CN106589125B - Monoclonal antibody of anti-lygus lucorum ecdysone receptor protein and application thereof - Google Patents

Abstract

The invention discloses A monoclonal antibody specifically binding with lygus lucorum AlEcR-A protein. The invention also discloses a mouse hybridoma cell strain with a preservation number of CCTCC NO: C2016206. the monoclonal antibody prepared by the invention can be specifically combined with lygus lucorum total protein and AlEcR-A recombinant protein. The invention also discloses application of the monoclonal antibody in comprehensive treatment of lygus lucorum. The invention also discloses a detection kit, which comprises the monoclonal antibody.

Description

Monoclonal antibody of anti-lygus lucorum ecdysone receptor protein and application thereof

Technical Field

The invention relates to the technical field of antibody engineering agricultural science, in particular to a monoclonal antibody of an anti-lygus lucorum ecdysone receptor protein, a hybridoma cell strain for generating the antibody and application of the monoclonal antibody.

Background

Since Bt cotton is planted in large areas in China in 90 years in the last century, target lepidoptera pests such as cotton bollworms (Helicoverpa armigera H) and the like are effectively controlled, the frequency and the use amount of chemical pesticides in cotton fields are greatly reduced, so that the ecological niches of the cotton field pests are subjected to a series of successive changes, and lygus lucorum (Apolygus lucorum mM) has risen to become main disaster-causing pests in Yangtze river, yellow river basin and Xinjiang cotton areas in China. At present, chemical pesticides are mainly used for controlling lygus lucorum, but various problems are brought about, such as reduction of control effect caused by drug resistance, environmental pollution, food safety and the like. Therefore, the prevention, control and disaster reduction of lygus lucorum needs to develop an effective prevention and control technology of a new target. Compared with chemical pesticides, the insect growth regulator pesticide has the advantages of high toxicity and environmental friendliness, and the action mechanism of the pesticide is mainly that insect ecdysone or juvenile hormone is interfered, so that insects can not ecdyse and die. The steroid hormone 20-hydroxyecdysone (20E) is the main active form of insect ecdysone and plays an important regulation and control role in the metamorphosis development process of insects. Research has shown that insect ecdysone receptors (EcR) are 20E target receptors, play a key role in ecdysone signaling, and become important targets for developing novel pesticides.

Therefore, the research on the monoclonal antibody of the ecdysone receptor protein of the lygus lucorum is particularly important in the comprehensive control of pests.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide A monoclonal antibody specifically binding to lygus lucorum AlEcR-A protein. The technical problem to be solved by the invention is to provide a mouse hybridoma cell strain.

The technical problem to be solved by the invention is to provide the application of the monoclonal antibody in preparing A detection tool for detecting AlEcR-A protein of lygus lucorum.

The technical problem to be solved by the invention is to provide the application of the monoclonal antibody in comprehensive treatment of lygus lucorum.

The invention also aims to solve the technical problem of providing a detection kit.

The technical scheme is as follows: in order to solve the problems, the technical scheme of the invention is to provide A monoclonal antibody specifically binding to lygus lucorum AlEcR-A protein, which is prepared from the monoclonal antibody with the preservation number of CCTCC NO: c2016206.

The invention also comprises a mouse hybridoma cell strain with a preservation number of CCTCC NO: c2016206, a hybridoma cell line 8H7, deposited in China Center for Type Culture Collection (CCTCC) at 12/1/2016, address: wuhan university collection center (opposite to the first subsidiary school of Wuhan university) in Wuchang district, Wuhan city, Hubei province, zip code: 430072.

the preparation method of the monoclonal antibody comprises the following steps:

1. constructing A pCzn1-AlEcR-A recombinant expression plasmid;

a primer is designed according to the gene sequence (SEQ ID NO: 1) of AlEcR-A for PCR amplification, restriction enzyme sites Nde I and XbA I are introduced at the same time, A His tag is introduced at the tail end, and an expression vector pCzn1 plasmid is inserted to construct A pCzn1-AlEcR-A recombinant expression plasmid.

2. Expressing and purifying pCzn1-AlEcR-A recombinant protein; see example 1 for details.

3. Preparing a monoclonal antibody; see example 1 for details.

The invention also comprises the application of the monoclonal antibody in preparing A detection tool for detecting the AlEcR-A protein of lygus lucorum.

Wherein, the detection tool is a kit, a chip or test paper.

The invention also comprises the application of the monoclonal antibody in comprehensive treatment of lygus lucorum.

The invention also comprises a detection kit, and the kit contains the monoclonal antibody.

Wherein, the detection kit is an immunohistochemical detection kit or an immunofluorescence detection kit.

Has the advantages that: compared with the prior art, the invention has the following advantages: the AlEcR-A gene of the invention can efficiently express A protein of about 55kD in EscherichiA coli Arctic express, and the recombinant protein mainly exists in the form of inclusion body, the strain containing the target gene is collected to carry out Ni-IDA affinity chromatography, and finally only 1 clear specific band appears near 55kD, which indicates that the purified target protein is obtained. The invention further obtains 1 cell strain which can be stably passaged and secrete the AlEcR-A protein monoclonal antibody through mouse immunization, cell fusion and ascites preparation, and the cell strain is named as 8H7, and the monoclonal antibody can be specifically combined with the total green plant bug proteins and the AlEcR-A recombinant proteins.

