CN108486066B - Hybridoma cell strain and monoclonal antibody secreted by hybridoma cell strain and resisting chlamydia abortus - Google Patents

Abstract

The invention belongs to the field of immunology, and particularly discloses a hybridoma cell strain and an anti-chlamydia-production monoclonal antibody secreted by the hybridoma cell strain. The invention uses the purified prokaryotic expression Chlamydia abortus main outer membrane recombinant protein (MOMP) as antigen immune mice, and obtains hybridoma CA1-MM01 secreting monoclonal antibody against Chlamydia abortus main outer membrane recombinant protein by screening positive clones by indirect ELISA method. The hybridoma cells are identified in the aspects of stability after continuous subculture and cryopreservation, subtype and titer of the monoclonal antibody, and the like, and the results show that the obtained hybridoma cells have strong and stable antibody secretion capacity and high titer of the monoclonal antibody. The obtained monoclonal antibody of the chlamydia abortus is marked by fluorescein and then is detected by a direct fluorescence method, and the experimental result shows that the obtained monoclonal antibody has high specificity and strong binding capacity to the chlamydia abortus.

Description

Hybridoma cell strain and monoclonal antibody secreted by hybridoma cell strain and resisting chlamydia abortus

Technical Field

The invention belongs to the field of immunology, and particularly relates to a hybridoma cell strain and an anti-chlamydia-production monoclonal antibody secreted by the hybridoma cell strain.

Background

Chlamydia abortus (c. abortus) is an obligate intracellular parasitic gram-negative microorganism between bacteria and viruses that can cause infections in various animals and humans, and is an important pathogen of zoonosis. The chlamydia abortus is the most common pathogen for infectious abortion of small ruminants (sheep and goats), and can also cause abortion of cattle, pigs and other livestock, and abortion or other diseases can occur after pregnant women are infected. The chlamydia abortus disease threatens intensive farming, causes great economic loss to animal husbandry production in many countries and regions, and simultaneously has influence on public health.

The detection and diagnosis technology of chlamydia is mainly based on molecular biological detection method and immunological detection method at present. The molecular biology diagnosis method is a Polymerase Chain Reaction (PCR) technology for detecting antigen established according to an amplification target sequence, the selection of the target sequence is established according to conserved genes such as MOMP gene, rRNA gene and the like, such as PCR, multiplex PCR, fluorescent quantitative PCR and the like for detecting chlamydia established by Wangcongming of Yangzhou university, however, although the technology is sensitive, the technology is difficult to popularize and apply in large-scale detection of a basic layer due to more interference factors, expensive equipment and higher technical level requirement.

The immunological detection method is a method for detecting an antigen or an antibody by using an antigen-antibody specific binding reaction, and a common chlamydia antigen kit includes: a direct immunofluorescence antibody detection technology (Imagen Chlamydai kit, ThermoFisher company) and an amplified ELISA kit (IDEIA PCE Chlamydia K603211-2, Oxoid) which are established on the basis of LPS. Antibody detection kits, such as monoclonal antibodies prepared with a Chlamydia trachomatis cross-reactive antigen and fluorescein labels, for detecting Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae antibodies (Savyon Diagnostics Ltd, israel). ELISA antibody detection kit (IDVET, France) using MOMP as coating antigen, and kit for detecting livestock Chlamydia abortus antibody (IDEXX, USA) using Chlamydia abortus polytype outer membrane protein 80-90kDa as coating antigen. The quality of the antigen and antibody detection kit circulating in the current market is uneven, so that the detection result is inaccurate, even misdiagnosis diseases are caused due to inaccurate detection result, and great economic loss is probably caused by delaying the diseases. The monoclonal antibody has the characteristics of high purity, good repeatability, strong antigen binding specificity and the like, and can greatly improve the test specificity and accuracy by applying the monoclonal antibody to ELISA and fluorescence detection methods. The Chlamydia abortus monoclonal antibody with high specificity and stability lays a foundation for preparing a high-quality Chlamydia abortus detection reagent or kit, and makes up the defects of low specificity and sensitivity and the like of the existing similar detection reagent or kit.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a hybridoma cell strain and an anti-chlamydia-influenza monoclonal antibody secreted by the hybridoma cell strain.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

in the first aspect, the purified prokaryotic expression Chlamydia abortus main outer membrane recombinant protein (MOMP) is used as an antigen to immunize mice, and positive clones are screened by an indirect ELISA method to obtain hybridoma CA1-MM01 which secretes monoclonal antibodies aiming at the Chlamydia abortus main outer membrane recombinant protein.

The invention provides a hybridoma cell strain CA1-MM01 which is classified and named as monoclonal antibody hybridoma cell strain of main outer membrane protein of Chlamydia abortus, and is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of Ministry of West Lu No.1 of Beijing republic of south China, institute of microbiology, China academy of sciences, postal code:

100101), the preservation date is 11 months and 22 days in 2017, and the preservation number is CGMCC No. 14882.

The hybridoma cell strain CA1-MM01 provided by the invention can be used for preparing a monoclonal antibody CA1-MM01H of the main outer membrane protein of the high-titer anti-abortion chlamydia.

The monoclonal antibody CA1-MM01H has the characteristics of high sensitivity and good specificity, and has good fluorescence characteristics.

