CN108486063B

CN108486063B - Hybridoma cell strain Anti-CLas McAb2, monoclonal antibody secreted by hybridoma cell strain Anti-CLas McAb2 and application of monoclonal antibody

Info

Publication number: CN108486063B
Application number: CN201810132721.6A
Authority: CN
Inventors: 丁芳; 约翰·哈通; 邓秀新; 彭抒昂; 洪霓; 王国平; 刘永忠
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-02-09
Filing date: 2018-02-09
Publication date: 2021-05-28
Anticipated expiration: 2038-02-09
Also published as: CN108486063A

Abstract

The invention provides a Hybridoma cell strain Anti-CLas McAb2, which is a mouse Hybridoma cell (Mus musculus Hybridoma) Anti-CLas McAb2, and the preservation number is CCTCC NO: C2017123. the invention also provides a preparation method of the hybridoma cell strain and a monoclonal antibody of the recombinant Huanglongbing bacterium CLas McAb2 secreted and generated by the hybridoma cell strain or a subcultured cell strain thereof. The invention also provides application of the monoclonal antibody of the recombinant Huanglongbing pathogen CLas McAb2, which comprises a detection kit and an immune colloidal gold detection test strip, and the detection is carried out by utilizing the specificity of the monoclonal antibody of the recombinant Huanglongbing pathogen CLas McAb 2. The method has the advantages of good specificity, strong anti-interference performance, low cost and wide application, and can quickly and effectively detect the yellow dragon germs existing in the citrus material.

Description

Hybridoma cell strain Anti-CLas McAb2, monoclonal antibody secreted by hybridoma cell strain Anti-CLas McAb2 and application of monoclonal antibody

Technical Field

The invention relates to a hybridoma cell strain and a monoclonal antibody, in particular to a hybridoma cell strain Anti-CLas McAb2, a monoclonal antibody secreted by the hybridoma cell strain and application of the monoclonal antibody.

Background

The citrus yellow shoot is a destructive disease which is the most difficult, most harmful and most threatening in citrus production, is called AIDS on citrus by people, and is widely distributed in almost all citrus main production areas in China. As a destructive disease, citrus infected plants often lose fruiting capacity or die within 2-3 years, and even cause garden damage. Rampant harm and spread of citrus yellow shoot cause serious threats to the stability and development of the citrus industry, and become a major obstacle to the development of the citrus industry.

At present, an effective control medicament and an ideal resistant variety are not available for the citrus yellow dragon disease, so that comprehensive control is a main method for controlling the disease at present. The strict plant quarantine is the premise of protecting the citrus in a disease-free area and a new area, the establishment of a virus-free nursery, the cultivation and the planting of virus-free nursery stocks are the basis for preventing the yellow dragon disease, and the key measures for preventing the disease from being epidemic are to eliminate the disease-transmitting psyllids in time and thoroughly dig out diseased trees. However, these efforts must be based on ascertaining the presence or absence of the xanthomonas bacterium in the citrus material. Therefore, the rapid and accurate detection and diagnosis of the huanglongbing pathogen is an important prerequisite for the comprehensive prevention and control of the disease at present.

In the prior art, various methods and technologies such as field diagnosis, identification of indicator plants, microscopic observation, serological detection, nucleic acid hybridization detection, PCR detection and the like have been developed for the detection and diagnosis of citrus greening disease. Although these methods have promoted a growing understanding of citrus greening disease, they have shown varying degrees of limitations in the practical production of citrus. For example, in the field diagnosis process, the disease symptoms are complicated and changeable and are easily confused with symptoms such as deficiency, virus diseases, phytotoxicity and the like, so that diagnosis errors are often caused; the efficiency of identification through indicating plants is affected by the problems of low grafting success rate, poor stability and the like, and the method is long in time consumption (the identification result can be obtained after several months or even longer); identification by means of microscopy, serology and the like requires specific instruments and equipment and professional experimental skills; nucleic acid hybridization and various PCR techniques, while capable of detecting Crohn's disease rapidly and accurately, require expensive instrumentation, reagent materials and skilled experimental skills as do methods such as microscopy, serology, etc. Therefore, a new technology or a new product applied to the citrus production practice is urgently needed to be developed, and the fast and accurate detection and diagnosis of the yellow dragon pathogen are realized.