Drawings

FIG. 1 is a partial sequence alignment chart;

FIG. 2 shows an electrophoresis chart of enzyme digestion identification; m: DL 10000; 1: plasmid 2 before digestion: carrying out enzyme digestion on the plasmid;

FIG. 3 SDA-PAGE analysis of pCzn1-AlEcR-A induced expression; m is a protein molecular weight standard; control (no IPTG induction, precipitation); 2:37 ℃ overnight culture (total protein 1); culture overnight at 3:37 ℃ (total protein 2); 4:37 ℃ overnight incubation (precipitation); 5:37 ℃ overnight culture (supernatant);

FIG. 4 molecular Sieve purification of pCzn1-AlEcR-A protein;

m, protein molecular weight standards; 1. unpurified inclusion bodies; 2. a hetero-protein not bound to the Ni column; 3. a protein of interest bound to a Ni column;

FIG. 5 Western-blot specific analysis of monoclonal antibodies; m, protein molecular weight standards; 1. 3-year-old lygus lucorum nymphs; 2. recombinant protein pCzn 1-AlEcR-A;

FIG. 6 uses the prepared monoclonal antibody to analyze the protein expression difference of AlEcR-A under the induction of ecdysone.

Detailed Description

The present invention will be further described with reference to the accompanying drawings.

The main reagents and consumables adopted in the invention are as follows: pCzn1 plasmid (purchased from Nanjing Belding Biotechnology Co., Ltd.), TOP10 strain (purchased from Nanjing Belding Biotechnology Co., Ltd.), Arcticepress strain (purchased from Nanjing Belding Biotechnology Co., Ltd.), restriction enzyme (purchased from TaKaRa), Pfu DNA polymerase (purchased from Zoonbio, product number PC12), Protein Marker (purchased from Thermo Co., Ltd.), Freund's adjuvant (purchased from Sigma Co., Ltd.), IPTG, Acr, Bis, Tris (purchased from Sigma Co., Ltd.), SDS (purchased from Amresco Co., Ltd.), TEMED (purchased from BIO-Rad Co., Ltd.), Tyrptone, Yeast Extract (purchased from OXEN Co., Ltd.), 0.22 μm sterile filter and dialysis bag (purchased from Millipore Co., Ltd.), Ni2+ IDA affinity chromatography gel (purchased from Novagen, Agarose (purchased from AGAROSE), DNA purification kit (purchased from YG), mini-YG affinity chromatography gel kit (purchased from YG Co., Ltd.),

High-glucose DMEM medium (purchased from Gibco), a cell counting plate (purchased from Improved Neubauer), liquid paraffin (Nanjing Belding Biotechnology Limited), PEG (purchased from sigmaa), a 96 cell culture plate (purchased from Costar, lot No. 3599), consumables such as a PCR tube and a gun head (purchased from Fisher), 6-8 week-old female BALB/c mice purchased from Yangzhou university college of veterinary medicine, 20E is a product of Sigma, SYBR Premix Ex Taq Kit is a TaKaRa product, iCycler iQ is a Bio-Rad product, and other reagents are domestic analytically pure or chemically pure;

allegra 21R desktop high speed refrigerated centrifuge (BECKMAN, USA), desktop high speed centrifuge (SORVAL, Germany), Biologic LP chromatography system, Mini Protean II vertical plate electrophoresis system, Gel Doc2000 imaging system, horizontal electrophoresis system (BIO-RAD, USA), PTC-200 gene amplification instrument (MJ Research, USA), 320-S pH meter (Mettler Toledo, USA), AR5120 electronic balance (AHOM, USA), multitempIII constant temperature water bath, Hofer M V-25 ultraviolet transmission instrument (Amersham Pharmacia, USA), snowflake ice machine (SAO, Japan), JY NY 92-2D ultrasonic cell crusher (NYNYO, Japan), ultra clean bench (ROSujing group, China), NANODP 2000 (Thermo).

In the initial experiment, the lygus lucorum was collected from Jiangsu Dafeng and Dongtai broad bean (Vicia faba L.) field in early spring and was subcultured indoors with kidney beans (Phaseolus vulgaris L.) under the conditions of temperature (25 +/-1) ° C, relative humidity 70 +/-5%, photoperiod 12L: 12D, and 10% honey water was supplemented in the adult stage.

EXAMPLE 1 preparation of anti-AlEcR-A protein monoclonal antibody

Firstly, construction of pCzn1-AlEcR-A recombinant expression plasmid

The gene AlEcR-A is connected into an expression vector pCzn1 through cloning sites Nde I and XbA I by A PAS (PCR-based Accurate Synthesis) method; the obtained recombinant plasmid pCzn1-AlEcR-A is transferred into A TOP10 clone strain; selecting positive clones for sequencing, comparing the sequencing result with an expected sequence, and intercepting part of the compared sequence as shown in figure 1; the sequencing result shows that the T4DNA is successfully connected with the expression vector pCzn1 subjected to double enzyme digestion and A T vector containing an AlEcR-A sequence, which indicates that the AlEcR-A is subcloned onto the pCzn1 vector and is named as pCzn1-AlEcR-A (figure 2); meanwhile, plasmid restriction identification (AlEcR-AD260/OD280:1.84) is carried out, and the restriction system is as follows:

the results of enzyme cleavage identification are shown in FIG. 2. The recombinant plasmid pCzn1-AlEcR-A with correct sequencing is transformed into an EscherichiA coli Arctic express strain: adding 1. mu.l of recombinant plasmid pCzn1-AlEcR-A into 100. mu.l of competent cells, and placing on ice for 20 min; thermally shocking at 42 deg.C for 90sec, and rapidly placing in ice for 5 min; adding 600 mul LB culture liquid; shaking at 37 deg.C and 220rpm for 1h, centrifuging, spreading on LB plate containing 50 μ g/ml Amp, and performing inverted culture at 37 deg.C overnight to obtain positive clone, i.e. recombinant genetically engineered bacteria.

