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Wheat yellow striate virus N gene recombination expression protein, method for preparing polyclonal antibodies and application thereof

A polyclonal antibody and stripe virus technology, applied in the field of agricultural biology, can solve the problems of complicated purification procedures, etc., and achieve the effect of simple and convenient operation, good application prospects and strong specificity

Inactive Publication Date: 2019-05-24
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the purification of plant viruses requires the propagation of a large number of toxin sources, and the purification procedure is relatively complicated, many studies have used prokaryotic expression to prepare antibodies

Method used

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  • Wheat yellow striate virus N gene recombination expression protein, method for preparing polyclonal antibodies and application thereof
  • Wheat yellow striate virus N gene recombination expression protein, method for preparing polyclonal antibodies and application thereof
  • Wheat yellow striate virus N gene recombination expression protein, method for preparing polyclonal antibodies and application thereof

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1. Gene optimization and synthesis, subcloning of wheat yellow streak virus N protein

[0033] According to the full-length gene sequence of WYSV (GenBank accession number MG604920), the protein encoded by ORF1 of the virus, Nucleocapsid protein (N), was analyzed by software, and the N protein gene was found to be 1635 nt in length, encoding 544 amino acids, and the predicted molecular weight and The isoelectric points are 59.8KDa and 8.81, respectively.

[0034] 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, the analysis found that the N protein of WYSV has no potential signal peptide cleavage site and transmembrane region. Using Optimum Gene TM Codon optimization technology (Nanjing KingScript Technology Co., Ltd.), finally select 365 amino acids (from the 94th to 1188nt of the original prote...

Embodiment 2

[0035] Embodiment 2. WYSV-N recombinant protein expression and purification

[0036] The recombinant expression plasmid pET-30a-(WYSV-N) was transformed into the BL21(DE3) expression strain by heat shock, and after sequencing verification, the BL21 bacterial solution 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 42kDa, which was consistent with the expected fusion pro...

Embodiment 3

[0037] The preparation of embodiment 3.N protein antibody

[0038] 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-N-IgG was purified from antiserum using protein A-Sepharose affinity column, according to 500×2 1 (1000) to 500×2 10 (512 000) serial ratio dilution, the experiment took pre-immune serum as the negative control and PBS buffer as the blank control, the results showed that the titer determined by indirect E...

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Abstract

The invention discloses a wheat yellow striate virus N gene recombination expression protein, a method for preparing polyclonal antibodies and application thereof. The method for preparing the polyclonal antibodies includes steps of synthesizing genes by optimization codons according to sequences of N genes of WYSV (wheat yellow striate viruses) for the first time and subcloning the genes into target vectors pET-30a for expressing Escherichia coli; carrying out prokaryotic expression on recombinant proteins and immunizing New Zealand white rabbits to obtain the WYSV-N protein polyclonal antibodies. The wheat yellow striate virus N gene recombination expression protein, the method and the application have the advantages that as shown by Western blot detection, the antibodies prepared by theaid of the method can be specifically bound with toxic insect samples, and accordingly the high specificity of the obtained antibodies is illustrated; whether vector insects carry the WYSV or not canbe quickly detected by the aid of immunofluorescence detection processes under laboratory conditions, and accordingly a foundation can be laid for research on WYSV prediction and forecast and psammotettix striatus L. and WYSV interaction mechanisms.

Description

Technical field [0001] The invention belongs to the technical field of agricultural biology, and specifically relates to applications such as a recombinant expression protein of wheat yellow stripe virus N gene, a polyclonal antibody preparation method and an immunofluorescence-labeled in situ hybridization virus detection system based on the antibody, thereby achieving rapid , for the purpose of efficiently detecting WYSV and clarifying the tissue distribution of the virus. Background technique [0002] Wheat yellow striate virus (WYSV) is a new nuclear rhabdovirus discovered during a disease field survey in Hancheng, Shaanxi Province in 2016. It is spread by the vector Psammotettix striatus L. in a persistent multiplication mode. , can be spread by leafhoppers to wheat, barley and other grass crops. The heterosandy leafhopper (is an agricultural insect belonging to the genus Psammotettix of the family Paralimnini, subfamily Deltocephalinae, family Cicadellidae, order Hemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/47C12N15/70C07K14/145C07K16/10C07K16/06C07K1/22G01N33/569
Inventor 刘艳付玉梅杜真真王锡锋
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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