The monoclonal antibody CA1-MM01H of the main outer membrane protein of the anti-chlamydia abortus secreted by the hybridoma cell strain CA1-MM01 can generate specific reactivity to the MOMP protein of the chlamydia abortus. Moreover, the good fluorescence property of the monoclonal antibody enables the monoclonal antibody CA1-MM01H to be suitable as a fluorescent antibody, so that a method for detecting Chlamydia abortus by direct fluorescence is established.

Therefore, the monoclonal antibody CA1-MM01H of the main outer membrane protein of the anti-chlamydia influenzae secreted by the hybridoma cell strain CA1-MM01 also belongs to the protection scope of the invention.

In the second aspect, in the process of obtaining the monoclonal antibody CA1-MM01H by using the hybridoma cell strain CA1-MM01, through condition exploration of in vitro culture of hybridoma cells, the fact that the adherence dependency of the hybridoma cells can be gradually reduced by applying a culture medium with a specific ratio is discovered, the domestication effect of suspension culture is achieved, the purpose of mass production of the hybridoma cells through suspension culture is finally achieved, the expression level of the monoclonal antibody is improved, and the separation and purification effects of the monoclonal antibody are improved.

Therefore, the invention also provides a method for preparing the monoclonal antibody CA1-MM01H from the hybridoma cell strain CA1-MM01, which comprises the following specific steps:

the hybridoma cells were recovered at 2.5X 10⁵cells/mL in a T75 flask containing 10% fetal bovine serum in RPMI1640 medium at 37 deg.C and 5% CO₂The culture was cultured in an incubator for 48 hours (at a cell density of about 90%), digested with trypsin and passaged at 2.5X 10⁵cells/mL in a T75 flask containing 8% fetal bovine serum in RPMI1640 medium at 37 deg.C and 5% CO₂Culturing in the incubator for 48 hr; the concentration of the fetal calf serum is reduced to 6% in the third passage, and the culture is carried out under the same other conditions; collecting hybridoma cells in logarithmic growth phase by trypsinization at the fourth subculture, centrifuging at 1000r for 10min, and centrifuging at 2.5 × 10⁵cells/mL density in 125mL shake flasks in 4mmol/L Gln, 4% fetal bovine serum RPM1640: SFM4CHO: OptiCHO (3.5:3.5:3) mixed media placed in 5% CO₂Shaking table culture at 37 deg.C and 125 r/min; after the cell state is stabilized every 48h for liquid change passage (about 3-4 times of continuous passage), the concentration of the fetal calf serum is reduced to 2 percent again, the culture medium is a mixed culture medium containing 4mmol/L Gln and 2 percent fetal calf serum RPM1640: SFM4CHO: OptiCHO (4:4:2), the hybridoma cells are continuously cultured for 2 generations in the same other culture environments, the hybridoma cells finally reach a suspension growth state, and the secretion expression amount of the monoclonal antibody reaches the highest and is about 150 mg/L.

Wherein, the culture medium and each reagent involved in the method are all commercially available products.

In a third aspect, the invention provides an application of the monoclonal antibody CA1-MM01H in preparation of a detection reagent or a kit for Chlamydia abortus.

Preferably, the reagent or the kit is a direct fluorescence detection reagent or a kit for Chlamydia abortus.

Furthermore, based on the good specificity and fluorescence characteristics of the main outer membrane protein monoclonal antibody (CA1-MM01H) of the Chlamydia abortus prepared in the above way, the antibody CA1-MM01H is used as a fluorescent antibody, so that the method for directly detecting Chlamydia abortus by fluorescence is established.

The method for detecting the Chlamydia abortus by direct fluorescence is characterized in that a fluorescein-labeled main outer membrane protein monoclonal antibody (CA1-MM01H) of the Chlamydia abortus is directly combined with a corresponding antigen in a tissue cell, and specific fluorescence is detected under a fluorescence microscope to detect the Chlamydia abortus.

The method comprises the following specific steps:

(1) preparing a pathological section: collecting suspected abortion materials, such as spleen, uterine mucosa, vaginal mucosa tissue, and larynx mucus, and cutting to obtain appropriate amount of the materials. If the tissue is the tissue, after the tissue needs to be ground at the temperature of 4 ℃, gentamicin is treated at room temperature for 30min, 1,000 r/min and centrifuged for 10min, the precipitate is discarded, the supernatant is used for infecting McCoy cells growing to a single layer (cover glass is placed in a 24-hole cell plate in advance), the infected cover glass is taken out and is fixed for 10-20 min by precooled methanol or absolute ethanol; or slicing the tissue into slices with the thickness of 0.1-0.3 mm, and pasting the slices on a glass slide soaked in cold methanol; or smearing the collected suspicious respiratory tract or vaginal swab into cold acetone, fixing, taking out, air drying, and storing at-20 deg.C for use;

(2) establishment of direct immunofluorescence diagnostic method: preparing fluorescent monoclonal antibody liquid, namely taking out a fluorescent labeled main outer membrane protein monoclonal antibody of the abortion chlamydia, diluting the monoclonal antibody with sterilized PBS (phosphate buffer solution) in a ratio of 1:500, simultaneously diluting evans blue with sterilized PBS in a ratio of 8:1, then proportioning and uniformly mixing the diluted monoclonal antibody and evans blue in a ratio of 7:1 to obtain final fluorescent anti-liquid, and standing at 4 ℃ for later use; taking out the prepared slide, firstly dripping PBS for soaking for 10min, then dripping about 25 mu L of fluorescence labeling abortion chlamydia major outer membrane protein monoclonal antibody, putting into a wet box, incubating for 30min at 37 ℃, taking out and washing for 5min with PBS, after washing, naturally drying for 5-10 min in a dark place, sealing with glycerol with volume fraction of 50%, and observing by a fluorescence microscope. Meanwhile, a chlamydia infection positive slide is used as a positive control, and an uninfected slide is used as a negative control.