The sequencing results are spliced as follows: the single-underlined region is the AlEcR-A gene region. The gray area is a restriction enzyme site;

TGCATCACCGCATGCGTGCTATGCACATCCAATTGTGGAGCGGATACATTGATGTGCTAGCGCATATCCAGTGTAGTAGGCAGTCCTCAGAGTTATCGTTGATACCCCTCGTAGTGCACATTCCTTTAACGCTTCAAAATCTGTAAAGCACGCCATATCGCCGAAAGGCACACTTAATTATTAAGAGGTAATACACC

ATGGGCGAGGAGAAGCGGGCAGACGATGACTGGATGGTGAGCGGTGGCCCCCCCAGAAACTATCAGACCAACGGGG GCTATCCGTCCCCAACTATGTCCAGCAACAGCTACGACCCTTACAGCCCCAACTCTAAACTAGGTCGTGAGGATTT GAGCCCGCCCAACTCGCTAAACGGCTACAGCGCCGATAGCTGTGATGGCTCGAAGAAGAAGAAGGGGACGGCCACC CGGCAGCAAGAAGAGCTCTGCCTCGTTTGCGGAGATCGAGCTTCGGGTTACCATTATAATGCTCTCACGTGCGAGG GCTGTAAGGGGTTCTTTAGACGGAGTATAACGAAAAACGCGGTCTACCAGTGCAAGTACGGGAACAACTGTGAAAT CGACATGTACATGCGGCGGAAATGTCAGGAATGTCGGCTCAAAAAGTGCCTCAGCGTCGGGATGAGGCCTGAATGT GTGGTACCGGAGTACCAATGCGCGGTGAAACGAAAAGAGAAGAAGGCGCAGAAGGAGAAGGACAAGCCTGTGAGCA CGACAACCATCCCCCCTGAGGCAGTCAAACCTGAGCCTGAACCCCACAGGGTCAGTTTCACATCAAGCCTCTTTCA ATCTGTGATTAAAGAGCCAACCAAACTGTTGCCGGAAAGCGGTGCTGAAGTTGCGCTCAAACATTCGCCCCCCTAC GGCATAAAACCGGTGAGCCCTGAGCAGCAGGAACTCATCAACAGGCTCGTCTACTTTCAAAGCGAGTACGAACACC CCTCGGAGGAAGATGTCCGGAGAATTAACTCACCTAACGAAGATGAGGAGCAGATAGACTTTAGGTTTAGGCATAT AACAGAAATCACAATACTCACCGTTCAACTTATCGTAGAGTTCGCGAAGAGGCTGCCGGGTTTCGATAAAATTATT AAAGAAGATCAAATAGCCTTACTAAAGGCGTGTTCAAGCGAAGTGATGATGCTGCGGACAGCGCGGAAGTACGACG CGAGTACGGACTCCATAGTGTTCGCGGACAACCAGCTGTACTCTCGAGAGTCGTACAACCTTGCCGGAATGGGGGA CGTGGTCGATGACATCCTCAAGTTCTGTCGGCACATGTACCGAATGAAGGTTGACAACGCCGAGTACGCCCTCCTT ACCGCCATCGTTATCTTTTCGGAGAGACCGTCCCTCATGGAGAGTTGGAAAGTCGAAAAGATCCAGGAGACCTACC TAGAAGCCCTCAAGTCCTACGTGGACAACCGAACGAAGTCTCGGTCTCCCACACTCTACGCTAAACTTCTTTCTGT CCTCACGGAGCTTCGAACCCTCGGAAACCAAAACTCCGAAATGTGCTTCTCCCTGAAACTTCAGAACAAGAAGCTA CCGCCCTTCCTAGCCGAGATCTGGGACGTCAACTCGTAA

TAGGTAATCTCTGCTTAAAAGCACAGAATCTAAGATCCCTGCCATTTGGCGGGGATTTTTTTATTTGTTTTCAGGAAATAAATAATCGATCGCGTAATAAAATCTATTATTATTTTTGTGAAGAATAAATTTGGGTGCAATGAGAATGCGCAGGCCGTTA

second, expression and purification of pCzn1-AlEcR-A recombinant protein

1. IPTG induced expression of pCzn1-AlEcR-A carrier fusion protein

1) The positive monoclonal on the transformation plate was selected and inoculated into a tube containing 50. mu.g/ml ampicillin (Amp) in 3ml of LB medium, and shaken overnight at 37 ℃ and 220 rpm;

2) the following day is as follows: 100 in 50 u g/ml Amp 30ml LB culture solution, 37 degrees C220 rpm shake until bacterial OD600 is 0.6-0.8 (about 2 h);

3) taking out 1ml of culture, centrifuging at 10000g of room temperature for 2min, discarding supernatant, and resuspending the thallus precipitate with 100 μ l of 1 Xloading buffer;

4) adding IPTG into the rest culture in the step 3) until the final concentration is 0.5mM, shaking at 37 ℃ and 220rpm for 4h, and inducing the expression of AlEcR-A His fusion protein;

5) 1ml of the culture obtained after the induction in step 4) was taken out, centrifuged at 12000g at room temperature for 2min, the supernatant was discarded, and the pellet was resuspended in 100. mu.l of 1 Xloading buffer.