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone, directed against only a particular epitope, and are referred to as monoclonal antibodies. Usually, hybridoma (hybridoma) antibody technology is used to prepare hybridoma, which is a method of fusing a sensitized B cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited proliferation ability into a B cell hybridoma based on cell fusion technology. By culturing a single hybridoma having such a characteristic into a cell population, a monoclonal antibody, which is a specific antibody against one epitope, can be produced. Comparison document 1: the patent number of CN102212134A entitled polyclonal antibody against outer membrane protein of citrus yellow shoot germ, and the preparation method and application thereof disclose a polyclonal antibody against outer membrane protein of citrus yellow shoot germ. The prepared polyclonal antibody can be used for detecting a sample infected with liberobacter citreum in the field. The patent discloses a polyclonal antibody, because the polyclonal antibody has the inevitable defect of poor specificity, and the outer membrane proteins of the citrus yellow dragon germ have various types, the best effect of detecting the citrus yellow dragon germ by using which outer membrane proteins as antigens to prepare monoclonal antibodies is not reported, and the research report on the aspect of the monoclonal antibodies aiming at the specific proteins of the citrus yellow dragon germ is not available at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides hybridoma cell strains Anti-CLas McAb2 and CLas McAb2 monoclonal antibodies, and the monoclonal antibody aiming at the CLas McAb2 membrane surface protein has the advantages of good specificity, strong Anti-interference performance, low cost and wide application, and can quickly and effectively detect the phoma flavipes existing in citrus materials.

The invention is realized by the following steps:

one of the purposes of the invention is to provide a Hybridoma cell strain Anti-CLas McAb2, which is a mouse Hybridoma cell (Mus musculus Hybridoma) Anti-CLas McAb2 with the preservation number of CCTCC NO: C2017123.

the invention also aims to provide a monoclonal antibody of the recombinant Huanglongbing pathogen CLas McAb2, which is a specific monoclonal antibody aiming at the protein of the recombinant Huanglongbing pathogen CLas McAb2, and the hybridoma cell strain Anti-CLas McAb2 or a subculture cell strain thereof is secreted and generated by the monoclonal antibody of the recombinant Huanglongbing pathogen CLas McAb 2.

The invention also aims to provide a preparation method of the hybridoma cell strain Anti-CLas McAb2, which comprises the following steps:

step 1, amplifying coding region genes of Xanthomonas campestris CLas McAb2, and loading the genes into a prokaryotic expression vector pET-28a through a specific primer containing a specific enzyme cutting site (BamH I/Xho I) to obtain a recombinant expression plasmid pET28a-McAb 2;

step 2, transforming the recombinant expression plasmid pET28a-McAb2 into an escherichia coli Rosetta strain, adding an inducer to induce the expression of the CLas McAb2 protein, and obtaining induced thalli;

and 3, collecting and crushing the induced bacteria, fusing spleen cells and myeloma cells of the immunized experimental animal and the immunized animal to obtain a hybridoma cell strain, and obtaining a positive cell strain only secreting a CLas McAb2 protein antibody from the hybridoma cell strain by a differential ELISA screening method.

The fourth purpose of the invention is to provide the application of the monoclonal antibody of the recombinant Huanglongbing bacterium CLas McAb2, and the monoclonal antibody of the recombinant Huanglongbing bacterium CLas McAb2 is used for detecting the Huanglongbing bacterium citrulli by an indirect ELISA method.

The fifth purpose of the invention is to provide an ELISA detection kit for Huanglongbing bacteria, which comprises any one of the monoclonal antibodies of the recombinant Huanglongbing bacteria CLas McAb 2.

The sixth purpose of the invention is to provide an immune colloidal gold test strip for huanglongbing, which comprises any one of the monoclonal antibodies of the recombinant huanglongbing CLas McAb 2.

The invention has the beneficial effects that: the method has the advantages of good specificity, strong anti-interference performance, low cost and wide application, and can quickly and effectively detect the yellow dragon germs existing in the citrus material.