Furthermore, the detection reagent or the kit for Chlamydia abortus prepared by the monoclonal antibody CA1-MM01H or the detection reagent or the kit containing the monoclonal antibody CA1-MM01H belong to the protection scope of the invention.

The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.

The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.

The invention has the beneficial effects that:

the invention uses the purified prokaryotic expression Chlamydia abortus main outer membrane recombinant protein (MOMP) as antigen immune mice, and obtains hybridoma CA1-MM01 secreting monoclonal antibody against Chlamydia abortus main outer membrane recombinant protein by screening positive clones by indirect ELISA method. The biological characteristics of the obtained monoclonal antibody are systematically identified from the aspects of the biological characteristics of the hybridoma after continuous subculture and cryopreservation, the type, the subclass, the titer and the like of the monoclonal antibody, and the result shows that the obtained hybridoma has strong and stable antibody secretion capability and high monoclonal antibody titer. The direct fluorescence detection experiment result shows that the obtained monoclonal antibody can be used for detecting the Chlamydia abortus r-MOMP protein, has strong specificity and fluorescence characteristic, and is suitable for being used as a fluorescence antibody to establish a direct fluorescence detection method. Lays a foundation for establishing an immunological detection method of chlamydia which is a pathogen of zoonosis.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of the recombinant Chlamydia abortus protein r-MOMP; wherein, 1: BL21(DE3) + pET-28a empty plasmid; 2: BL21(DE3) + pET-28a + MOMP recombinant plasmid; m: protein molecular weight standards.

FIG. 2 shows that the recombinant protein can be combined with a chlamydia specific antibody, and a specific band does not appear in control negative fetal bovine serum, when Western-blot detection is carried out on swine abortion chlamydia positive serum; 1: BL21(DE3) null bacteria; 2: BL21(DE3) + pET-28 a; 3: BL21(DE3) + pET-28a + MOMP recombinant plasmid expression product; m protein molecular weight standard

FIG. 3 shows the Western blot detection results of the monoclonal antibody CA1-MM 01H; wherein, A: a swine abortion chlamydia positive serum; b: the hybridoma cell line CA1-MM01 secretes the supernatant.

FIG. 4 is a chromosomal map of CA1-MM01 hybridoma cells.

FIG. 5 is a graph showing detection of an abortion disease by direct immunofluorescence of a fluorescently labeled monoclonal antibody (CA1-MM 01H); a: commercial kit test (IMAGENTM CHLAMYDIA, Oxoid Ltd, uk) for chlamydia infection patterns; b: the direct immunofluorescence detection method established by the monoclonal antibody (CA1-MM01H) provided by the invention is used for detecting the chlamydia infection pattern of the cells.

Detailed Description

The present invention is further illustrated by the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of Chlamydia abortus r-MOMP protein monoclonal antibody

1. Preparing materials and reagents:

abortion chlamydia: the strain of the porcine abortion chlamydia CP12 is purchased from a strain storeroom of China veterinary medicine inspection institute;

bacterial DNA extraction kit, plasmid extraction kit: purchased from Beijing, Total gold biology, Inc.; e.coli DH5 α: stored in the room;

pMD18-T Vector: purchased from TaKaRa Bio-engineering (Dalian) Co., Ltd;

coli BL21(DE 3): storing in the laboratory;

pet-28a prokaryotic expression vector: purchased from Novagen corporation;

isopropylthio- β -D-galactoside (IPTG): purchased from Merk corporation;

low molecular weight protein Marker: purchased from Beijing Santai Biotechnology, Inc.;

urea (analytical grade), coomassie brilliant blue R-250, SDS, DAB, BPB and ponceau S: all purchased from beijing kao ze biotechnology.

HAT selective medium, HT selective medium, fetal bovine serum, DMEM high-sugar medium, RPMI1640 medium, SFM4CHO, OptiCHO: all from Gibco;

horse Radish Peroxidase (HRP) labeled rabbit anti-mouse IgG, Fluorescein (FITC) labeled rabbit anti-mouse IgG antibody: all purchased from Abcm company;

experimental animals: pure line BALB/c mice, female, purchased from the laboratory animals department of the department of medicine, Beijing university;

SepHadex Gln: purchased from Sigma company;

2. preparation of monoclonal antibody hybridoma cell strain of main outer membrane protein of chlamydia abortus

