Rabbit polyclonal antibody for specific detection of areca yellow phytoplasma as well as preparation method and application of rabbit polyclonal antibody

Abstract

The invention relates to a rabbit anti-polyclonal antibody for specifically recognizing areca yellowed phytoplasma as well as a preparation method and a use method of the rabbit anti-polyclonal antibody. According to the present invention, the gene sequence of the IMP protein is found from the genome of the betel nut yellow phytoplasma previously detected by the inventor, the codon optimization of the prokaryotic expression is performed to obtain the (40-154aa) nucleotide region of the gene synthesis IMP protein, and the rabbit anti-polyclonal antibody is further prepared; the antibody can be used for well detecting and distinguishing areca catechu yellowing materials infected with the phytoplasma at the protein level, overcomes the technical problems of tedious detection process, high detection cost, high detection technical difficulty, long time consumption and the like of the current infected phytoplasma, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a novel rabbit polyclonal antibody for specifically detecting betel nut yellowing phytoplasma, a preparation method and application thereof. Background Art

[0002] Betel nut (Areca catechu L.) is a single-growth-point evergreen tree of the genus Areca in the palm family. Betel nut is an important medicinal plant and the first of the four major southern medicinal herbs in my country. Its fruit has the effects of strengthening teeth and killing bacteria, eliminating accumulation and digestion, eliminating athlete's foot and expelling insects. In addition to its good medicinal value, betel nut has gradually become a chewing hobby for people after cigarettes, alcohol and caffeinated beverages, and is loved by consumers. At present, there are about 1 to 1.2 billion people in the world who love to eat betel nut, including 100 million in China. Due to its good medicinal value and hobby use, as more and more people deepen their understanding of betel nut, and like and consume it, the market demand continues to expand, and the industry prospects are broad.

[0003] Areca nut yellowing disease is a deadly infectious disease of betel nut. The disease was first discovered in India in 1914 and also appeared in Tunchang County, Hainan, my country in 1981. Since then, betel nut yellowing disease has spread to almost all betel nut planting areas in India and Hainan, my country, and has also occurred in a small number of betel nut planting areas in other countries. The typical symptoms of betel nut yellowing disease are: in the early stage of the disease, the leaf tips of the 2-3 last leaves of the plant first show yellowing, and then the yellowing symptoms worsen year by year, and finally the whole plant leaves turn yellow, and the yellowing symptoms are more obvious in the dry season; the whole plant leaves cannot unfold normally, resulting in a smaller crown; the axillary buds are water-soaked, dark black, with a light brown center at the base; the flower spikes are short and cannot unfold normally; a small number of black fruits are produced, which cannot be eaten; most plants die 5-7 years after the disease.

[0004] Areca nut yellowing disease caused by phytoplasma is a devastating disease in the production of betel nut in Hainan, my country. Phytoplasma is a type of prokaryotic pathogen that parasitizes the phloem of plants and cannot be isolated and purified, making it difficult to culture artificially. In addition, phytoplasmas are low in content and unevenly distributed in woody plants, and their concentration varies with the seasons, making them difficult to detect. Currently, there are nested-PCR technology, real-time fluorescence quantitative PCR technology, loop-mediated isothermal amplification, and droplet digital PCR (ddPCR) technology, among which nested-PCR technology is the most commonly used. The design of nested-PCR primers is based on the conserved 16SrDNA sequence of phytoplasmas, among which R16mF2 / R16mR1 and R16mF2n / R16mR2 are universal primers for detecting phytoplasmas. In addition, with the successful design of P1 / P7 primers, the combination of P1 / P7 with the above two pairs of primers is also a commonly used technique. So far, phytoplasmas have been detected in betel nut leaves, flower buds, roots, inflorescences and other tissues. Therefore, the establishment of highly sensitive, efficient and rapid phytoplasma detection methods is crucial in the research of all aspects of phytoplasma diseases, especially in disease monitoring, seedling quarantine and integrated prevention and control, where it occupies an irreplaceable position and plays a key role.

[0005] Immunodominant membrane proteins are important components of phytoplasma membrane proteins, which mainly interact with certain host skeleton proteins to achieve their stable parasitic effect. Therefore, studying the characteristics and functions of immunodominant membrane proteins of the yellowing phytoplasma of Areca nut can lay a theoretical and application foundation for further analyzing the parasitic and pathogenic mechanisms of phytoplasmas and effective prevention and control. Therefore, we urgently need to establish a simple and low-cost method for efficient detection of yellowing phytoplasmas of Areca nut, which is convenient for the rapid detection of field disease surveys by the majority of planting personnel, especially for the rapid detection of areca nut nurseries, so that pathogens can be quickly discovered, the spread of diseases can be prevented from the source of planting, and the normal production of the areca nut planting industry can be guaranteed. Summary of the invention

[0006] In response to the technical problems of complicated detection process, high detection cost, great detection technology difficulty and long time consumption of the above-mentioned betel nut yellowing phytoplasma, the present invention proposes a rabbit anti-polyclonal antibody, preparation method and application thereof that has a reasonable design, simple method, convenient operation and can detect betel nut yellowing disease without the need for professional knowledge, which can specifically identify betel nut yellowing phytoplasma.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides an optimized betel nut yellowing phytoplasma immune membrane protein gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The second aspect of the present invention provides a protein encoded by the above gene, whose amino acid sequence is shown in SEQ ID NO.2.