The preservation date of the hybridoma cell strain is 2017, 10 and 11 months, and the preservation number is CCTCC NO: C2017123. named hybridoma cell strain Anti-CLas McAb2, the name of Latin literature is Mus musculus, the name of the preservation unit is China center for type culture Collection, the address is Wuhan university, Wuhan City, China, the zip code: 430072.

drawings

FIG. 1 is a flow chart of the preparation of the monoclonal antibody against the recombinant Xanthomonas campestris CLas McAb2 provided in example 1 of the present invention;

FIG. 2 is a plasmid map of the vector pET-28a provided in example 1 of the present invention;

FIG. 3 shows the SDS-PAGE detection of purified CLas McAb2 protein according to example 1 of the present invention;

FIG. 4 is a diagram showing the SDS-PAGE detection of the purified monoclonal antibody of the recombinant Huanglongbing bacterium CLas McAb2 in ascites in accordance with embodiment 2 of the present invention;

FIG. 5 shows that the specificity of the monoclonal antibody of the recombinant Xanthomonas campestris CLas McAb2 was verified by Western-blot experiment provided in embodiment 3 of the present invention.

Detailed Description

EXAMPLE 1 establishment of hybridoma cell line Anti-CLas McAb2

Firstly, preparing experimental materials and reagents

Before the operation, the sterilized consumables required in the following table were placed in a clean bench for irradiation sterilization. Pipettes of various specifications and matched gun heads, homogenizers, scissors, tweezers, glass plates, foam pads, needles, cell screens, filter paper and 96-hole cell culture plates (sterile).

TABLE 1

Second, Experimental methods

1. Preparation of recombinant expression plasmid pET28a-McAb2

Firstly, designing a specific primer aiming at McAb2 (the nucleotide sequence of the recombinant Clas McAb2 protein is shown as SEQ ID NO: 1) of the yellow dragon germ, designing an upstream primer and a downstream primer according to the specific structure of a carrier, wherein the adopted enzyme cutting site is BamH I/Xho I, and the upstream primer and the downstream primer are respectively 5' -CGGGATCCATGATCGATAACTTGGATACG-3 '(the nucleotide sequence is shown in SEQ ID NO: 2) and 5' -CCCTCGAGAGTATTAGCACGACATC-3' (the nucleotide sequence is shown in SEQ ID NO: 3), PCR technology is used to amplify McAb2 coding region gene (high fidelity enzyme) from the genome of Huanglongbing pathogen, the amplified product is recovered and purified and then is loaded into a vector pET-28a to obtain a recombinant expression plasmid pET28a-McAb2, the plasmid is transferred into TOP10 strain by a heat shock transformation method for propagation, and after sequence verification, the recombinant plasmid pET28a-McAb2 is transferred into Rosetta expression strain.

Blank control group: empty vector plasmid pET-28a without McAb2 gene. The plasmid map of the vector pET-28a is shown in FIG. 2.

2. Preparation of immune antigens

(1) Bacterial liquid activation: the recombinant expression plasmid pET28a-McAb2 is inoculated with a liquid culture medium and shaken, 20ul of the bacterial liquid is taken to be activated in 2ml LB culture medium containing Kan + (37 ℃, 16 h); blank control group: the same treatment was carried out with the empty vector plasmid pET-28a, which did not contain the McAb2 gene.

(2) Inducible expression test: individual colonies were picked into LB medium containing Kan +, and when shaken to OD600 of 0.6, expression was induced with 0.8mM IPTG (37 ℃ C., 4 h). Meanwhile, the expression of McAb2/Rosetta is not induced. After induction, the cell is centrifuged at 12000rpm for 1min, the cell sediment is collected, after PBS heavy suspension, equal volume 2 xSDS-PAGE loading buffer is added for boiling and sample preparation, and 12 percent SDS-PAGE is used for analyzing the expression condition. McAb2/Rosseta is normally expressed as judged by SDS-PAGE results. Blank control group: the same induction was performed on the empty vector plasmid pET-28a, which did not contain the McAb2 gene.

(3) The activated bacterial solution was subjected to scale-up culture, and when the OD600 was 0.6, 0.2mM IPTG was added for induction (20 ℃ C., 16 hours), and the cells were collected. Ultrasonic bacteria breaking is carried out for 15min, power is 390W, working is carried out for 2s and stopped for 4s, 9000rpm is carried out, centrifugation is carried out for 10min, supernatant is collected, denaturation buffer solution is used for resuspension and precipitation, samples are respectively prepared, and the expression state of the samples is analyzed through 12% SDS-PAGE detection. Blank control group: the empty vector plasmid pET-28a without McAb2 gene was subjected to the same amplification treatment. The expression is mainly in the pellet [ inclusion body ] judged by the SDS-PAGE result and the volume.

