Patent Application
20230160883
This application claims priority to and the benefit of Chinese Application No. 202111399298.4 filed Nov. 19, 2021. The entire disclosure of Chinese Application No. 202111399298.4 is incorporated herein by reference.
The present invention relates to the technical field of plant virus detection, in particular to a Tomato zonate spot virus (TZSV) detection reagent.
Tomato zonate spot virus (TZSV) belongs to Orthotospovirus genus, it was first discovered in China in 2005 and is mainly distributed in Yunnan and Guangxi provinces and other regions. It affects tobacco, pepper, tomato and other crops. The virus can cause spotted wilt disease, and even fail to harvest in serious cases, resulting in significant economic losses to agricultural production. Jin Yan Tian et al. prepared a monoclonal antibody to TZSV N protein and a test strip. However, there is still no commercial test strip for TZSV in market.
Purpose of the present invention is to provide a colloidal gold detection reagent for tomato zonate spot virus (TZSV).
In order to achieve the purpose of the invention, Optimized gene encoding the N protein of TZSV virus (SEQ ID NO:2) was constructed into Escherichia coli expression system, and N protein of TZSV virus was obtained by expression and purification; then, 6-week-old female BALB/c mice were immunized with the TZSV N protein, and splenocytes of the immunized mice were fused with myeloma cells SP2/0.
Two specific hybridoma cell lines were obtained by screening, whose depository numbers are CCTCC No. C202160 and CCTCC No. C202196, respectively.
Specific anti-TZSV monoclonal antibody could be obtained from above two hybridoma cell lines.
Anti-TZSV monoclonal antibody could be obtained as follows: resuscitating hybridoma cells, preparing ascites, and purifying ascites to obtain specific monoclonal antibody.
The monoclonal antibody obtained by the invention can be used to prepare tomato zonate spot virus detection reagent.
In an example of the invention, a tomato zonate spot virus (TZSV) colloidal gold test strip (TZSV hand-held colloidal gold rapid test strip) is provided. the test strip consists of a sample pad, a gold labeled pad, a nitrocellulose membrane, an absorbent pad and a backplane; sample pad, gold labeled pad, nitrocellulose membrane and absorbent pad (absorption pad) are successively superimposed and pasted on the backplane, and each part is superimposed between each other by 2-3 mm; the gold labeled pad was coated with colloidal gold labeled anti-TZSV monoclonal antibody; nitrocellulose membrane is provided with a test line and a quality control line (T line and C line), the test line coated with anti-TZSV monoclonal antibody, the quality control line coated with anti-mouse IgG secondary antibody; Wherein, the anti-TZSV monoclonal antibody (murine anti-TZSV monoclonal antibody) is generated by hybridoma cell lines with depository numbers of CCTCC No. C202160 and CCTCC No. C202196.
The hybridoma cell lines of anti-TZSV monoclonal antibody were obtained as follows: gene encoding N protein of TZSV virus (SEQ ID NO:2) was optimized and constructed into Escherichia coli expression system, and the recombinant TZSV virus was obtained by expression and purification.; the 6-week-old female BALB/c mice were immunized with the recombinant TZSV virus, and then splenocytes of the immunized mice were fused with myeloma cells SP2/0 to obtain specific monoclonal antibody cell lines.
Preferably, the sample pad is a glass cellulose film.
Preferably, concentration of colloidal gold labeled anti-TZSV monoclonal antibody coated on the gold labeled pad is 0.3-0.5 mg/ml.
Preferably, concentration of anti-TZSV monoclonal antibody coated on the test line is 0.5-1.5 mg/ml.
Preferably, concentration of anti-mouse IgG secondary antibody coated on the quality control line is 1 mg/ml.
Preferably, size of the sample pad is 3 mm × 15 mm, size of the gold labeled pad is 3 mm × 3 mm, size of the nitrocellulose membrane is 3 mm × 28 mm, and size of the absorbent pad is 3 mm × 19 mm.
Preferably, space between the test line and the quality control line is 6-6.5 mm.
Preferably, the backplane (bottom plate) is a PVC board.
Structure diagram of the TZSV virus hand-held colloidal gold rapid test strip of the invention is shown in
Secondly, the invention provides use of the test strip in detecting TZSV virus.
Specific detection procedure and principle of the TZSV handheld colloidal gold rapid test strip of the invention are as follows:
first, put the rapid test strip vertically into test sample, not exceeding position of arrow (
Therefore, judgment standard of results of the TZSV handheld colloidal gold rapid test strip of the present invention is:
The TZSV virus hand-held colloidal gold rapid test strip of the invention has good anti-interference effect on leaf and other samples, and can be widely used in rapid detection of TZSV, with the following unique advantages:
The following examples are used to illustrate the invention without limiting scope of the invention. Unless otherwise specified, technical means used in the examples are conventional means well known to a person skilled in the art, and the raw materials used are commercially available goods.