[0010] The third aspect of the present invention provides a rabbit anti-polyclonal antibody that specifically recognizes betel nut yellowing phytoplasma, the antibody can be used to detect betel nut yellowing disease, and the preparation method of the antibody comprises the following steps:

[0011] (1) The immunodominant membrane protein gene sequence of the betel nut yellowing disease phytoplasma annotated in the betel nut yellowing disease phytoplasma genome was predicted by SnapGene software, and its amino acid sequence information was used for antigen preparation analysis. The sequence 40-155aa region without the transmembrane region of the protein was selected for subsequent prokaryotic expression vector construction (such as Figure 1 shown);

[0012] (2) The IMP (40-154aa) gene sequence without the transmembrane region was constructed into a prokaryotic expression vector (pET28a) (e.g. Figure 2 The constructed recombinant plasmid was transformed into a prokaryotic expression strain (DE3) for prokaryotic expression and purification of the target protein (as shown in Figure 3 shown);

[0013] (3) Purify the recombinant protein using a prokaryotic expression system to more than 4 mg, with a protein purity greater than 85% and a soluble protein concentration between 1-5 mg / mL. Sent to Wuhan Abotek Biotechnology Co., Ltd. for the preparation of rabbit anti-polyclonal antibodies, select four SPF (Specific Pathogen-Free) grade Japanese large-eared white rabbits, use complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) subcutaneous multi-point injections for antigen immunization, and finally collect the serum of the immunized rabbits for antibody purification;

[0014] (4) The prepared rabbit anti-polyclonal antibody was used for Western Blot detection of the protein level of Areca catechu yellowing phytoplasma.

[0015] The specific steps are:

[0016] Step 1: Sample processing: 200 mg of betel nut sample showing yellowing symptoms was fully ground using liquid nitrogen;

[0017] Step 2: Total protein extraction: After grinding, add 500 μL of protein extraction buffer, vortex for 1 min, rotate at 4 °C for 30 mins, centrifuge at 12000 g for 1 min, and take the supernatant for later use;

[0018] Step 3: Gel separation: Pipette 20 μL of the extracted total protein and add 5 μL of 5X protein loading buffer, incubate in a 100°C water bath for 10 mins, and take 10 μL for SDS-PAGE gel separation;

[0019] Step 4: Transfer the completed protein gel to a membrane (PVDF membrane);

[0020] Step 5: Blocking: Place the transferred PVDF membrane in a blocking buffer (TBST buffer containing 5% (w / v) skim milk powder) and incubate at room temperature for 30 minutes;

[0021] Step 5: Primary antibody incubation: dilute the prepared rabbit polyclonal antibody against the immunodominant membrane protein of Areca catechu yellowing phytoplasma to an appropriate working concentration (1:2000) using antibody diluent, prepare 5-10 ml and add it to the incubation box, and place the blocked PVDF membrane in the incubation box and incubate at 4°C overnight;

[0022] Step 6: Wash the membrane: discard the antibody solution in the incubation box, add 5-10 ml of TBST buffer, place on a shaker at room temperature to wash for 5-10 minutes and discard the TBST buffer in the box, repeat 3 times;

[0023] Step 7: Secondary antibody incubation: Use antibody diluent to prepare 5-10 ml of alkaline phosphatase-labeled goat anti-rabbit (1:10000) and add it to the incubation box, and incubate at room temperature for 2-4 hours;

[0024] Step 8: Wash the membrane: same as step 6;

[0025] Step 9: Exposure: Add an appropriate amount of luminescent liquid to the membrane, let it stand for 5 minutes, and then expose it with the instrument.

[0026] As a preferred embodiment: in step 1, the sample is placed in a 2 ml EP tube, 2-3 4 mm steel balls are placed in the tube, and the tube is placed in a grinder at 70 Hz and shaken for 1 min, so that the grinding is more complete.

