Expression and purification method of sugarcane yellow leaf virus movement protein and preparation of polyclonal antiserum thereof
A sugarcane yellow leaf virus, expression and purification technology, applied in the field of preparation of polyclonal antiserum, to achieve great application value and market prospects, high accuracy and high sensitivity
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Embodiment 1
[0032] Embodiment 1, construction of recombinant plasmid pDB.His.MBP-ScYLV-MP
[0033] 1. Using the pMDC32-ScYLV-MP cloning plasmid containing the ScYLV-MP gene as a template, the 5'- CATATG TCAGAAGACGCGCTAACCGT-3' (the underline is the recognition site of restriction endonuclease Nde I) and 5'- CTCGAG TAAAGTCTTTTCCCCCTG-3' (the underline is the recognition site of the restriction endonuclease Xho I) was used as a primer for PCR amplification, and a 469bp PCR amplification product was obtained.
[0034] 2. Ligate the PCR amplification product obtained in step 1 with the cloning vector pMD19-T (simple) to obtain the recombinant plasmid pMD19-T-ScYLV-MP.
[0035] 3. Take the recombinant plasmid pMD19-T-ScYLV-MP, digest it with restriction endonucleases NdeI and XhoI, and recover a 450bp digested fragment.
[0036] 4. Take the vector pDB.His.MBP, digest it with restriction endonucleases Nde I and Xho I, and recover the vector skeleton of about 6.5 kb.
[0037] 5. Ligate the res...
Embodiment 2
[0040] Embodiment 2, expression and purification of fusion protein
[0041] 1. The recombinant plasmid pDB.His.MBP-ScYLV-MP was introduced into Escherichia coli Rosseta (consistent with the strain in the material) to obtain a recombinant bacterium, which was named Rosseta-ScYLV-MP.
[0042] 2. After completing step 1, take the single clone of Rosseta-ScYLV-MP, inoculate it into 10 mL of LB liquid medium (containing 50 μg / mL kanamycin), shake it at 37°C and 220 rpm for 12 hours, and obtain a culture solution.
[0043] 3. After completing step 2, take the culture liquid, inoculate it into LB liquid medium (containing 50 μg / mL kanamycin) at a volume ratio of 1:1000, and culture it with shaking at 37°C and 220 rpm until OD 600nm The value is 0.6 to 0.8. Add IPTG at a final concentration of 0.1 mM, induce culture with shaking at 18 °C and 180 rpm for 18 h, centrifuge at 4 °C and 5000 rpm for 6 min, and collect bacterial pellet 1.
[0044] Take the culture solution, inoculate it i...
Embodiment 3
[0050] Embodiment 3, the preparation of polyclonal antiserum
[0051] 1. Take the concentrated protein solution and add physiological saline to dilute to 200-500 μL to obtain a fusion protein solution.
[0052] 2. Mix 1 volume part of the fusion protein solution (containing 400 μg of fusion protein) and 1 volume part of Freund's complete adjuvant, and emulsify to obtain Mixture A. Mix 1 volume part of fusion protein solution (containing 200 μg of fusion protein) and 1 volume part of Freund's incomplete adjuvant, and emulsify to obtain mixture B.
[0053] 3. Take 1 New Zealand white rabbit with a body weight of about 2 kg, and subcutaneously inject (8-10 points) the mixed solution A on the back.
[0054] 4. On the 14th day after completing step 3, subcutaneously inject (8-10 points) the mixture B on the back.
[0055] 5. On the 24th day after completing step 3, subcutaneously inject (8-10 points) the mixture B on the back.
[0056] 6. On the 34th day after completing step 3,...
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