification of epitope:

as shown in FIG. 5, the p72 protein (1 aa to 647 aa) was first divided into 3 segments (F1: 1aa to 500aa; F2:20aa to 303aa; F3:430aa to 647 aa), the gene sequences thereof were synthesized and prokaryotic expression plasmids were constructed. The synthesized plasmid was transformed into competent cell BL21, induced to express and purified. The epitope identification of the ASFV p72 monoclonal antibody is carried out by a Western blot method (Western blot), and the result shows that the epitope of the monoclonal antibody is positioned between 20aa and 303aa.

Further determination of epitope:

f2 is further divided into 3 segments (F4: 20 aa-150 aa; F5:100 aa-220 aa; F6:170 aa-303 aa), the gene sequence is synthesized and a prokaryotic expression plasmid is constructed. The epitope of the monoclonal antibody is located between 20aa and 99aa as a result of induction expression, purification and identification under the above conditions.

Further accurate identification of epitope:

the gene sequence of 20aa to 99aa is further divided into 3 segments (F7: 20aa to 59aa; F8:40aa to 79aa; F9:60aa to 99 aa), and the prokaryotic expression plasmid is constructed. The results of the induction expression, purification and identification under the conditions show that the epitope of the monoclonal antibody is located between 20aa and 39aa.

The gene expression sequences of 20aa to 39aa are further divided into 3 segments (F10: 20aa to 29aa; F11:25aa to 34aa; F12:30aa to 39 aa), and prokaryotic expression plasmids are constructed. As a result, it was found that the monoclonal antibody did not react with F10, F11 and F12, and it was confirmed that the epitope of the monoclonal antibody was located at 20aa to 39aa.

The gene sequences of F1-F12 are shown in SEQ ID NO 3-SEQ ID NO 14.

EXAMPLE 8 conservation analysis of the epitope recognized by the monoclonal antibody to ASFV p72 protein

13 ASFV p72 gene sequences (China/2018/AnhuiXCGQMK 128995; china_ASFV-SY18_2018KP055815;China_LN_2018MK333181;Russia_Georgia_2007FR682468;Portugal_OURT_88.3_1988MN318203;Spain_BA71_1971KP055815;Italy_26544_OG10_2010KM102979;Malawi_Tengani_1962 AY261364;Namibia_Warthog_1980AM712239;Uganda_R35_2015MH025920;Uganda_R8_2015MH025916;Kenya_1950AM712239;Malawi_Lil_1983AM712239) including the major representative strains of genes I, II, IV, V, VIII, IX, X were downloaded from the GenBank database. The 13 p72 amino acid sequences were aligned using MEGA-X software. As a result, it was found that amino acids 20aa to 39aa of the different ASFV strains were conserved, as shown in FIG. 6.

The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention, so that all equivalent changes or modifications of the structure, characteristics and principles described in the claims should be included in the scope of the present invention.

Claims

1. An epitope peptide of African swine fever virus p72 protein is characterized in that the epitope peptide is a 20-39 peptide segment of the African swine fever virus p72 protein, and the amino acid sequence is ²⁰ ILAQDLLNSRISNIKNVNKS ³⁹ 。

2. An antigenic composition comprising at least the epitope peptide of claim 1.

3. A hybridoma cell line which secretes a monoclonal antibody against the p72 protein of african swine fever virus, characterized in that the hybridoma cell line specifically secretes a monoclonal antibody against the epitope peptide of claim 1.

4. A monoclonal antibody against the p72 protein of african swine fever virus, wherein the monoclonal antibody is specific for the epitope peptide of claim 1.

5. Use of the epitope peptide of claim 1, the antigen composition of claim 2, the hybridoma cell line of claim 3, and the monoclonal antibody of claim 4 in preparing a kit for diagnosing or detecting african swine fever virus.