[0027] Compared with the prior art, the advantages of the present invention are:

[0028] The invention provides a rabbit anti-polyclonal antibody for specifically identifying the betel nut yellowing phytoplasma, which has significant specificity in the detection of the betel nut yellowing phytoplasma, thereby avoiding the false negative and false positive results that are very likely to occur in traditional detection methods, and ensuring the accuracy of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 : Schematic diagram of the expanded membrane region of the immunodominant membrane protein of the Areca nut yellows phytoplasma, wherein 10-40aa is the expanded membrane region of the immunodominant membrane protein of the Areca nut yellows phytoplasma. In order to better prepare specific antibodies, the 40-135aa region of the immunodominant membrane protein of the Areca nut yellows phytoplasma is selected.

[0031] Figure 2 : SDS-PAGE gel image of the immunodominant membrane protein 40-135aa of the prokaryotic expression of Areca catechu yellowing phytoplasma, where M is a low molecular weight marker; from left to right, 5μL, 10μL, and 15μL of the purified IMP (40-154aa) protein were loaded. Figure 3 : Schematic diagram of Western Blotting test results of tissue samples infected with Areca nut yellows phytoplasma, wherein M is a low molecular weight marker; AYLP_IMP (40-154aa) is the test result of the immunodominant membrane protein of Areca nut yellows phytoplasma purified by prokaryotic expression without the membrane expansion region; H1 and H2 are the test results of healthy Areca nut tissue samples; S1 and S2 are the test results of tissue samples infected with Areca nut yellows phytoplasma; Anti-RBCL is the internal standard antibody for detecting the quality of total protein extraction, and Anti-IMP is the prepared rabbit anti-polyclonal antibody against the immunodominant membrane protein of Areca nut yellows phytoplasma. DETAILED DESCRIPTION

[0032] Example 1 Preparation of rabbit anti-polyclonal antibodies that specifically recognize the betel nut yellow phytoplasma

[0033] In order to prepare antibodies against the immunodominant membrane protein (IMP) of the betel nut yellowing phytoplasma, the gene sequence of the IMP protein was first found from the genome of the betel nut yellowing phytoplasma previously detected by the inventors, and sent to Qingke Biotechnology Co., Ltd. for codon optimization for prokaryotic expression to obtain the optimized nucleotide region (SEQ ID NO.1) for synthesizing the IMP protein. After obtaining the optimized gene sequence, PCR amplification was performed by designing primers with restriction sites, and the primer sequences are as follows:

[0034] IMP_NdeI_40-F: 5'-CATATGCCGAAGAAAGTTTGGGCGAAACAGG-3' (SEQ ID NO. 3);

[0035] IMP_SalI_135-R: 5'-GTCGACTTATTTGGTCAGGTTGTTAACTTTCTCTTGCAG-3' (SEQ ID NO. 4).

[0036] PCR reaction system: 2×KOD OneTM PCR Master Mix 10 μL, 1 μL each of 10 μM amplification primers IMP_NdeI_40-F and IMP_SalI_135-R, 1 μL of gene synthesis DNA template, and sterile distilled water to 20 μL.

[0037] PCR reaction conditions: 98°C, 30 s; 98°C, 10 s, 52°C, 5 s, 68°C, 5 s, 35 cycles; 68°C, 5 min.

[0038] The amplified PCR product and the pET-28a prokaryotic expression plasmid were double-digested. The double-digestion system is as follows:

[0039]

[0040]

[0041] After reacting at 37°C for 30 minutes to 1 hour, the PCR products after double enzyme digestion and the plasmid vector after enzyme digestion were run on a gel for identification, and then the gel was cut and recovered and purified. After recovery, the target fragment was connected to the expression vector pET-28a. The connection system is as follows:

[0042]

[0043] The ligation product was transformed into E. coli DH5a competent cells, and the positive clones were selected and sent to a sequencing company for sequencing. The protein sequence of the immunodominant membrane protein (IMP) of the yellow phytoplasma of Areca catechu is shown in SEQ ID NO.2.

[0044] The obtained recombinant plasmid with correct sequencing was transformed into E. coli BL21 competent cells for prokaryotic expression. After tag purification, the obtained IMP protein (40-154aa) reached more than 4 mg, with a protein purity greater than 85% and a soluble protein concentration between 1-5 mg / mL. The gel run identification (such as Figure 3 ), and then sent the prepared protein to Wuhan Abotek Biotechnology Co., Ltd. for the preparation of rabbit anti-polyclonal antibodies. Four SPF (Specific Pathogen-Free) Japanese large-eared white rabbits were selected, and complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were used for subcutaneous multi-point injection for antigen immunization. Finally, the serum of the immunized rabbits was collected for antibody purification. The detailed process is as follows:

[0045]

[0046]

[0047] Finally, the prepared rabbit anti-polyclonal antibody was used for Western Blot detection of the protein level of Areca catechu yellowing phytoplasma.

[0048] Example 2 Functional verification of rabbit anti-polyclonal antibodies

[0049] To verify the applicability and specificity of the obtained antibodies, Western blotting was performed on the antibodies.