(4) Protein purification: the inclusion bodies dissolved in the denaturing buffer are taken, the target protein is purified by means of sephadex separation, ion exchange or the like, and the purified protein is replaced by the buffer and dissolved in a PBS solution, as shown in FIG. 3, and the protein CLas McAb2 with high purity is obtained by SDS-PAGE. Wherein, the main components and concentrations of buffer in the purified protein solution are shown in the following table 2. Blank control group: the empty vector plasmid pET-28a, which does not contain the McAb2 gene, was subjected to the same purification treatment.

TABLE 2

Composition (I)	Na₂HPO₄	NaH₂PO₄	NaCl	KCl
					Concentration (mmol/L)	8	1.47	137	2.7

3. Immunization of mice

10 pure-line BALB/C female mice with quick action at 4-6 weeks are selected and divided into two groups, namely 5 mice in each of an experimental group and a control group, subcutaneous multipoint injection is respectively adopted to immunize the mice for the first time, the immunization dose of each mouse in the control group is 10 ul/mouse, the immunization interval of the experimental group is 7-15 days, and the immunization time interval of each time is 7-15 days.

TABLE 3

Serial number	Time	Working node	Work content
					1	2016.6.17	Collecting negative blood before immunization	Negative serum is more than or equal to 10 ul/mouse
2	2016.6.17	First immunization	Immune antigen 100 ug/mouse
				3	2016.7.2	Second immunization	Immune antigen 100 ug/mouse
4	2016.7.16	Third immunization	Immune antigen 100 ug/mouse
				5	2016.7.30	The fourth immunization	100 ug/tail vein

4. Fusion of cells

The method comprises the following specific steps: after the tail vein is immunized for 3 days for the fourth time, a small amount of blood is collected, and serum is separated and frozen at the temperature of 20 ℃ below zero to serve as a positive control in screening. Killing an immune mouse according to a biological safety method, soaking and disinfecting the immune mouse by 75% alcohol for 5min, taking spleen cells aseptically, fusing the spleen cells with myeloma cells SP2/0 in a logarithmic growth phase under the action of PEG (MW4000), taking Balb/c mouse abdominal cavity macrophages as feeder cells, suspending the fused cells and feeder cells by using an HAT culture medium, subpackaging 96 pore plates, and culturing in a 6% CO2 incubator at 37 ℃. Adding fresh HAT culture medium after 5 days, culturing with HAT complete culture medium after 10 days, and periodically observing, changing solution and detecting.

5. Screening of specific Positive cell lines

10 days after cell fusion, the hybrid cells were observed under a microscope to form a small colony. Fresh HAT complete medium was replaced, 100. mu.l/well. And (3) carrying out positive screening on the wells with the hybridoma cells by adopting an ELISA detection indirect enzyme-linked immunosorbent assay, using lysates obtained after induction of pET-28a and pET28a-McAb2 bacteria as detection antigens, and enabling cell well supernatants to be screened to simultaneously react with the lysates, only react with lysate coated wells obtained after induction of pET28a-McAb2 bacteria, namely specifically react with CLas McAb2 protein to be determined as candidate strains. And (3) judging the wells as positive wells when the ratio of the OD450 value of the supernatant to the OD450 value of the blank control well is more than 2.1, wherein the hybridoma cells in the wells can be called as positive cell strains. By this principle, a cell line which is specific to the antibody against the protein of CLas McAb2 can be obtained, and thus, a cell culture supernatant or ascites can be obtained by this method.

The OD value of the monoclonal antibody measured by ELISA detection indirect enzyme-linked immunosorbent assay is 2.896.

