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A kind of polyclonal antibody based on wheat yellow stripe virus g protein, preparation method and application thereof

A polyclonal antibody and stripe virus technology, applied in the field of agricultural biology, can solve the problems of expensive, unsuitable for the detection of large quantities of samples, and unable to complete the identification, etc., to achieve strong specificity, good application prospects, both practicability and rapid sexual effect

Active Publication Date: 2020-06-02
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as far as WDV and the new virus WYSV are concerned, since the two viruses are transmitted by the leafhopper and have similar symptoms, biological identification cannot complete the identification; molecular biological methods represented by PCR are fast, It is highly sensitive, but requires expensive instruments and molecular biology reagents, and is not suitable for the detection of large quantities of samples; serology has the advantages of fast, simple, high-throughput, and is suitable for large-scale sample detection in the field, so it is widely used Diagnosis of plant viruses, analysis of epidemic patterns, prediction and early warning, scientific prevention and control, and disease-resistant breeding, etc.

Method used

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  • A kind of polyclonal antibody based on wheat yellow stripe virus g protein, preparation method and application thereof
  • A kind of polyclonal antibody based on wheat yellow stripe virus g protein, preparation method and application thereof
  • A kind of polyclonal antibody based on wheat yellow stripe virus g protein, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1. Gene optimization and synthesis, subcloning of wheat yellow streak virus G protein

[0044] According to the full-length gene sequence of WYSV (GenBank accession number MG604920), software was used to analyze the protein encoded by ORF5 of the virus, glycoprotein Glycoprotein (G), and it was found that the full-length G protein gene was 1986 nt, encoding 661 amino acids, and the predicted molecular weight and The electrical points are 74.9KDa and 5.30, respectively.

[0045] Using the online software SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ) and TMHMMServer v.2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ) to predict the signal peptide of the protein And the transmembrane region, after analysis, it was found that the G protein of WYSV has no potential signal peptide cleavage site, but contains two transmembrane regions, which are located at positions 7 to 24 and positions 611 to 633 at the 5' end of the gene, respectively. Using Optimum Gene TM Codon...

Embodiment 2

[0046] Example 2. Recombinant protein expression and purification

[0047] The recombinant expression plasmid pET-30a-(WYSV-G) was transformed into the BL21(DE3) expression strain by heat shock, and after sequencing verification, the BL21 bacterial fluid containing the correct recombinant expression plasmid was inoculated in the culture medium containing 50ug / mL In 4 mL LB liquid medium of kanamycin, culture at 37°C and 200rpm until OD600 is 0.6-0.8, add IPTG to a final concentration of 0.3mmol / L, induce at 15°C for 16h, then continue to induce at 37°C for 4h Collect 1mL of the bacterial liquid, and centrifuge to collect the bacterial cells. Detected by 12% SDS-PAGE gel electrophoresis, the protein expression in the whole bacteria, supernatant and precipitate were detected respectively. It was found that the specific expression of the target protein was detected in the induced sample, and the size was about 65kDa, which was consistent with the expected fusion protein size, wh...

Embodiment 3

[0048] Example 3. Antibody Preparation

[0049] After the purified soluble protein was emulsified with complete Freund's adjuvant, the New Zealand male rabbits were subcutaneously immunized at multiple points, and the second immunization was carried out 10 days later, and the immunization was boosted every other week, and the booster immunization was emulsified with incomplete Freund's adjuvant. Thigh intramuscular injection method. A small amount of serum was taken 5 days after each immunization, and the titer of the antibody was determined by indirect ELISA. When the titer reached 1:100,000 or more, blood was taken and the serum was separated. Anti-WYSV-G-IgG was purified from antiserum by using protein A-Sepharose affinity column, according to 500×2 1 (1000) to 500×2 10 (512000) serial doubling dilution, the experiment uses pre-immune serum as the negative control and PBS buffer as the blank control respectively, the results show that the titer measured by indirect ELISA ...

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Abstract

The invention discloses a polyclonal antibody based on WYSV (Wheat Yellow Striate Virus), a preparation method and the application of the polyclonal antibody. The preparation method for the polyclonalantibody comprises the following steps: according to the sequence of the G gene of WYSV, firstly, optimizing a codon to synthesize the G gene, and carrying out subcloning on the gene to a target carrier pET-30a expressed by escherichia coli; carrying out prokaryotic expression on recombinant protein, a New Zealand white rabbit is immunized to prepare the polyclonal antibody of the WYSV G protein.Western blot detection indicates that the prepared antibody, the G recobinant protein and a susceptible wheat sample can be subjected to specific binding so as to indicate that the obtained antibodyhas high specificity. On the basis of the prepared polyclonal antibody, a DIBA (Dot Immunobinding Assay) method for detecting the WYSV is established, whether a field grass crop is infected by WYSV viruses or not can be specifically, sensitively and quickly detected.

Description

technical field [0001] The invention belongs to the technical field of agricultural biology, and in particular relates to a WYSV polyclonal antibody, a preparation method, an antibody-based immune detection kit and its application, so as to achieve the purpose of fast and efficient detection of WYSV. Background technique [0002] Wheat is one of the main food crops in my country, and its safe production is related to people's livelihood. In recent years, the occurrence of viral diseases on wheat has been serious, especially the wheat yellow dwarf disease caused by barley yellow dwarf virus (Barley yellow dwarf virus, BYDV) and wheat yellow mosaic virus (WYMV) caused by wheat yellow mosaic virus. disease, resulting in a severe reduction in wheat production and huge economic losses. In addition, wheat dwarf virus disease caused by wheat dwarf virus (WDV) is transmitted by the medium leafhopper (Psammotettix striatus L.) in a persistent non-proliferative manner, and the typica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/70G01N33/569
CPCC07K16/10C12N15/70G01N33/56983
Inventor 刘艳杜真真付玉梅王锡锋
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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