1. Sequence synthesis: According to protein sequence, optimized sequence was synthesized and cloned into vector pET30a, and the sequence was synthesized by Sangon Bioengineering (Shanghai) Co., LTD. Amino acid sequence of tomato zonate spot virus protein was shown as SEQ ID NO: 1. Nucleic acid sequence of the optimized tomato zonate spot virus gene is shown as SEQ ID NO:2.
2. Strain activation: PET30a-TZSV plasmid was transformed into BL21 (DE3) and coated on LB solid medium (Kanamycin concentration of 50 µg/mL). Next day, monoclonal colonies were selected and added into 5 mL LB liquid medium (Kanamycin concentration 50 µg/mL) and cultured at 37° C. for 12 h-14 h. Original sequence of protein was SEQ ID NO: 1.
3. Small test expression: next day, strains were added into 5 mL LB liquid medium at 1:50 (Kanamycin concentration of 50 µg/mL) and cultured at 37° C. until OD=0.4-0.6. After centrifugation, 1 mL of bacterial solution was absorbed and centrifuged for using as control before induction. 4 mL of bacterial solution was added with 0.8 mM IPTG, induced and expressed at 25° C. for 6 h, after that, the bacterial solution was centrifuged at 8000 RPM and 4° C. for 1 min and collected.
4. Identification of protein expression form: above expressed bacteria were added to 1 mL crushing solution for ultrasonic lysis. Lysis conditions: temperature ice bath, power 40%, ultrasonic 2 s, 2 s interval, time 30 min. Supernatant and precipitate were collected after centrifugation at 12000 rpm and 4° C. for 1 min. Result of SDS-PAGE showed that target protein was mainly expressed in soluble form.
1. Strain activation: pET30a-TZSV colonies were selected from solid plates and added into 5 mL LB liquid medium (Kanamycin concentration 50 µg/mL), and incubated at 37° C. for 12 h-14 h.
2. Small test expression: next day, strains were added into 800 mL LB liquid medium at 1:50 (Kanamycin concentration of 50 µg/mL), cultured at 37° C. to OD=0.4-0.6, and IPTG was added at 0.8 mM. After induction and expression at 25° C. for 6 h, bacteria was centrifuged at 8000 RPM and 4° C. for 15 min and collected.
3. Strain lysis: 100 mL crushing solution was added for ultrasonic lysis. Lysis conditions: temperature ice bath, power 60%, ultrasonic 2 s, interval 2 s, time 15 min. Supernatant and precipitate were collected after centrifugation at 12000 RPM and 4° C. for 15 min.
4. Purification of supernatant: The collected supernatant was purified by high affinity Ni resin, and the flowing through fluid and eluent were collected. Results of SDS-PAGE showed that 50 mM imidazole elution had best purity of protein. The imidazole was removed by dialysis with 50 mM imidazole eluent, and effect of dialysis was detected by SDS-PAGE, and results showed that purity and concentration of protein after dialysis were feasible. After detection, finally, purity of target protein was more than 90%, concentration was 2 mg/mL, and protein amount was 10 mg.
Expression conditions of pet30a-tzsv protein were as follows: IPTG concentration was 0.8 mM, induction temperature is 25° C., induction time is 6 h. Protein was mainly expressed in supernatant, which was purified by Ni column and dialyzed to remove imidazole, and in final, TZSV N protein was obtained with concentration of 2 mg/mL, purity more than 90%, protein of 10 mg.
1. Immunogen preparation: The expressed and purified protein was mixed and emulsified with equal volume of Freund’s adjuvant to form a water-in-oil state for immunizing mice.
2. Immunization strategy: 4 Balb/c mice were immunized with protein for 3 times subcutaneously, with an interval of 4 weeks, and finally titer was detected by indirect ELISA.
3. Cell fusion: two weeks after the last immunization, intraperitoneal injection of antigen was performed for booster immunization, and cell fusion was performed three days later. Mice were sacrificed by cutting their neck, soaked in 70% ethanol for 30 min for disinfection, and their abdominal cavity was cut open in the ultra-clean table. Spleen was removed, ground, and passed through 80 mesh screen to obtain splenocytes. SP2/0 myeloma cells were added, and cell fusion was carried out under action of PEG4000.
4. Fusion screening: The fused cells were spread into a 96-well plate and cultured with HAT medium. After three days, medium was changed to HT medium. After 10 days, cell culture supernatant was taken for detection.
5. Cloning and strain building: use the limited dilution method to clone the positive wells, 10 days later, the positive clones continue to be cloned by the limited dilution method, until the clones are all positive, positive cell lines can be established. Finally, 5 positive cell lines were obtained.