6. Subclone selection

And (3) respectively subcloning the 3 positive cell strains screened in the step (6) by adopting a limiting dilution method, removing the false positive cell strains after two successive subcloning, and finally obtaining hybridoma cell strains which respectively secrete specific monoclonal antibodies only aiming at the CLas McAb2 protein. Namely the hybridoma cell strain provided by the invention has the preservation date of 2017, 10 and 11 months and the preservation number of CCTCC NO: C2017123. named hybridoma cell strain Anti-CLas McAb2, the name of Latin literature is Mus musculus, the name of the preservation unit is China center for type culture Collection, the address is Wuhan university, Wuhan City, China, the zip code: 430072.

example 2: preparation of monoclonal antibodies

1. Preparation of ascites

Injecting 0.5ml of Freund incomplete adjuvant into the abdominal cavity of BALB/c mice with the age of more than 6 weeks; three days later, hybridoma cells diluted with PBS or incomplete medium were intraperitoneally inoculated, 1-5X 10 per mouse⁶0.5 ml; after 3 days, observing the ascites generation condition of the mouse every day, if the abdomen is obviously enlarged and the skin is tense when the mouse touches the abdomen, namely, a 10ml needle can be used for collecting the ascites; the ascites fluid was centrifuged (12000rpm for 10min), the supernatant was collected and frozen in a freezer at-20 ℃.

2. Ascites antibody purification

(1) Serum pretreatment, namely filtering the serum by using a 0.22-aperture filter, mixing the filtered serum with PBS (pH7.4) with the same volume, and adjusting the pH to be 7.8, wherein the affinity hanging column has better effect;

(2) a step of balancing affinity column, which is to wash the column by 10 times volume of PBS (pH7.4) and connect and fix the lower end of the column with the liquid inlet of the nucleic acid protein detector;

(3) loading: when the PBS balances the column, the numerical value of the nucleic acid protein detector is adjusted to be stabilized at about 0, the sample is added into the sample loading column bed, the numerical value on the nucleic acid protein detector is slowly raised, and the liquid is collected;

(4) after the loading is finished, washing the protein by PBS (pH7.2), and stopping collecting the flow-through liquid when the value of the nucleic acid protein detector is reduced to an equilibrium value and is not reduced any more;

(5) antibody elution: when the numerical value of the nucleic acid protein detector is kept to be lower and stable, when the liquid level of the column bed is quickly drained, the eluent (0.1M Gly-HCl, pH2.7) is gently added into the column bed, and the column bed is not flushed as much as possible; when the value on the nucleic acid protein detector rapidly rises, the eluted liquid is rapidly received in different tubes in sequence, and is rapidly neutralized by a neutralization solution (0.5M Tris-Cl, pH8.0), multiple tubes are sequentially connected when the eluted liquid is received, and the sequence is recorded;

(6) after the elution is finished, when the value is lowered and does not change any more, washing the equilibrium column by PBS with at least 5 times of the volume of the column bed at the flow rate of 2-4 ml/min;

(7) preserving the column bed with preservation solution (PBS containing 20% ethanol), sealing the upper and lower ends of the column, and preserving at 4 deg.C;

(8) dialyzing the antibody against PBS (pH7.2) for 3 times, each time for 4 hr or more;

(9) the antibody concentration was measured at 2mg/ml, and the purity of the antibody was analyzed by SDS-PAGE.

As shown in FIG. 4, the first lane in FIG. 4 is Marker, the second lane and the third lane are negative controls for BSA, and the fourth lane is monoclonal antibody secreted by the cell strain of Anti-CLas McAb2 provided by the present invention, i.e., monoclonal antibody against CLas McAb2 protein.

EXAMPLE 3 characterization of monoclonal antibodies

1. Monoclonal antibody potency detection

(1) Coating: McAb2 antigen was coated at 1ug/ml, 100 ul/well, overnight at 4 ℃.

(2) And (3) sealing: patting the liquid in the ELISA plate, adding 150ul of blocking solution (1% BSA in TBST) per well, and incubating at 37 ℃ for 60 min.

(3) A first antibody: the liquid in the ELISA plate was patted dry at 350 ul/well/time, washed 3 times with TBST, patted dry, and the antibody purified in example 2 was diluted 1:3000 and 3 times (1% BSA dilution), 100 ul/well and incubated at 37 ℃ for 60 min.

(4) Secondary antibody: patting the liquid in the ELISA plate to 350 ul/hole/time, washing for 3 times by TBST, patting to dry, adding 100 ul/hole of HRP goat anti-mouse (diluted by 1:3000 confining liquid), and incubating for 60min at 37 ℃.