[0050] The materials used in the experiment of the present invention are:

[0051] Areca nut yellowing samples were collected from areca nut plantations in Qionghai and Wanning, Hainan. The positive phytoplasma materials were detected by nested PCR and sequenced as phytoplasmas and the retained material samples. The samples without positive bands were healthy samples without phytoplasma infection. Main reagents:

[0052] KOD OneTM PCRMaster Mix was purchased from Toyobo, DNA marker, Gel red nucleic acid dye, and protein marker were purchased from Polymer, and DNA gel recovery kit was purchased from Quanshijin. Primers and IMP gene were synthesized by Qingke Biotechnology Co., Ltd.

[0053] After grinding, add 500 μL of protein extraction buffer, vortex for 1 min, rotate at 4 °C for 30 mins, centrifuge at 12000 g for 1 min, and take the supernatant for later use;

[0054] The specific steps of Western Blot detection are as follows:

[0055] Step 1: Sample treatment: 200 mg of betel nut sample showing yellowing symptoms was placed in a 2 ml EP tube and 2-3 4 mm grinding steel beads were placed in the tube and completely frozen with liquid nitrogen. The tube was then placed in a grinder and ground and shaken at 70 Hz for 1 min.

[0056] Step 2: Total protein extraction: After grinding, add 500 μL of protein extraction buffer, vortex for 1 min, rotate at 4 °C for 30 mins, centrifuge at 12000 g for 1 min, and take the supernatant for later use;

[0057] Step 3: Gel separation: Pipette 20 μL of the extracted total protein and add 5 μL of 5X protein loading buffer, incubate in a 100°C water bath for 10 mins, and take 10 μL for SDS-PAGE gel separation;

[0058] Step 4: Transfer the completed protein gel to a membrane (PVDF membrane);

[0059] Step 5: Blocking: Place the transferred PVDF membrane in a blocking buffer (TBST buffer containing 5% (w / v) skim milk powder) and incubate at room temperature for 30 minutes;

[0060] Step 6: Primary antibody incubation: dilute the prepared rabbit polyclonal antibody against the immunodominant membrane protein of Areca catechu yellowing phytoplasma to an appropriate working concentration (1:2000) using antibody diluent, prepare 5-10 ml and add it to the incubation box, and place the blocked PVDF membrane in the incubation box and incubate at 4°C overnight;

[0061] Step 7: Wash the membrane: discard the antibody solution in the incubation box, add 5-10 ml of TBST buffer, place on a shaker at room temperature to wash for 5-10 minutes and discard the TBST buffer in the box, repeat 3 times;

[0062] Step 8: Secondary antibody incubation: Use antibody diluent to prepare 5-10 ml of alkaline phosphatase-labeled goat anti-rabbit (1:10000) and add it to the incubation box, and incubate at room temperature for 2-4 hours;

[0063] Step 9: Washing the membrane: Same as step 6;

[0064] Step 10: Exposure: Add an appropriate amount of luminescent liquid to the membrane, let it stand for 5 minutes, and then expose it with the instrument.

[0065] Test results such as Figure 3 As shown, the rabbit anti-polyclonal antibody against the immunodominant membrane protein IMP of the betel nut yellowing phytoplasma prepared in the present invention can well detect and distinguish the betel nut yellowing materials infected with phytoplasmas at the protein level. This shows that the antibody of the present invention can be applied to the detection of the betel nut yellowing phytoplasma pathogen.

[0066] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An optimized betel nut yellowing phytoplasma immune membrane protein gene, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A betel nut yellowing phytoplasma immune membrane protein, characterized in that: The amino acid sequence of the membrane protein is shown in SEQ ID NO.

2.

3. A rabbit anti-polyclonal antibody that specifically recognizes Areca catechu yellowing phytoplasma, characterized in that: The method for preparing the antibody comprises the following steps: (1) The immunodominant membrane protein gene sequence of the betel nut yellowing disease phytoplasma annotated in the betel nut yellowing disease phytoplasma genome was predicted by SnapGene software, and the amino acid sequence information was used for antigen preparation analysis, and the sequence 40-155aa region without the transmembrane region of the protein was selected for prokaryotic expression vector construction; (2) constructing the IMP gene sequence without the transmembrane region into a prokaryotic expression vector, and transforming the constructed recombinant plasmid into a prokaryotic expression strain for prokaryotic expression and purification of the target protein; (3) Purify the recombinant protein using a prokaryotic expression system, prepare rabbit anti-polyclonal antibodies, and finally collect rabbit serum for antibody purification.

4. Use of the gene according to claim 1, the protein according to claim 2 and / or the antibody according to claim 3 in the preparation of a product for detecting betel nut yellowing phytoplasma.

5. The use according to claim 4, characterized in that The products include reagents, kits, gene chips, etc.

Images