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Development and Application of Papaya Ringspot Virus Watermelon Strain Attenuated Vaccine

A ring spot virus and attenuated vaccine technology, applied in the field of genetic engineering, can solve the problems of poor protection and no protection of PRSV strains

Active Publication Date: 2021-07-20
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have used nitrous acid to mutagenize the virulent papaya ringspot virus strain HA in Hawaii, USA to obtain its attenuated strain HA5-1 and conduct cross-protection and control of PRSV. However, the protective effect of the weak strain has strain-specific problems. , For example, the HA5-1 weak strain has a stronger protective effect on the Hawaiian wild strain than the Chinese Taiwan strain, has a poor protective effect on the PRSV strain in South China, and has no protective effect on the Thai strain and the Mexican strain.
However, there is no report on mutagenesis and screening of attenuated strains of papaya ringspot virus watermelon strains for cross-protection research

Method used

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  • Development and Application of Papaya Ringspot Virus Watermelon Strain Attenuated Vaccine
  • Development and Application of Papaya Ringspot Virus Watermelon Strain Attenuated Vaccine
  • Development and Application of Papaya Ringspot Virus Watermelon Strain Attenuated Vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Site-directed mutagenesis

[0044] Using the full-length invasive cDNA clone pCamPRSV-W-GFP of the papaya ringspot virus watermelon strain with a green fluorescent protein (green fluorescent protein, GFP) tag as a template, the mutation was carried out by PCR, and the DNA polymerase used was Phusion high-security true polymerase.

[0045] Mutation PCR reaction system:

[0046]

[0047] In the above mutation PCR reaction system, primer 1 and primer 2 can be the primers shown in SEQ ID No.1 and SEQ ID No.2, or the primers shown in SEQ ID No.3 and SEQ ID No.4, respectively,... Or the primers shown in SEQ ID No.19 and SEQ ID No.20 are used to mutate amino acids at different positions of HC-Pro.

[0048] PCR reaction program: 98°C pre-denaturation for 30s; 98°C denaturation for 10s, Tm no+3°C annealing for 20s, 72°C extension for 9min30s, 15 cycles; 98°C denaturation for 10s, Tmpp+3°C annealing for 20s, 72°C extension for 30min, Store at 4°C. Tm no and Tmpp...

Embodiment 2

[0050] Example 2: Inoculation of virus

[0051] Transform pCamPRSV-W-GFP and the mutant plasmid into Agrobacterium GV3101 competent cells. After colony PCR verification, pick a single spot and inoculate it in 2 mL containing kanamycin (50 μg / mL), rifamycin (50 μg / mL), In the liquid LB medium of tetracycline (5 μg / mL), shake culture at 180 r / min at 28° C. for 24 h. Add 500 μL of bacterial liquid to 5 mL of LB medium containing 10 mM 2-(N-morpholine)-ethanesulfonic acid (MES) and 20 μM acetosyringone (AS) and the same antibiotics as in the previous step, at 28 °C 200 r / min Shaking culture to the logarithmic growth phase takes about 12 hours. The bacteria were collected by centrifugation, and the resuspension (10mM MgCl 2 , 10mM MES, 0.15mM AS) gently resuspend the cells, adjust the OD 600 The value is 0.5-0.6, and it is allowed to stand at 28°C for 3 hours. Use a 10 μL pipette tip to pierce a small hole on the back of the cotyledon of the plant, use a 1mL needle-free medical...

Embodiment 3

[0052] Example 3: Symptom observation and virus accumulation level detection

[0053] On the 15th day after inoculation, the symptoms of the inoculated plants were observed, and the leaves of the system were collected to extract RNA and protein. RT-PCR was used to detect whether the mutant successfully infects the host, and the level of virus accumulation was detected by Western blotting. The wild-type PRSV-W with GFP tag caused severe symptoms such as mosaic, chlorosis, mottling and deformity on the leaves of muskmelon (Cucumis melo), and obvious green fluorescence was visible under ultraviolet light; while the melon plants inoculated with the mutant No obvious symptoms, weak fluorescence under ultraviolet light, distributed in lumps along the veins or in the mesophyll, even no fluorescence ( figure 1 ). The test results showed that the mutants did not revert to wild type, and the inventors named these attenuated mutants as gC26A, gC57A, gK125D, gK126D, gN137A, gG317K, gP328...

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Abstract

The invention discloses a PRSV-W attenuated vaccine and its application. Based on PRSV-W full-length invasive cDNA clone, the present invention introduces mutations in the HC-Pro gene of PRSV-W through site-directed mutation technology, and clarifies the amino acid sites that regulate the pathogenicity of the virus, and obtains the pathogenicity The PRSV-W attenuated mutant with significantly decreased force; the present invention finds that attenuated vaccines K125D, G317K and N137A have good cross-protection effect on PRSV-W virulent strains, and can protect melon and zucchini plants from being infected by virulent strains .

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a papaya ringspot virus watermelon strain attenuated vaccine and application thereof. Background technique [0002] Virus diseases are common in the production of Cucurbitaceae crops, which can significantly reduce the quality and yield of crops, and even lead to extinction in severe cases. At present, nearly 50 viruses have been identified that are harmful to Cucurbitaceae crops. Among them, Papaya ringspot virus-watermelon strain (PRSV-W) belongs to Potavirus Y and mainly infects Cucurbitaceae crops such as zucchini and melon. , causing symptoms such as leaf curling, deformity, mosaic, blisters, and plant dwarfing, and even fruit deformity. PRSV-W has occurred in cucurbitaceous crops in Shandong, Henan, Guangdong, Sichuan and other places in my country, and has become an important virus harmful to cucurbitaceous crops. [0003] Planting disease-resistant varieties...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C07K14/08A01G13/00
CPCA01G13/00C07K14/005C12N7/00C12N2770/34021
Inventor 李向东黄显德田延平王玉耿超
Owner SHANDONG AGRICULTURAL UNIVERSITY
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