6) The 12% SDS-PAGE analysis showed that the target protein was mainly present in the precipitate, and the results are shown in FIG. 3. SDS-PAGE shows that the concentration of the sodium dodecyl sulfate is 0.5 mmol.L^-1The IPTG-induced pCzn1-AlEcR-A recombinant plasmid can specifically Express A protein of about 55kD, and the theoretical size is consistent with that, which shows that the AlEcR-A gene is successfully expressed in EscherichiA coli Arctic Express cells, but the IPTG-induced pCzn1-AlEcR-A recombinant plasmid does not have the protein expression of the band. In addition, the recombinant AlEcR-A protein induced to be expressed is mainly distributed in the sediment, and is hardly expressed in the supernatant, which indicates that the AlEcR-A recombinant protein mainly exists in the form of inclusion bodies.

2. Ni-column affinity purification of fusion proteins

1) Centrifuging the culture bacteria liquid subjected to induced expression at low temperature of 6000g for 10min, and carrying out precipitation, heavy suspension and ultrasonic crushing on thalli in a lysine buffer; centrifuging the ultrasonically-broken cell lysate at 4 ℃ and 10000g for 20min, and collecting the precipitate; inclusion bodies were washed 3 times with inclusion body wash (20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1% Triton X-100, pH 8.0); the inclusion bodies were dissolved in a dissolution Buffer (20mM Tris, 5mM DTT, 8M urea, pH8.0) in proportion, the above solution was added dropwise to 20mM Tris-HCL and 5mM EDTA Buffer (pH 7.8) Buffer, gradually diluted in stepwise gradients with slow stirring, and the protein solution was put into dialysis bags and dialyzed overnight in PBS (pH7.4) solution.

2) Loading the inclusion body solution to a Ni-IDA-Sepharose CL-6B affinity chromatography column pre-balanced by Ni-IDABinding-Buffer at the flow rate of 0.5ml/min by using a low-pressure chromatography system;

3) flushing with Ni-IDA Binding-Buffer at a flow rate of 0.5ml/min until the effluent OD280 value reaches the baseline;

4) then flushing with Ni-IDA Washing-Buffer (20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1ml/min until the effluent OD280 value reaches the baseline;

5) finally, eluting the target protein by using Ni-IDA Elution-Buffer (20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0) at the flow rate of 1ml/min, and collecting the effluent;

6) adding the collected protein solution into a dialysis bag, dialyzing overnight by using PBS (PH7.4), and determining the concentration of the protein to be 1.3mg/mL by using a protein quantitative kit;

7) an appropriate amount of the dialysate was subjected to 12% SDS-PAGE analysis, and the results are shown in FIG. 4. The purification of the pCzn1-AlEcR-A gene recombinant protein after affinity purification by A Ni column was analyzed by SDS-PAGE electrophoresis. After the elution and dialysis of the buffer solution, the purity of the inclusion body containing the pCzn1-AlEcR-A gene recombinant protein is improved to A greater extent, and only an obvious specific band is present near 55kD, and no other band is seen, which indicates that the purified AlEcR-A recombinant protein is obtained. The theoretical molecular weight of the protein is: about 54.95KD (containing HIS-tag), the translated amino acid sequence is as follows:

MNHKVHHHHHHMMGEEKRADDDWMVSGGPPRNYQTNGGYPSPTMSSNSYDPYSPNSKLGREDLSPPNSL NGYSADSCDGSKKKKGTATRQQEELCLVCGDRASGYHYNALTCEGCKGFFRRSITKNAVYQCKYGNNCEIDMYMRRK CQECRLKKCLSVGMRPECVVPEYQCAVKRKEKKAQKEKDKPVSTTTIPPEAVKPEPEPHRVSFTSSLFQSVIKEPTK LLPESGAEVALKHSPPYGIKPVSPEQQELINRLVYFQSEYEHPSEEDVRRINSPNEDEEQIDFRFRHITEITILTVQ LIVEFAKRLPGFDKIIKEDQIALLKACSSEVMMLRTARKYDASTDSIVFADNQLYSRESYNLAGMGDVVDDILKFCR HMYRMKVDNAEYALLTAIVIFSERPSLMESWKVEKIQETYLEALKSYVDNRTKSRSPTLYAKLLSVLTELRTLGNQN SEMCFSLKLQNKKLPPFLAEIWDVNS

preparation of monoclonal antibody

1. Animal immunization

Selecting 5 female BALB/c mice with the age of 6-8 weeks, mixing the purified AlEcR-A protein with Freund's adjuvant in the same volume, injecting 50-100 mu g/mouse subcutaneously in the abdomen and back, emulsifying the recombinant protein AlEcR-A with Freund's incomplete adjuvant every 14 days for 2 times, and boosting the immunity once every 2-3 weeks (dose is doubled). And (3) blood sampling detection after the four-immunization, determining the titer of antiserum against AlEcR-A protein by an indirect ELISA method, performing western blotting detection (screening result is shown in figure 5) by using the antiserum after the four-immunization when the titer is more than 1:10,000 (4-immunization result is shown in table 1), and selecting 1-2 positive mice for cell fusion arrangement when the recombinant protein is positive.