6. Expanded culture: monoclonal cells of the established strain were expanded cultured and stored by freezing.
1. Preparation of ascites: one week in advance, mice were intraperitoneally injected with mineral oil, and a certain number of cells were injected into mice., about 10 days later, ascites was collected and centrifuged at 4000 rpm to obtain supernatant as ascites of monoclonal antibody.
2. Monoclonal antibody purification: ascites were centrifuged for 15 min (4000 rpm, room temperature), supernatant was taken, and saturated ammonium sulfate was slowly added drop by drop under stirring at 4° C. add to semi-saturation; stirring was continued for 30 min, centrifugation was performed for 30 min (13000 rpm, 4° C.), and supernatant was discarded; precipitate was dissolved in an appropriate amount of PBS(0.01 M, pH7.4), and slowly added with saturated ammonium sulfate drop by drop to 33% under stirring at 4° C.; continue stirring was continued for 30 min, centrifugation was performed for 30 min (13000 rpm, 4° C.), and supernatant was discarded. Precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4), dialyzed at 4° C. overnight, antibody content was determined, and frozen at -20° C. for reserve. After ammonium sulfate precipitation, Protein G column was used for purification. New column was first passed with 5 mL ultrapure water, and then balanced with 5 mL 0.4 M PB buffer (pH 7.0) to purify the column; antibody was passed through the column with requirement of slow passing through the column in process, in order to better bind antibody protein on binding site; the column was subsequently equilibrated with 10 mL 0.4 M PB buffer (pH 7.0); the antibody at the binding site was eluted with 5 ml of 0.1 M glycine-hydrochloric acid buffer (pH 2.7) and glycine was neutralized by adding 1 M Tris-HCl (pH 8.0) to maintain a neutral pH suitable for antibody preservation.
1. Titer detection Titer of purified antibody was detected by indirect ELISA method, and data were shown in Table 2:
2. Subtype detection Mouse antibody subtype detection kit was used for subtype detection of antibodies, and results were shown in Table 3 and Table 4:
3. Antibody pairing The obtained antibody was screened by gold labeled method of double antibody sandwich, and the antibody pairs which could be paired were screened out.
Among them, FL484-03 and FL484-10 had the best pairing effect, so this pair of antibodies was selected as antibody for preparing subsequent colloidal gold rapid test strip, and this pair of antibodies was deposited.
4. Strip specificity detection Cross reaction of TMV, PVY, TSWV, ChiVMV and other common tobacco viruses was detected, and results showed that there was no cross reaction between the antibodies and other plant viruses. The result was shown in
Experimental conclusion: The test strip provided by the invention has specificity for TZSV, and no specific reaction to other viruses like TMV, PVY, TSWV and ChiVMV
TZSV hand-held colloidal gold rapid test strip provided in this example (TZSV virus immunoassay test card) consists of a sample pad, gold labeled pad, nitrocellulose membrane, absorbent pad (absorption pad) and a backplane; sample pad, gold labeled pad, nitrocellulose membrane and absorbent pad are successively superimposed and pasted on the backplane, and each part is superimposed between each other by 2.5 mm; the gold labeled pad was coated with colloidal gold labeled anti-TZSV monoclonal antibody; nitrocellulosic membrane is provided with a test line and a quality control line (T line and C line), the test line is coated with anti-TZSV monoclonal antibody, the quality control line is coated with anti-mouse IgG secondary antibody.
Wherein, the anti-TZSV monoclonal antibody was secreted and produced by hybridoma cell lines with depository numbers of CCTCC No. C202160 and CCTCC No. C202196.
Concentration of anti-mouse IgG secondary antibody coated on the quality control line was 1 mg/ml. Size of the sample pad is 3 mm × 15 mm, size of the gold labeled pad is 3 mm×3 mm, size of the nitrocellulose membrane is 3 mm×28 mm, and size of the absorption pad is 3 mm×19 mm.
Space between the test line and the quality control line is 6-6.5 mm.
The backplane (bottom plate) is a PVC board.
Production method of the test card is as follows:
1. 200 µl blank sample solution (pH 7.4, 0.2 M/L PBS solution) was added into the sample cup, and the preparation method was as follows: 8 g sodium chloride, 3.35 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium dihydrogen phosphate and 0.2 g potassium chloride was dissolved by double steamed water to a constant volume of 1 L. The test strip was inserted into the sample cup rapidly, and the color development results were observed in the result observation area after 8 minutes of reaction at room temperature. The results show that T line did not produce color in the observation area, while C line did.
2. 100 µl of 0.1 µg/ml TZSV virus standard (prepared with above PBS solution) was added into the sample cup, and the same procedure as the blank sample was performed.