(1) Preparation of recombinant protein r-MOMP of Chlamydia abortus

Preparation and cloning of Chlamydia abortus OmpA Gene:

the freeze-dried powder of the porcine chlamydia abortus CP12 strain is diluted by 1ml of sterilized normal saline, and is inoculated to 6-day-old SPF chick embryos for strain propagation. Collecting yolk membranes of dead chick embryos in 3-6 days, adding 1mL of PBS, and grinding into suspension; centrifuging at 3000rpm for 10min, discarding the precipitate, and keeping the supernatant, wherein the supernatant is chlamydia bacterial liquid, and extracting chlamydia genome according to the steps of the bacterial DNA extraction kit. OmpA gene primers (P1, P2 below) were designed and synthesized with reference to the OmpA gene sequence of Chlamydia abortus S26/3 strain registered in GenBank: CR 848038:

P1(F):5’—ATG AAA AAA CTC TTG AAA TCG G—3’(SEQ ID No.3)，

P2(R):5’—TTA GAA TCT GAA TTG AGC ATT CAT—3’(SEQ ID No.4)。

the primers were synthesized by Beijing Olympic Biotechnology, Inc.

And (3) amplifying a target fragment, namely the OmpA gene, by using the extracted chlamydia genome as a template. Connecting the amplified OmpA gene with a pMD18-T vector, transferring the recombinant plasmid into Escherichia coli DH5 alpha competence, and culturing positive clonal bacteria screened by an LB/Amp plate in an incubator at 37 ℃ for 12-16 h. The recombinant bacteria which are identified as suspected positive by the PCR of the plasmid and the double enzyme digestion method of the recombinant plasmid are sent to Shanghai Boya biotechnology company for further sequencing verification. The sequence is shown as SEQ ID No.1, and the coded protein is shown as SEQ ID No. 2.

Prokaryotic expression construction and identification of OmpA gene:

pMD18-T-OmpA recombinant plasmid is used as a template, primers P3 and P4 are used for amplification, then the plasmid is connected with a pET-28a prokaryotic expression vector, escherichia coli BL21(DE3) is transformed, and after positive colonies are identified, sequencing is carried out by Boya biotechnology company.

P3(F):5’-GGG GTA CCG GAA TTC CTG GTC CCG CGT TTG CCT GTA GGG AAC CCA GC-3’(SEQ ID No.5)，

P4(R):5’-TT CCG CGG CCG CTA TGG CCG ACG TCG ACG CGT TTA GAA TCT GAA TTG AGC-3’(SEQ ID No.6)。

(2) Inducible expression of recombinant MOMP protein (r-MOMP):

the positive strain BL21(DE3) containing the recombinant plasmid was cultured in LB medium to the middle of logarithmic growth (i.e., the concentration of the strain reached OD600 of 0.4-0.6), IPTG (final concentration of 1.0mmol/L) was added thereto, and the mixture was taken out at 37 ℃ for 3 hours. And detecting the recombinant protein by using SDS-PAGE and Western-blot. SDS-PAGE showed smooth expression of recombinant protein of theoretical size (see FIG. 1), and the target protein size was equivalent to theoretical [ MW 40.0 (target protein) +3.83kDa (vector pET-28a) ═ 43.8kDa ]. Western-blot detection is carried out on the recombinant protein by using standard positive serum of the aborted chlamydia pig, and the result shows that the recombinant protein can be combined with a chlamydia specific antibody under the denaturation condition, while a specific band does not appear in the negative fetal calf serum of the control (see figure 2).

(3) Large-scale expression and purification of recombinant protein (r-MOMP):

the recombinant bacteria are inoculated in a liquid LB culture medium for enlarged expression, the bacteria are collected by centrifugation at 6000r, the bacteria are cracked by ultrasonic waves after PBS is resuspended, and the inclusion bodies are collected by centrifugation at 10000r for 40 min. The experiment utilized urea solutions of different concentrations to dissolve and wash bacterial inclusion bodies: resuspending the inclusion bodies with deionized water (1g:1mL ratio), eluting other miscellaneous proteins with 5M urea solution to retain the inclusion body components; fully dissolving the inclusion body by using 10M urea solution, then dialyzing and renaturing the inclusion body, putting the recombinant protein dissolved solution into a dialysis bag, sealing, putting the dialysis bag into a beaker filled with 1L of 8M urea solution, dialyzing for 10 hours at 4 ℃, and then replacing urea with the same concentration to dissolve for 2 times and then dialyzing; then dialyzing with urea solution with gradient concentration, sequentially 6M, 4M, 2M and 1M, and finally dialyzing with 0.1M Tris-HCl (pH8.5) for 10 h; detecting the dialyzed state by SDS-PAGE after the end, finally obtaining purified protein, determining the concentration, subpackaging, freezing and pumping to prepare freeze-dried powder, and storing at-80 ℃ for later use.