Results of ELISA for serum titers in mice after 4 immunizations: after immunization, the mice all generate high titer antibodies, and the specific results are as follows:

coating antigen: protein AlEcR-A

Coating concentration: 5. mu.g/ml, 100. mu.l/well

Coating buffer solution: phosphate buffer (PBS, pH7.4)

Secondary antibody: goat anti-mouse-HRP, 1/5000

TABLE 1 Indirect Elisa results of mouse antiserum after 4 immunizations

Initial dilution 1:500

The titer, i.e., sample OD/blank OD > -2.1 highest dilution.

2. Cell fusion

1) Myeloma cell preparation

One week prior to fusion, SP2/0 cells were expanded in DMEM medium containing 10% FBS. At the time of confluency, the cells grew out of approximately 6T 25 cell culture flasks, and SP2/0 cells were harvested into 50ml centrifuge tubes at the day of confluency and centrifuged at 1000rpm for 5 min. The supernatant was discarded, and then 20ml of DMEM basal medium was added, and the cells were blown off and counted.

2) Spleen cell preparation

Serum ELISA titers after four immunizations were 1: mice above 10000 were immunized 3 days before fusion and 100 μ g of antigen was intraperitoneally injected. Mice to be fused were euthanized by cervical dislocation on the day of fusion. Soaking in 75% ethanol for 5min, aseptically taking out spleen, and placing spleen into culture dish containing 10ml DMEM basic culture. The spleen was removed from the screen and placed in another dish, transferred to the screen, and ground using a syringe. DMEM was added to the screen and the screen was washed to collect more splenocytes into the dish. The cells were transferred to a 10ml centrifuge tube, and the spleen cells were washed twice with serum-free DMEM, centrifuged at 1000rpm for 5min, and the spleen cells were collected and counted.

3) Cell fusion

And mixing myeloma cells and spleen cells, so that the number ratio of the myeloma cells to the spleen cells is 1: preferably 20. The cells were placed in 50ml centrifuge tubes, diluted with DMEM basal medium, and then centrifuged at 1000rpm for 5 min. The supernatant was discarded. The tubes were shaken to homogenize the cells. 0.8ml of 50% PEG was slowly added for 90 seconds, and then 20-30ml of DMEM medium was added to stop the PEG, and the fused cells were placed in a water bath at 37 ℃ for 10 minutes. 1000rpm for 5min, the supernatant was discarded and HAT DMEM medium (purchased from Wuhan Pronace technologies, Ltd.) was added to the supernatant, and the fused cells were plated in 96-well plates at 100. mu.l per well. The cell culture plate was then placed in CO₂Culturing in an incubator. The cloning rate of the hybridoma cells is over 50 percent, a small amount of cell fragments exist, and the cell growth state is good when the hybridoma cells are checked 4 days after fusion. The screening assay was started 10 days after fusion.

3. Fusion screening and subcloning

1) Fusion screening

The day before the assay, 5. mu.g/ml antigen was coated with PBS on ELISA plates overnight. And (3) sucking 100 mu l of cell supernatant per well on the next day, performing ELISA detection, judging positive wells according to an ELISA result (the positive wells are judged if the OD value of the sample wells/the OD value of the negative wells are more than or equal to 2.1), picking the positive wells detected on the whole plate by using a single-channel pipettor, performing secondary confirmation detection, further confirming the positive wells, and performing subcloning on the determined positive well cells.

And fusion screening results: 2# mice with higher titer and positive WB detection are selected for cell fusion, and the results of screening positive clones by ELISA are shown in the following table 2:

coating antigen: protein AlEcR-A

Coating concentration: 5. mu.g/ml, 100. mu.l/well

Coating buffer solution: phosphate buffer (PBS, pH7.4)

Secondary antibody: goat anti-mouse-HRP, 1/5000

Table 2: elisa results of cell supernatants after 2# mice fusion

① size board

② size board

③ size board

④ size board

⑤ size board

2) Subcloning

And (3) blowing cells in the positive holes, counting, adding N/4ml (N refers to the number of the positive cloning holes) of DMEM culture medium into the centrifuge tube, taking 100 mu l of cell suspension into the centrifuge tube, uniformly blowing, then reserving 1ml, supplementing DMEM to 4ml, uniformly blowing, and reserving 100 mu l (about 2 drops) of DMEM at the bottom of the tube. Adding DMEM into a centrifuge tube to 5ml, dropwise adding the DMEM into the first three rows of a 96-well plate after uniformly mixing, keeping 1.8-2ml of DMEM at the bottom of a drop tube of each hole, replenishing DMEM into 5ml, dropwise adding the DMEM into D, E, F three rows of the 96-well plate after uniformly blowing, keeping 1.5-1.8ml of DMEM at the bottom of the tube, replenishing DMEM into 2.8-3ml of DMEM, dropwise adding the DMEM into G, H rows of the 96-well plate after uniformly blowing, keeping a drop in each hole, observing under a microscope after 7-10 days, detecting a hole with clone growth, marking a hole with a monoclonal cell, picking a monoclonal cell which is positive as much as possible to perform subcloning again, and picking out the hole with the monoclonal cell to perform expanded culture for a fixed strain after detecting that the.