(5) Color development: patting the liquid in the ELISA plate to be 350 ul/hole/time, washing for 3 times by TBST, patting the plate to be dry, adding 100 ul/hole of single substrate TMB, and developing for 3-5 min.

(6) And (4) terminating: stop solution was added at 50 ul/well and the OD450 read.

Experiments show that the titer of the monoclonal antibody of the recombinant Huanglongbing CLas McAb2 is 1: 7.29X 10⁵。

2. Identification of monoclonal antibody specificity

The target protein was subjected to SDS-PAGE, and the negative serum was used as a negative control. And after electrophoresis is finished, carrying out a Western-blot experiment, transferring the protein onto an NC membrane by adopting a wet transfer method, and then carrying out exposure and color development after the steps of skimmed milk powder sealing, primary antibody incubation, secondary antibody incubation and the like.

As shown in fig. 5, lane 1 is a negative control, lane 2 is a positive reaction, wherein the 19KD band is a positive band (the size of the CLas McAb2 protein is 19KD), and the result shows that the prepared monoclonal antibody can specifically bind to the target protein in the citrus leaves positive for huanglongbing, but not react with the healthy citrus leaf control.

Example 4 application of monoclonal antibody of recombinant Xanthomonas campestris CLas McAb2

1. The prepared recombinant Huanglongbing McAb2 monoclonal antibody is applied to preparation of ELISA kit/immune colloidal gold detection test paper, products in various forms such as immunofluorescence, immunoturbidimetry, chemiluminescence and the like.

(1) An ELISA kit of the recombinant Huanglongbing McAb2, which is selected from any one of the following:

a. the kit comprises a kit body, an ELISA plate arranged in the kit body and reagents stored in the kit body, wherein the reagents comprise a monoclonal antibody of recombinant Huanglong pathogen McAb2, a standard product, namely purified recombinant Huanglong pathogen McAb2 protein, a detection antibody or a competitive marker;

b. the kit comprises a kit body, an ELISA plate which is arranged in the kit body and is coated with a monoclonal antibody of the recombinant Huanglong pathogen McAb2, and reagents stored in the kit body, wherein the reagents comprise a standard product, namely purified recombinant Huanglong pathogen McAb2 protein, a detection antibody or a competitive marker.

(2) The immune colloidal gold test paper strip of huanglong pathogen, including sample pad, gold mark pad, nitrocellulose membrane and absorption pad, the sample pad with the gold mark pad closely link to each other, the gold mark pad with the nitrocellulose membrane closely link to each other, the nitrocellulose membrane with the pad that absorbs water closely link to each other the nitrocellulose membrane on be provided with the detection line, the detection line be close to the gold mark pad, the detection line keep away from the nitrocellulose membrane of one side of gold mark pad on be provided with the quality control line, wherein, by the collection number is CCTCC NO: the monoclonal antibody of the recombinant Huanglong pathogen CLas McAb2 produced by the hybridoma cell strain of C2017123 is combined with colloidal gold to form a gold-labeled antibody, the gold-labeled antibody is sprayed on a combination pad to form a gold-labeled pad, and the gold-labeled pad is prepared by mixing the following components in percentage by weight, wherein the collection number is CCTCC NO: and the monoclonal antibody of the recombinant Huanglongbing pathogen CLas McAb2 produced by the hybridoma cell line of C2017123 is used as a detection antibody, and the detection antibody is sprayed on a nitrocellulose membrane to form a detection line.

2. The prepared antibody or detection kit is used for detecting the citrus yellow dragon germ in the plant material by an indirect ELISA method, and the operation steps are as follows:

(1) taking a mature citrus branch to be detected, a midrib, a petiole or a root of a leaf, transversely cutting a sample to be detected by using a sharp disposable blade, simultaneously treating healthy citrus leaves and citrus leaves infected with yellow dragon disease as negative control and positive control respectively, and transferring the balanced and uniform transverse section onto a nitrocellulose membrane for about 8-12 seconds;

(2) sealing the transferred nitrocellulose membrane in SuperBlock sealing solution at room temperature for 2 hours;

(3) adding the monoclonal antibody of the recombinant Huanglong pathogen CLas McAb2 diluted according to the proper proportion, and incubating for 1.5 hours at 37 ℃ and 120 rpm;

(4) PBST buffer washing 3 times, each time 10 minutes;

(5) adding enzyme-labeled secondary antibody diluted according to the proper proportion, and incubating at 37 ℃ and 120rpm for 1.0 hour;

(6) PBST buffer washing 3 times, each time 10 minutes;

(7) adding a substrate, and developing for 30 minutes in a dark place;

(8) observing and recording the color reaction, wherein if the color is positive reaction (infected with yellow dragon germs), the non-color reaction is negative reaction (not infected with yellow dragon germs).