3. Preparation of monoclonal antibody hybridoma cell strain for chlamydia abortus

Animal immunization:

and (3) taking the purified and concentrated r-MOMP recombinant protein freeze-dried powder, diluting the powder with a proper amount of PBS (phosphate buffer solution), and immunizing a Balb/c mouse with the age of 6 weeks for 6 times. The first immunization protocol was: the r-MOMP recombinant protein and Freund's complete adjuvant are emulsified in a volume ratio of 1:1, the dosage is 200 mu g/mouse, and abdominal multipoint subcutaneous injection is carried out. 2 nd immunization: two weeks later, the third immunization one week later, 2 nd and 3 rd times of Freund's incomplete adjuvant at a volume ratio of 1:1, at a dose of 100. mu.g/mouse, and by abdominal multipoint subcutaneous injection. The 4 th and 5 th immunizations were performed at intervals of one week, and immunized with antigen solution at a dose of 100. mu.g/mouse, i.p.. One week after the 5 th immunization, the tail of the mouse is subjected to small blood sampling, the antibody titer is detected, and the titer is selected to be 10 orders of magnitude higher⁷Three days before fusion, the mice are boosted and injected with antigen solution in an abdominal cavity, and the dosage is 100 mu g/mouse.

Cell fusion:

SP2/0 myeloma cells were prepared by early resuscitation. Spleen of mice after the last booster immunization was aseptically taken, and spleen cell suspension prepared by gently grinding and prepared to contain 1X 10⁸Spleen cells and 2X 10⁷Mixed suspension of myeloma cells, and adding fusion agent PEG4000 for fusion. The fused cells were suspended uniformly in HAT selection medium and added to a 96-well cell plate at 100. mu.L/well in 5% CO₂Culturing in an incubator.

Screening and subcloning of hybridoma cells:

after the fused cells grow to cover the bottoms of the wells of the cell plates 1/4-1/3 in a clonal manner, detecting the culture supernatant of the clonal growth by using an established indirect ELISA method. The strong positive cell wells were subcloned by limiting dilution, and the negative wells were tested 3 days later and discarded if they were still negative. After 4 times of screening and cloning culture, 3 hybridoma cells which can stably secrete the main outer membrane protein monoclonal antibody of the Chlamydia abortus and have high titer are obtained.

4. In vitro large-scale preparation of monoclonal antibody (CA1-MM01H) against major outer membrane protein of Chlamydia abortus

Through the condition exploration of in vitro culture hybridoma cells, the adherence dependence of the hybridoma cells can be gradually reduced by using a culture medium with a specific ratio, the domestication effect of suspension culture is achieved, the aim of mass production of the hybridoma cells through suspension culture is finally achieved, the expression quantity of the monoclonal antibody is improved, the separation and purification effects of the monoclonal antibody are improved, and the operation is as follows:

the hybridoma cells were recovered at 2.5X 10⁵cells/mL in a T75 flask containing 10% fetal bovine serum in RPMI1640 medium at 37 deg.C and 5% CO₂The culture was cultured in an incubator for 48 hours (at a cell density of about 90%), digested with trypsin and passaged at 2.5X 10⁵cells/mL in a T75 flask containing 8% fetal bovine serum in RPMI1640 medium at 37 deg.C and 5% CO₂The incubator of (4) was cultured for 48 hours. At the third passage, the concentration of fetal calf serum was decreased to 6%, and the culture was performed under the same conditions as above. Collecting hybridoma cells in logarithmic growth phase by trypsinization at the fourth subculture, centrifuging at 1000r for 10min, and centrifuging at 2.5 × 10⁵cells/mL are cultured in 125mL shake flasks in a specific ratio of 4mmol/L Gln in 4% fetal bovine serum at RPM of 164%0: SFM4CHO: OptiCHO (3.5:3.5:3) mixed medium, and shake-cultured at 37 ℃ and 125r/min with 5% CO 2. After the cell state is stabilized every 48h for liquid change passage (about 3-4 times of continuous passage), the fetal calf serum concentration is reduced to 2 percent again, the specific ratio of the culture medium is 4mmol/L Gln, the RPM1640 of 2 percent fetal calf serum is: SFM4CHO: OptiCHO (4:4:2), other culture environments are the same as above, the continuous culture is carried out for 2 generations, the hybridoma cells finally reach a suspension growth state, and the secretion expression quantity of the monoclonal antibody reaches the highest and is about 150 mg/L.

5. Identification and purification of monoclonal antibody against major outer membrane protein of Chlamydia abortus (CA1-MM01H)

(1) Identification of monoclonal antibody (CA1-MM 01H):

and (3) monoclonal antibody subtype identification: the monoclonal antibodies secreted by 3 hybridoma cells are IgG1 type as shown by the agar diffusion test determination by using a mouse monoclonal antibody typing kit of Sigma company: CA1A-1MM01H, CA1A-2MM02H, CA1A-3MM 03H.

And (3) measuring the titer of the culture supernatant of the hybridoma: the titer of the hybridoma cell culture supernatant was detected by indirect ELISA. The result shows that the monoclonal antibody CA1A-1MM01H (IgG1 type) of the C.abortus r-MOMP protein has the highest antibody titer, and the hybridoma cell strain secreting the antibody is preserved in the China general microbiological culture Collection center.