Results after subcloning of cell lines: 8 positive cells (with high OD values in Table 2, indicated by grey areas) were selected for subcloning, and 11 positive cells were screened by ELISA and the results are shown in Table 3 below:

coating antigen: protein AlEcR-A

Coating concentration: 5. mu.g/ml, 100. mu.l/well

Coating buffer solution: phosphate buffer (PBS, pH7.4)

Secondary antibody: goat anti-mouse-HRP, 1/5000

Table 3: elisa results of cell supernatants after strain determination:

cell line name	OD value (450nm)
		3D6	2.710
3G5	2.388
		5D9	2.333
7D9	2.648
		7G3	2.816
7G4	3.009
		8B8	2.884
8D11	3.243
		8H7	3.122
8H9	3.118
		8G12	3.043

After A mouse is immunized by using A recombinant protein pCzn1-AlEcR-A expressed by escherichiA coli, after 10d of cell fusion, the positive cloning rate of ELISA detection after antigen coating reaches 100%, and the OD450nm value of hybridomA cell supernatants collected at different periods is almost unchanged. Obtaining 1 monoclonal antibody hybridomA cell strain which can stably secrete anti-AlEcR-A protein and is named as 8H7, wherein the preservation number of the cell strain is CCTCC NO: c2016206, which was deposited in China Center for Type Culture Collection (CCTCC) at 12 months and 1 day of 2016.

The cell line was amplified and cultured to obtain a fixed strain, and the specificity of the antibody was determined.

4. Ascites preparation, agarose affinity medium Protein A purified antibody and antibody identification

1) Preparation of ascites

From table 3 above, this OD was not the highest, 8D11, 3.243, but 8D11 cells of this monoclonal cell did not fuse well, so 8H7 was selected. Finally 8H7 monoclonal cells are selected for ascites preparation, 0.2mL Freund's incomplete adjuvant is injected into the abdominal cavity of the mouse, and a proper amount of the positive hybridoma cells are injected after 7 days, and then ascites is collected.

2) ProteinA purified antibody

The ascites fluid obtained from 8H7 was purified using agarose affinity medium Protein A. Taking Protein A agarose affinity medium, filling a chromatographic column, mixing ascites and PBS uniformly according to a ratio of 1:1, slowly loading the mixture, eluting the mixture by using glycine elution buffer solution after the antibody is combined to obtain the required purified antibody, immediately dialyzing the mixture in the PBS at 4 ℃ overnight, and detecting the purity, the concentration and the titer every other day; wherein, the concentration of the antibody is: 0.5 mg/ml; antibody volume: 10.0 ml. And (3) identifying the purity of the antibody: after purification the antibody was subjected to SDS-PAGE and stained with Coomassie Brilliant blue, see FIG. 6.

3) Identification of antibody

The antiserum indirect ELISA titer detection method specifically comprises the following steps: 1) designing a coating plate according to the experimental requirement, and marking the lath; 2) coating: diluting AlEcR-A protein with PBS coating solution according to 5 μ g/ml, mixing well, adding into lath, each hole 100 μ l, and refrigerating overnight at 4 deg.C; wherein, the coating antigen: an AlEcR-A protein; coating concentration: diluting according to 5 mu g/ml, 100 mu l/well; coating buffer solution: phosphate buffered saline (PBS, pH 7.4); 3) and (3) sealing: after coating, the coating solution was discarded, the plate was washed 3 times, 200. mu.l of blocking solution was added to each well, and the plate was incubated at 37 ℃ for 1 hour. Taking out the enzyme label plate, discarding the internal liquid, and washing the plate for 1 time; 4) primary anti-reaction: diluting the purified antibody by 1/500, 2 times, wherein each well is 100 μ l, and keeping the temperature in a thermostat at 37 ℃ for 1 h; 5) secondary antibody reaction: taking out the enzyme label plate, discarding the internal liquid, washing the plate for 3 times, adding 100 mul of diluted enzyme-labeled secondary antibody and enzyme-labeled secondary antibody into each hole: goat anti-mouse-HRP, 1/5000. A thermostat at 37 ℃ for 1 hour; 6) color development: taking out the enzyme label plate, discarding the inner liquid, washing the plate for 4 times, adding 100 μ l of TMB color development solution into each hole, determining the color development time according to the color depth, generally 37 deg.C, 15 min; 7) termination the reaction was terminated by adding 100. mu.l of 1M HCl solution to each well. The titer of the sample was determined by reading immediately on the microplate reader at 450nm and the dilution corresponding to wells having an OD greater than 2.1 times the OD of the negative control set (see Table 4).

TABLE 4 Indirect Elisa results of antibodies

Initial dilution 1:500

Titer, i.e.the highest dilution at which sample OD/blank OD > is 2.1

4) Western-blot detection of specificity of monoclonal antibody

The Western-blot assay was performed according to the method of Song et al (2012) with minor routine modifications. And carrying out SDS-PAGE analysis on the prokaryotic expression AlEcR-A recombinant protein and the freshly hatched 3-year nymph of lygus lucorum. And sealing after transferring A PVDF membrane, wherein the primary antibody is the AlEcR-A recombinant protein monoclonal antibody, the secondary antibody is goat anti-mouse HRP marked by horseradish peroxidase, and ECL (electron cyclotron resonance) color development is performed.

Referring to fig. 5, the prepared AlEcR-A recombinant protein monoclonal antibody can be specifically combined with the total green plant bug protein and the recombinant protein, and has no cross reaction with other proteins.