The results show that C line and T line produce color simultaneously in the observation area. Experimental conclusion: The TZSV virus detection reagent of the invention can quickly detect TZSV virus, and has high sensitivity and convenient operation.
1. 200 µl of negative leaf sample extract was added into the sample cup and performed according to the same procedure as the standard (same as step 1 of example 4). The results show that only the control line C produced color in the observation area, while the test line T did not.
2. 200 µl positive leaf sample extract was added into the sample cup, and performed according to the same procedure as negative sample.
The results show that both the control line C and the test line T produce color in the observation area. Experimental conclusion: The TZSV virus hand-held colloidal gold speed test strip of the invention has good anti-interference effect on leaf and other samples, and can be widely used in rapid detection of TZSV, with high sensitivity and convenient operation.
This example performed theoretical evaluation on hydrophilicity or hydrophobicity, signal peptide, transmembrane domain, basic structure of N protein in TZSV Yunnan strain, plasmid construction program was designed according to results of the evaluation with method of full gene synthesis to construct plasmid, clone into expression vector, further transform into a competent escherichia coli cells, culture, induced express, collect bacteria, purify protein. Finally, 5 mg recombinant TZSV-N protein with purity >90% was obtained and verified by SDS-PAGE.
Gene sequence with optimized codon was shown in SEQ ID NO.2, with 6 bases at 5 ‘end and 6 bases at 3’ end as restriction sites.
Amino acid sequence of prokaryotic expressed TZSV-N Protein was shown in SEQ ID NO.1. Protein Length=282 AA, MW= 31 KDa (34 KDa with label), Predicted pI= 8.6.
Protein expression and verification
After purification, fusion protein can be expressed in a large amount in supernatant, and it has been proved that the maximum amount of the fusion protein can be purified by elution of imidazole at a concentration of 200-500 mM. It’s preliminarily to determine that the fusion protein was purified successfully when results of SDS-PAGE showed that there were obvious bands in the corresponding position of theoretical molecular weight ±5 kDa. Electrophoresis results of pET28a-TZSV-N protein purification are shown in
TZSV N protein was successfully expressed and purified by pET28a vector. After purification, 10 ml of target protein was obtained at a concentration of 0.5 mg/ml, whose total weight was 5 mg, and was packed according to 1 ml/tube.
Protein storage buffer: 20 mM Tris-Cl, 50 mM NaCl, 1 mM EDTA, pH=8.0.
Four 6-week-old female BALB/ C mice were immunized for three times by subcutaneous multipoint injection. Antigen dosage of each mouse was 25 µg, the first immunization was emulsified with same volume of Freund’s complete adjuvant, the second immunization was emulsified with same volume of Freund’s incomplete adjuvant, and the third immunization was mixed with same volume of normal saline and injected intraperitoneally. Specific immunization procedures are shown in Table 5.
Twenty days after the second immunization, blood was collected from tail vein and antibody titers were measured by an indirect method to determine whether antibodies against antigen had been generated. Titers of four mice were compared, and the mouse with higher antibody titer was finally selected for cell fusion (mouse number: M210045). Three days before fusion, booster immunization was performed directly with antigen and with same dose as before.
A total of 37 positive monoclonal hybridoma cell lines were screened and established. The hybridoma cells were collected, and mouse ascites were prepared and collected, the 1000-fold dilution of TZSV positive samples collected in field was used as sample to be tested, and antibody titer was determined by indirect ELISA method. The titer can be as high as 1:1024000. Results of monoclonal antibody titer detection are shown in Table 6.
Antibody with good performance in preliminary experiment was selected to be paired with polyclonal antibody, the results in Table 7 showed that the monoclonal antibody with No. 3#, 9#, 10, 21, 37 could detect recombinant protein with a sensitivity of more than 10 PPB when they were paired with polyclonal antibody, and all of them could detect TZSV tobacco leaf samples that were pathogenetic in field; 9# monoclonal antibody was selected for subsequent enzyme-labeled product optimization.
9# monoclonal antibody was selected as the coated antibody, and standard product with gradient concentration was prepared through condition optimization. Linear range of 10-160 ppb was selected as detection range (Table 8), and standard curve was obtained through linear regression (
Firstly, sensitivity of test strip was tested. As shown in
TMV, PVY, CMV, TVBMV, ChiVMV, TSWV and TZSV infected leaves which were identified as positive by RT-PCR were used as test samples to detect specificity of TZSV test strip. As shown in
Evaluation on stability of TZSV strip: Test strip was placed in an environment of 4° C., room temperature and 37° C. for 6 months, during this period, test strip was taken out to test 100-fold dilution of negative control and TZSV positive sample every other month, and results are shown in Table 9, during the stability evaluation period of 6 months, the test strip maintained good stability at three temperatures, and no false positives or false negatives occurred.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111399298.4 | Nov 2021 | CN | national |