Carrying out Western-blot detection on hybridoma cell culture supernatant: the freeze-dried powder of the porcine chlamydia abortus CP12 strain is diluted by 1ml of sterilized normal saline, and is inoculated to 6-day-old SPF chick embryos for strain propagation. Collecting yolk membranes of dead chick embryos in 3-6 days, adding 1mL of PBS, and grinding into suspension; centrifuging at 3000rpm for 10min, taking the supernatant as an immunoblotting test antigen, and respectively carrying out centrifugation on standard positive serum of a pig with chlamydia abortus as a primary antibody and a monoclonal antibody CA1MM01H to be detected by using a confining liquid (5% skimmed milk powder, prepared by TBST) 1:1000 dilution, secondary antibody, diluted with antibody (PBS, ph7.2 solution) 1:1000 dilutions of HRP-labeled rabbit anti-pig IgG and HRP-labeled goat anti-mouse IgG.

Numbering	A	B
			Antigens	CP12 bacterial liquid	CP12 bacterial liquid
Sample loading amount	1μg	1μg
			A primary antibody	Chlamydia abortus positive serum for pig	Suspension culture supernatant of CA1-MM01H
Primary dilution resisting ratio	1：1000	1:1000

The result shows that in the immunoblotting test, after DAB development, a band of about 41KDa is on the PVDF transfer membrane, which is consistent with the theoretical size of the Major Outer Membrane Protein (MOMP) of Chlamydia abortus, and the prepared monoclonal antibody has specificity and immunogenicity (FIG. 3).

Identification of chromosome of hybridoma cell-colchicine method. Culturing cells for 24h, adding colchicine to make final concentration 0.2mg/L, culturing for 4h, digesting and collecting cells, centrifuging at 2000r/min for 10min, collecting cell precipitate, adding 0.075mol/L KCl10mL preheated at 37 deg.C into the precipitate, blowing and sucking uniformly, and placing in 37 deg.C water bath for 140min to make cells swell. Centrifuging at 2000r/min for 10min, removing supernatant, fixing cell precipitate with 10mL of a mixture of 3:1 (methanol and glacial acetic acid) for 30min, centrifuging at 2000r/min for 10min, repeating the centrifuging for 3 times, removing supernatant, taking cell precipitate smear, naturally drying, staining with Giemsa, and performing oil-lens examination. Hybridoma CA1-MM01 has 100-108 chromosomes (FIG. 4). The number of chromosomes was 104, indicating that the subcultured cells were not mutated.

(2) Purification of monoclonal antibody (CA1-MM01H)

Collecting suspension culture supernatant of the abortive chlamydia monoclonal antibody hybridoma cell strain, adding 40mL of PBS, slowly adding 80mL of 4 ℃ saturated ammonium sulfate solution (dropwise adding under the stirring of a magnetic stirrer or a glass rod) to form 50% saturated solution, and standing at 4 ℃ for precipitation overnight.

4 ℃, 8000r/min, and centrifuging for 15 min.

② after discarding the supernatant, the precipitate obtained adding PBS 40mL dissolved, then adding saturated ammonium sulfate 20mL (dropwise method is the same to the first), to make 33.3% saturated solution, 4 degrees C precipitation overnight (this step is repeated three times).

③ 4 ℃, 8000r/min, centrifuging for 15min, discarding the supernatant, leaving the precipitate, adding a small amount of 3-5mL, pH7.20.01mol/L

Dissolving PBS (floating up and down according to the precipitation amount), washing the centrifuge tube by 1-2mL PBS, mixing the two, putting the mixture into a dialysis bag, soaking the dialysis bag in PBS with the pH of 8.00.005 mol/L, dialyzing at 4 ℃ for overnight desalination, and replacing the PBS every 4-6 h.

Fifthly, taking out the liquid in the dialysis bag, centrifuging for 10min at the temperature of 4 ℃ at 6000r/min, discarding the precipitate, and keeping the supernatant (the rotating speed can be properly adjusted by observation).

Sixthly, measuring the protein concentration.

Seventhly, storing the purified antibody at the temperature of minus 20 ℃ for later use.

EXAMPLE 2 establishment of direct immunofluorescence assay for monoclonal antibody (CA1-MM01H)

1. Preparing a reagent:

preparation of carbonate solution: 0.5mol/L of carbonate solution with pH of 9.5, 3.7g of sodium bicarbonate, 0.6g of sodium carbonate (anhydrous) and 100mL of distilled water; preparation of PBS eluent: pH7.2, 0.01mol/L Phosphate Buffer Solution (PBS), hydrogen phosphateDisodium salt (Na)₂HPO₄·2H₂O)27g, sodium chloride (NaCl)8.5g, sodium dihydrogen phosphate (NaH)₂PO₄·2H₂O)0.39g, distilled to 1000 mL. Fluorescein Isothiocyanate (FITC): purchased from Sigma, SepHadex G200 SepHadex: purchased from beijing solibao corporation.

2. Fluorescence labeling monoclonal antibody method

According to the measured protein content, 1/20 mass of FITC (F7250, Sigma) with the protein content is weighed and dissolved in 0.5mol/L of prepared carbonate solution with the pH value of 9.5, the mixture is stirred gently to avoid generating bubbles when being dissolved, the protein and the FITC are mixed in a beaker, the beaker is placed in a magnetic stirrer, the mixture is stirred overnight in a refrigerator at 4 ℃ in dark at a slow speed, and the marked protein is collected.