Application example

The prepared AlEcR-A recombinant protein monoclonal antibody is used for analyzing the influence of ecdysone (20E) on the expression level of AlEcR-A protein. With the extension of the 20E treatment time, the expression level of the AlEcR-A protein of the lygus lucorum shows A gradually rising trend. In addition, compared with the distilled water treatment group (CK), the 20E-treated lygus lucorum AlEcR-A protein expression level has certain enrichment effect. See fig. 6.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

SEQUENCE LISTING

<110> agricultural science and academy of Jiangsu province

<120> monoclonal antibody of ecdysone receptor protein of lygus lucorum and application thereof

<130>SG20161214001

<160>2

<170>PatentIn version 3.3

<210>1

<211>1407

<212>DNA

<213> AlEcR-A Gene

<220>

<221>CDS

<222>(1)..(1407)

<400>1

atg ggc gag gag aag cgg gca gac gat gac tgg atg gtg agc ggt ggc 48

Met Gly Glu Glu Lys Arg Ala Asp Asp Asp Trp Met Val Ser Gly Gly

1 5 10 15

ccc ccc aga aac tat cag acc aac ggg ggc tat ccg tcc cca act atg 96

Pro Pro Arg Asn Tyr Gln Thr Asn Gly Gly Tyr Pro Ser Pro Thr Met

20 25 30

tcc agc aac agc tac gac cct tac agc ccc aac tct aaa cta ggt cgt 144

Ser Ser Asn Ser Tyr Asp Pro Tyr Ser Pro Asn Ser Lys Leu Gly Arg

35 40 45

gag gat ttg agc ccg ccc aac tcg cta aac ggc tac agc gcc gat agc 192

Glu Asp Leu Ser Pro Pro Asn Ser Leu Asn Gly Tyr Ser Ala Asp Ser

50 5560

tgt gat ggc tcg aag aag aag aag ggg acg gcc acc cgg cag caa gaa 240

Cys Asp Gly Ser Lys Lys Lys Lys Gly Thr Ala Thr Arg Gln Gln Glu

65 70 75 80

gag ctc tgc ctc gtt tgc gga gat cga gct tcg ggt tac cat tat aat 288

Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr Asn

85 90 95

gct ctc acg tgc gag ggc tgt aag ggg ttc ttt aga cgg agt ata acg 336

Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Ile Thr

100 105 110

aaa aac gcg gtc tac cag tgc aag tac ggg aac aac tgt gaa atc gac 384

Lys Asn Ala Val Tyr Gln Cys Lys Tyr Gly Asn Asn Cys Glu Ile Asp

115 120 125

atg tac atg cgg cgg aaa tgt cag gaa tgt cgg ctc aaa aag tgc ctc 432

Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu

130 135 140

agc gtc ggg atg agg cct gaa tgt gtg gta ccg gag tac caa tgc gcg 480

Ser Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Tyr Gln Cys Ala

145 150 155 160

gtg aaa cga aaa gag aag aag gcg cag aag gag aag gac aag cct gtg 528

Val Lys Arg Lys Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Pro Val

165 170 175

agc acg aca acc atc ccc cct gag gca gtc aaa cct gag cct gaa ccc 576

Ser Thr Thr Thr Ile Pro Pro Glu Ala Val Lys Pro Glu Pro Glu Pro

180 185 190

cac agg gtc agt ttc aca tca agc ctc ttt caa tct gtg att aaa gag 624

His Arg Val Ser Phe Thr Ser Ser Leu Phe Gln Ser Val Ile Lys Glu

195 200 205

cca acc aaa ctg ttg ccg gaa agc ggt gct gaa gtt gcg ctc aaa cat 672

Pro Thr Lys Leu Leu Pro Glu Ser Gly Ala Glu Val Ala Leu Lys His

210 215 220

tcg ccc ccc tac ggc ata aaa ccg gtg agc cct gag cag cag gaa ctc 720

Ser Pro Pro Tyr Gly Ile Lys Pro Val Ser Pro Glu Gln Gln Glu Leu

225 230 235 240

atc aac agg ctc gtc tac ttt caa agc gag tac gaa cac ccc tcg gag 768

Ile Asn Arg Leu Val Tyr Phe Gln Ser Glu Tyr Glu His Pro Ser Glu

245 250 255

gaa gat gtc cgg aga att aac tca cct aac gaa gat gag gag cag ata 816

Glu Asp Val Arg Arg Ile Asn Ser Pro Asn Glu Asp Glu Glu Gln Ile

260 265 270

gac ttt agg ttt agg cat ata aca gaa atc aca ata ctc acc gtt caa 864

Asp Phe Arg Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr Val Gln

275 280 285

ctt atc gta gag ttc gcg aag agg ctg ccg ggt ttc gat aaa att att 912

Leu Ile Val Glu Phe Ala Lys Arg Leu Pro Gly Phe Asp Lys Ile Ile

290 295 300

aaa gaa gat caa ata gcc tta cta aag gcg tgt tca agc gaa gtg atg 960

Lys Glu Asp Gln Ile Ala Leu Leu Lys Ala Cys Ser Ser Glu Val Met

305 310 315 320

atg ctg cgg aca gcg cgg aag tac gac gcg agt acg gac tcc ata gtg 1008

Met Leu Arg Thr Ala Arg Lys Tyr Asp Ala Ser Thr Asp Ser Ile Val

325 330 335

ttc gcg gac aac cag ctg tac tct cga gag tcg tac aac ctt gcc gga 1056

Phe Ala Asp Asn Gln Leu Tyr Ser Arg Glu Ser Tyr Asn Leu Ala Gly

340 345 350

atg ggg gac gtg gtc gat gac atc ctc aag ttc tgt cgg cac atg tac 1104

