A kind of yolk antibody of Macrobrachium rosenbergii wild field virus capsid protein and its application
A technology of Noda virus and Macrobrachium rosenbergii, applied in the direction of anti-virus immunoglobulin, immunoglobulin from eggs, etc., can solve the problem of not involving egg yolk antibody, achieve good immune protection effect, high titer, and broad application prospects Effect
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Embodiment 1
[0021] Example 1: Construction of recombinant plasmid pGEX-5x-1-MrNV-CP of Macrobrachium rosenbergii field virus capsid protein gene
[0022] Using the total RNA extracted from the muscle of Macrobrachium rosenbergii Noda virus-infected Macrobrachium rosenbergii as a template, the downstream primer MrNV-NotI-BW: 5'-ATAGCGGCCGCCTAATTATTATTGCCGACGATAG-3' was used to perform RT-PCR reaction on the capsid protein gene of Macrobrachium rosenbergii Noda virus. To generate the first-strand cDNA, the reagents for the RT-PCR reaction are: reverse transcriptase, dNTP, and buffer; the conditions are: 75°C, 5min; 42°C, 1 hour; 80°C, 10min. Subsequently, using the obtained cDNA as a template, the upstream primer MrNV-SalI-FW: 5'-CATGTCGACATGGCTAGAGGT AAACAA AA-3' and the downstream primer MrNV-NotI-BW: 5'-TAGCGGCCGCCTAATTATTGCCGACGATAG-3' were used for PCR amplification to obtain a double strand DNA. The reagents for PCR reaction are: high-fidelity DNA polymerase, dNTP, buffer; PCR amplifica...
Embodiment 2
[0023] Example 2: Expression of recombinant plasmid pGEX-5x-1-MrNV-CP in E. coli
[0024] The pGEX-5x-1-MrNV-CP recombinant plasmid was transformed into Escherichia coli. The next day, a single colony on the transformed plate was selected and placed in LB liquid medium containing 50g / mL kanamycin at 37°C, 200 revolutions / min. Incubate on a constant temperature shaker. When the OD value of E. coli reaches 0.4-0.6, add IPTG with a final concentration of 1 mmol / L and induce 4-8 hours at 25-37°C. Centrifuge at 6000 rpm for 10 min to collect the bacteria. The bacterial cells were crushed under high pressure, and the broken suspension was centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatant and the precipitate were collected. Use inclusion body purification technology to extract protein from the collected precipitate. The specific steps are as follows:
[0025] (1) Use 20mL buffer A (50mM Tris-HCl, 5mM EDTA, pH 8.0) to fully suspend the pellet, mix well, and centrifuge ...
Embodiment 3
[0030] Example 3: Preparation of Macrobrachium rosenbergii field virus yolk antibody
[0031] 1. Antigen preparation: Fully emulsify the recombinant protein and equal volume of adjuvant (after full emulsification, place the emulsified product in water to see if it is dispersed. If it is not dispersed, it means that the emulsification is sufficient). The first immunization injection is made by GST-MrNV- A vaccine emulsified by CP recombinant protein and an equal volume of complete Freund's adjuvant, and the second and third immunization injections are vaccines emulsified by GST-MrNV-CP recombinant protein and incomplete Freund's adjuvant.
[0032] 2. Immunization: select 10 healthy hens, take 1ml of vaccine and inject 0.25ml at each point on both wings and breast muscles of the hen, and immunize every 2 weeks. The specific immunization procedures are shown in Table 1 below. Start collecting eggs, store them at 4°C for later use, and select the eggs before immunization as the negativ...
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