3. Removing impurities with dextran gel

Preparation of reagent-prepared PBS eluent: pH7.2, 0.01mol/L Phosphate Buffer Solution (PBS), disodium hydrogen phosphate (Na)₂HPO₄·2H₂O)27g, sodium chloride (NaCl)8.5g, sodium dihydrogen phosphate (Na)₂HPO₄·2H₂O)0.39g, distilled to 1000 mL. According to the different sizes of the protein and the fluorescein molecules and the different elution rates, the fluorescein which is not combined with the protein is removed, and the separation effect is achieved.

3.1 column packing: adding at least 20mL of distilled water into the Sephadex G200 SepHadex gel per gram for swelling, weighing 18G of the Sephadex G200 SepHadex G gel, adding 500mL of distilled water for soaking for 24h, pouring out impurities on the upper layer after the dextran is settled, adding prepared PBS, uniformly blowing the Sephadex G200 SepHadex G gel by using a liquid transfer gun, slowly adding the Sephadex G gel into a previously cleaned common chromatographic column along the column wall to avoid generating bubbles, continuously adding PBS into the column during the period to ensure that the SepHadex G gel is completely soaked in PBS buffer solution, and carrying out sample adding and purification after the elution balance of the PBS with about twice the column volume.

3.2 sample adding: before sample addition, excess PBS on the gel in the column is first discharged until the liquid level in the column is flush with the gel surface (or a very thin liquid layer remains). Then a pipette or a long gun head is used for dropwise adding the sample at a position as close as possible to the liquid level without allowing the solution to wash the gel loose and float, after the sample is added, the lower opening is opened to slowly discharge the liquid until the liquid level is equal to the gel level, the original container for containing the sample is washed by a small amount of distilled water for 2-3 times, and the elution can be carried out after all the sample enters the chromatographic column.

3.3 column passing: the protein marked by fluorescence is dropwise added into the chromatographic column, the marked protein can firstly pass through the chromatographic column with light fluorescence color, fluorescein without the marked protein can be hung on the gel column to slowly flow down, and the protein which is firstly left as the mark is collected.

3.4, collecting: the sample is collected after completely entering the elution column, and is collected once every 4mL, and is numbered according to the time sequence, the collected sample is placed under an ultraviolet lamp to see whether fluorescence exists, if a tube begins to generate fluorescence, the protein marked with FITC begins to elute, and the protein layer marked with the above icon can be observed by naked eyes to have faint yellow and is eluted quickly, and the layer is collected intensively (more collection can be carried out in advance and delayed to prevent loss).

4. Detecting the sample by a direct fluorescence detection method:

4.1 tissue sample treatment: taking suspected chlamydia abortus disease material, taking a proper amount of fresh uterine mucosa and vaginal mucosa, preparing into a smear, fixing for 10 minutes by cold acetone, and storing in a dark place for examination.

4.2 cell sample treatment: grinding the pathological tissue at 4 ℃, treating the pathological tissue at 200UI/mL gentamicin for 30min at room temperature, centrifuging for 10min, discarding the precipitate, infecting McCoy cells growing to a single layer with the supernatant (a cover glass is placed in a 24-hole cell plate in advance), infecting for 48h, taking out the cover glass, fixing for 10min with precooled acetone or methanol, taking out, airing, and storing at-20 ℃ for later use;

4.3 diluting the collected FITC labeled monoclonal antibody and PBS according to the equal times of 1:10, 1:100, 1:500 and 1:1000, diluting evans blue by using sterilized PBS according to the proportion of 8:1, then mixing the diluted monoclonal antibody and evans blue uniformly according to the proportion of 7:1 to obtain the final fluorescent antibody liquid, and standing at 4 ℃ for later use; taking out the prepared slide, firstly dripping PBS for soaking for 10min, then dripping about 25 mu L of fluorescence labeling abortion chlamydia major outer membrane protein monoclonal antibody, putting into a wet box, incubating for 30min at 37 ℃, taking out and washing for 3 times with PBS, each time for 5min, after washing, naturally drying for 5-10 min in a dark place, sealing with 50% glycerol, and placing under a fluorescence microscope for detection. Meanwhile, a chlamydia infection positive slide is used as a positive control, and an uninfected slide is used as a negative control. And comparing the coincidence rate of the direct immunofluorescence diagnostic result and the detection result of the kit coated by the chlamydia specific Lipopolysaccharide (LPS).

50 collected samples were tested using a fluorescently-labeled monoclonal antibody (CA1-MM01H) and Chlamydia abortus was distributed in the cytoplasm with round or oval, uniformly fluorescent yellow-green, regularly shaped, luminescent particles under a fluorescent microscope. The results are shown in table 1 and figure 5 in comparison with the results of the chlamydia-specific Lipopolysaccharide (LPS) coated commercial kit assay (IMAGENTM CHLAMYDIA, Oxoid Ltd, uk).

TABLE 1C comparison of immunofluorescence assay results of LPS coating and direct fluorescence assay diagnosis of abortus major outer membrane recombinant protein monoclonal antibody (CA1-MM01H)

Pathological material	MOMP	LPS	Rate of agreement
				Positive for	49	50	98％
Negative of	1	0	100％

In conclusion, the C.abortus CA1A-1MM01H monoclonal antibody can be used for rapidly diagnosing the Chlamydia abortus disease.