The method can rapidly detect the citrus yellow dragon germs, and is simple, convenient and economical; the method is rapid and sensitive; no special instrument is needed; special training is not needed, and general fruit growers can operate the fruit growers.

a. Sensitivity of Anti-CLas McAb2 monoclonal antibody in detection of citrus greening disease sample

Collecting leaves of ponkan, local early, Cyte orange, sweet orange and other citrus varieties in the field, and 10 samples of each variety of catharanthus roseus infected with Scutellaria tabaci and healthy catharanthus roseus by grafting, and respectively using the outer membrane protein polyclonal antibody in the comparison document 1 and the prepared recombinant Bgacaja 2 monoclonal antibody as primary antibodies for detection by the indirect ELISA method. The results are shown in Table 4 below, and the detection rate of the recombinant Huanglongbing McAb2 monoclonal antibody prepared by the invention is greatly higher than that of the outer membrane protein polyclonal antibody in the comparison document 1.

TABLE 4 comparison of detection rates of monoclonal antibody of the present invention and polyclonal antibody in reference 1

b. Anti-CLas McAb2 monoclonal antibody for detecting specificity of citrus greening disease sample

The symptoms of the citrus leaves caused by certain nutrient deficiency, virus diseases and the like are similar to the symptoms of citrus yellow shoot diseases, so that people confuse the citrus yellow shoot diseases and the citrus yellow shoot diseases when identifying the citrus yellow shoot diseases in the field, and therefore the prepared monoclonal antibody is used for detecting the symptom leaves in the experiment to determine that the monoclonal antibody can only specifically detect the citrus yellow shoot diseases. Collecting leaves with symptoms of zinc deficiency, manganese deficiency, magnesium deficiency and the like and leaves infected with citrus leaf crushing virus and tristeza virus in the field, grinding the leaves by using a coating buffer solution, coating an enzyme label plate, and performing an indirect ELISA experiment by using the prepared antibody. The results are shown in Table 5, and it can be seen that the prepared antibody only specifically detects the citrus yellow dragon germ, but does not have positive reaction with samples with zinc deficiency, magnesium deficiency, manganese deficiency, citrus leaf shattering disease, recession disease and the like.

TABLE 5 identification of the detection specificity of the Anti-CLas McAb2 monoclonal antibody

Note: "+" indicates specific reaction, "-" indicates no specific reaction

c. Detection of Anti-CLas McAb2 monoclonal antibody on citrus phomopsis isolate from different regions

The method comprises the steps of collecting samples infected with citrus huanglongbing in Jiangxi, Fujian, Guangdong, Guangxi, Hunan, Yunnan, Hainan and the like, grinding the samples by using a coating buffer solution, coating an enzyme label plate, and performing indirect ELISA (enzyme-linked immuno sorbent assay) by using the prepared Anti-CLas McAb2 monoclonal antibody, wherein the results show that the Anti-CLas McAb2 monoclonal antibody can specifically identify pathogenic bacteria in citrus huanglongbing samples from different geographical sources in China and has good detection effects (see Table 6).