Met Gly Asp Val Val Asp Asp Ile Leu Lys Phe Cys Arg His Met Tyr

355 360 365

cga atg aag gtt gac aac gcc gag tac gcc ctc ctt acc gcc atc gtt 1152

Arg Met Lys Val Asp Asn Ala Glu Tyr Ala Leu Leu Thr Ala Ile Val

370 375 380

atc ttt tcg gag aga ccg tcc ctc atg gag agt tgg aaa gtc gaa aag 1200

Ile Phe Ser Glu Arg Pro Ser Leu Met Glu Ser Trp Lys Val Glu Lys

385 390 395 400

atc cag gag acc tac cta gaa gcc ctc aag tcc tac gtg gac aac cga 1248

Ile Gln Glu Thr Tyr Leu Glu Ala Leu Lys Ser Tyr Val Asp Asn Arg

405 410 415

acg aag tct cgg tct ccc aca ctc tac gct aaa ctt ctt tct gtc ctc 1296

Thr Lys Ser Arg Ser Pro Thr Leu Tyr Ala Lys Leu Leu Ser Val Leu

420 425 430

acg gag ctt cga acc ctc gga aac caa aac tcc gaa atg tgc ttc tcc 1344

Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn Ser Glu Met Cys Phe Ser

435 440 445

ctg aaa ctt cag aac aag aag cta ccg ccc ttc cta gcc gag atc tgg 1392

Leu Lys Leu Gln Asn Lys Lys Leu Pro Pro Phe Leu Ala Glu Ile Trp

450 455 460

gac gtc aac tcg taa 1407

Asp Val Asn Ser

465

<210>2

<211>468

<212>PRT

<213> AlEcR-A protein

<400>2

Met Gly Glu Glu Lys Arg Ala Asp Asp Asp Trp Met Val Ser Gly Gly

1 5 10 15

Pro Pro Arg Asn Tyr Gln Thr Asn Gly Gly Tyr Pro Ser Pro Thr Met

20 25 30

Ser Ser Asn Ser Tyr Asp Pro Tyr Ser Pro Asn Ser Lys Leu Gly Arg

35 40 45

Glu Asp Leu Ser Pro Pro Asn Ser Leu Asn Gly Tyr Ser Ala Asp Ser

50 55 60

Cys Asp Gly Ser Lys Lys Lys Lys Gly Thr Ala Thr Arg Gln Gln Glu

65 70 75 80

Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr Asn

85 90 95

Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Ile Thr

100 105 110

Lys Asn Ala Val Tyr Gln Cys Lys Tyr Gly Asn Asn Cys Glu Ile Asp

115 120 125

Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu

130 135 140

Ser Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Tyr Gln Cys Ala

145 150 155 160

Val Lys Arg Lys Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Pro Val

165 170 175

Ser Thr Thr Thr Ile Pro Pro Glu Ala Val Lys Pro Glu Pro Glu Pro

180 185 190

His Arg Val Ser Phe Thr Ser Ser Leu Phe Gln Ser Val Ile Lys Glu

195 200 205

Pro Thr Lys Leu Leu Pro Glu Ser Gly Ala Glu Val Ala Leu Lys His

210 215 220

Ser Pro Pro Tyr Gly Ile Lys Pro Val Ser Pro Glu Gln Gln Glu Leu

225 230 235 240

Ile Asn Arg Leu Val Tyr Phe Gln Ser Glu Tyr Glu His Pro Ser Glu

245 250 255

Glu Asp Val Arg Arg Ile Asn Ser Pro Asn Glu Asp Glu Glu Gln Ile

260 265 270

Asp Phe Arg Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr Val Gln

275 280 285

Leu Ile Val Glu Phe Ala Lys Arg Leu Pro Gly Phe Asp Lys Ile Ile

290 295 300

Lys GluAsp Gln Ile Ala Leu Leu Lys Ala Cys Ser Ser Glu Val Met

305 310 315 320

Met Leu Arg Thr Ala Arg Lys Tyr Asp Ala Ser Thr Asp Ser Ile Val

325 330 335

Phe Ala Asp Asn Gln Leu Tyr Ser Arg Glu Ser Tyr Asn Leu Ala Gly

340 345 350

Met Gly Asp Val Val Asp Asp Ile Leu Lys Phe Cys Arg His Met Tyr

355 360 365

Arg Met Lys Val Asp Asn Ala Glu Tyr Ala Leu Leu Thr Ala Ile Val

370 375 380

Ile Phe Ser Glu Arg Pro Ser Leu Met Glu Ser Trp Lys Val Glu Lys

385 390 395 400

Ile Gln Glu Thr Tyr Leu Glu Ala Leu Lys Ser Tyr Val Asp Asn Arg

405 410 415

Thr Lys Ser Arg Ser Pro Thr Leu Tyr Ala Lys Leu Leu Ser Val Leu

420 425 430

Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn Ser Glu Met Cys Phe Ser

435 440 445

Leu Lys Leu Gln Asn Lys Lys Leu Pro Pro Phe Leu Ala Glu Ile Trp

450 455 460

Asp Val Asn Ser

465

Claims (6)

1. The monoclonal antibody specifically binding to lygus lucorum AlEcR-A protein is characterized by comprising A monoclonal antibody with A preservation number of CCTCC NO: c2016206.

2. A mouse hybridoma cell strain with a preservation number of CCTCC NO: C2016206.

3. use of the monoclonal antibody of claim 1 for the preparation of A detection tool for the detection of lygus lucorum AlEcR- A protein.

4. The use of claim 3, wherein the detection means is a kit, chip or strip.

5. A test kit comprising the monoclonal antibody of claim 1.

6. The detection kit according to claim 5, wherein the detection kit is an immunohistochemical detection kit or an immunofluorescence detection kit.

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