Example 3 stability and specificity detection of hybridoma cell secreted antibody (CA1-MM01H)

1. Hybridoma cell secreted antibody stability detection

1) Subculturing the hybridoma cells, collecting cell culture supernatants one by one, detecting the antibody titer of the hybridoma cells by an indirect ELISA method every 5 generations, and observing whether the antibody titer is stable;

2) the hybridoma cells are thawed and subcultured continuously after being frozen in liquid nitrogen for 6 months, and the antibody titer of the supernatant of the cell culture solution is detected by 5 times of transmission by an indirect ELISA method.

2. Hybridoma cell secreted antibody specific detection

Respectively taking PEDV, PPV, PRV, CSFV, PRRS, PCV pathogenic solution and porcine abortion chlamydia CP12 bacterial solution as antigens, coating an enzyme label plate under the same condition, and taking primary antibody as a positive hybridoma cell culture supernatant, a cell culture solution containing serum and serum-free; the secondary antibody is HRP marked rabbit anti-mouse IgG, the OD450mn value is read on the enzyme labeling instrument, each sample is repeated for 3 times to calculate the average value, and the specificity of the monoclonal antibody is detected by adopting an indirect ELISA method.

3. Results

3.1 hybridoma cell secreted antibody stability detection assay

1) The hybridoma CA1-MM01 was serially passaged for 20 generations, and the results of detection by indirect ELISA at every 5 generations showed that the hybridoma cells still stably secreted the Chlamydia abortus monoclonal antibody at 20 generations, as shown in Table 2.

TABLE 2 hybridoma CA1-MM01 stability test results

Number of passages	1	5	10	15	20
						Antibody titer OD450Value (1:10240)	0.388	0.374	0.367	0.365	0.352

2) Shelf life test of hybridoma cells

The hybridoma cells are frozen and stored in liquid nitrogen for 6 months, the antibody titer of the hybridoma cells is detected by recovering, culturing and subculturing to 20 generations and taking supernatant of cell culture solution every 5 generations, and the measurement result shows that the recovered hybridoma cell strains still have good secretion after 6 months of freezing storage, the antibody level is stable, and the titer is not obviously reduced, and the result is shown in Table 3.

TABLE 3 stability test results of hybridoma CA1-MM01 after cryopreservation

Number of passages	1	5	10	15	20
						Antibody titer OD450Value (1:10240)	0.369	0.356	0.342	0.331	0.320

3.2 hybridoma cell secretion antibody specificity detection assay

Respectively taking PEDV, PPV, PRV, CSFV, PRRS, PCV pathogen solution and porcine chlamydia abortus CP12 bacterial solution as antigens, coating an enzyme label plate by the same concentration (5 mu g/mL) under the same condition, and secreting supernatant 1 from hybridoma CA1-MM01 strain: the indirect ELISA detection is carried out by diluting the hybridoma cells at a ratio of 1000, and the result shows that the monoclonal antibody secreted by the hybridoma CA1-MM01H strain only has strong specificity to the porcine chlamydia abortus CP12 strain and has no cross reaction detection result to other common pathogens of pigs, which is shown in Table 4.

TABLE 4 hybridoma CA1-MM01 monoclonal antibody Indirect ELISA specific detection (OD)₄₅₀) Value of

4. Conclusion

The stability test and the specificity test of the hybridoma cell strain CA1-MM01 show that when the hybridoma cells are frozen and stored and continuously passaged for 20 generations, the target antibody can still be stably secreted, and the titer of the secreted antibody is still kept at a stable level without obvious reduction.

It should be understood that the technical solutions of the above embodiments, in which the amounts of reagents or raw materials used are proportionally increased or decreased, are substantially the same as those of the above embodiments.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims (8)

1. A hybridoma cell strain CA1-MM01 is preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation date is 11 months and 22 days in 2017, and the preservation number is CGMCC number 14882.

2. The monoclonal antibody CA1-MM01H secreted by the hybridoma cell line CA1-MM01 of claim 1.

3. The monoclonal antibody according to claim 2, which specifically immunoreacts with the MOMP protein of chlamydia abortus, the MOMP protein having the amino acid sequence shown in SEQ ID No. 2.

4. A method for suspension culture of the hybridoma cell line of claim 1, comprising: the hybridoma cell line CA1-MM01 of claim 1, which is subcultured in serum-containing medium and the serum concentration is decreased at each passage.

5. The method according to claim 4, wherein the subculture is carried out using a medium containing 10% serum, 8% serum, 6% serum, 4mmol/L Gln and 4% serum, and 4mmol/L Gln and 2% serum in this order.

6. The method according to claim 5, wherein the subculture is carried out using RPMI1640 medium containing 10% fetal bovine serum, RPMI1640 medium containing 8% fetal bovine serum, RPMI1640 medium containing 6% fetal bovine serum, RPM1640 containing 4mmol/L Gln and 4% fetal bovine serum, mixed medium of SFM4CHO: OptiCHO, RPM1640 containing 4mmol/L Gln and 2% fetal bovine serum, SFM4CHO: OptiCHO, in this order.

7. Use of the monoclonal antibody CA1-MM01H of claim 2 or 3 in the preparation of a Chlamydia abortus detection reagent or kit.

8. A detection reagent or kit comprising the monoclonal antibody CA1-MM01H according to claim 2 or 3.

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