TABLE 6 detection results of Anti-CLas McAb2 monoclonal antibody on P.citrifolia isolates in different regions

Note: "+" indicates specific reaction, "-" indicates no specific reaction

The vector plasmid pET-28a described above is collectively referred to as pET-28a (+).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> university of agriculture in Huazhong

<120> hybridoma cell strain Anti-CLas McAb2, monoclonal antibody secreted by same and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 420

<212> DNA

<213> Huanglong pathogen (liberibacter)

<400> 1

atcgataact tggatacgaa gatatcatct cctgacacag tattaaatga gagttctttg 60

caggagcaat tttcttcatc tgttggtgat tctgtgtttt ttgataccag ttcttattcc 120

attcgcccag cagatattca agtgttatct aatttaggat cttggcttga aaaacatgat 180

tgtgattttc ttatagaagg tcatgcagat gaactaggtt cccgcaacag tagtattgct 240

ttgggacttc gtcgtgctta tgcagttttt aattattttg tagctagagg gattagcgca 300

tctcgcatga aggttacttc atatggaaag gagatgccat ctgtatatgg tcatgatgag 360

gatgcttatg cgaaaaatcg tcgtgcgatc gtttttttaa agggatgtcg tgctaatact 420

<210> 2

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgggatccat gatcgataac ttggatacg 29

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccctcgagag tattagcacg acatc 25

Claims

1. The Hybridoma cell strain Anti-CLas McAb2 is mouse Hybridoma cell (Mus musculus Hybridoma) Anti-CLas McAb2, and the preservation number is CCTCC NO: C2017123.

2. a preparation method of the recombinant protein CLas McAb2 for preparing the hybridoma cell strain Anti-CLas McAb2 of claim 1, comprising the following steps:

step 1, amplifying coding region genes of Xanthomonas campestris CLas McAb2, and loading the coding region genes into a prokaryotic expression vector pET-28a through a specific primer containing a specific enzyme cutting site to obtain a recombinant expression plasmid pET28a-McAb 2;

step 2, transforming the recombinant expression plasmid pET28a-McAb2 into an escherichia coli Rosetta strain, and adding an inducer to induce the expression of the CLas McAb2 protein;

wherein, the nucleotide sequence of the coding region gene of the CLas McAb2 is shown as SEQ ID NO: 1 is shown.

3. The method according to claim 2, wherein the primers for amplifying the gene encoding the Xanthomonas campestris CLas McAb2 in step 1 are the same as the primers for amplifying the gene encoding the Xanthomonas campestris Clas McAb2

5′-CGGGATCCATGATCGATAACTTGGATACG-3′、

And 5'-CCCTCGAGAGTATTAGCACGACATC-3'.

4. The monoclonal antibody of recombinant xanthomonas campestris CLas McAb2 is secreted by the hybridoma cell line Anti-CLas McAb2 or a passaged cell line thereof of claim 1.

5. The monoclonal antibody of claim 4, which is prepared by culturing the hybridoma cell line or injecting the hybridoma cell line into an experimental animal to obtain ascites or supernatant containing cells having an antibody specific to the CLas McAb2 protein, and further separating and purifying to obtain the specific monoclonal antibody of the recombinant Clas McAb2 protein.

6. Use of a monoclonal antibody against recombinant xanthomonas campestris CLas McAb2 for detecting xanthomonas citri by an indirect ELISA method using the monoclonal antibody against recombinant xanthomonas campestris CLas McAb2 according to any one of claims 4 to 5.

7. The use of the monoclonal antibody against the recombinant xanthomonas campestris CLas McAb2 as claimed in claim 6, wherein the detection of xanthomonas citri is performed by the following steps:

(1) taking a mature citrus branch to be detected, a midrib, a petiole or a root of a leaf, transversely cutting a sample to be detected by using a sharp disposable blade, and transferring the transverse section onto a nitrocellulose membrane in a balanced and uniform manner for 8-12 seconds;

(3) adding the monoclonal antibody of the recombinant Huanglongbing bacterium CLas McAb2 according to any one of claims 4-5 diluted according to the appropriate ratio, and incubating at 37 ℃ at 120rpm for 1.5 hours;

(4) PBST buffer washing 3 times, each time 10 minutes;

(6) PBST buffer washing 3 times, each time 10 minutes;

(7) adding a substrate, and developing for 30 minutes in a dark place;

(8) observing and recording the color reaction, wherein a color signal is positive and represents that the citrus yellow dragon bacteria are contained.

8. An ELISA detection kit for Huanglongbing bacteria, which comprises the monoclonal antibody of the recombinant Huanglongbing bacteria CLas McAb2 according to any one of claims 4 to 5.

9. An immune colloidal gold test strip for huanglongbing bacteria, which comprises the monoclonal antibody of the recombinant huanglongbing bacteria CLas McAb2 of any one of claims 4 to 5.