Yeast surface display of prawn white spot syndrome virus VP28 and application
A technology of white spot syndrome and surface display, applied in the field of genetic engineering, to achieve the effect of simple and easy cultivation method, good stability, and convenient mass production
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Embodiment 1
[0038] Embodiment 1: VP28 gene amplification
[0039] The PCR primers were synthesized by Shanghai Bioengineering Co., Ltd., and the following reaction system was established in a PCR tube:
[0040] 10×Buffer 2.5μl; dNTP 2.0μl; WSSVDNA template 1.0μl; 28-a (10pmol / μl) 1.0μl; 28-s (10pmol / μl) 1.0μl; rTaq enzyme (5U / μl) 0.5μl; ddH 2 O 17 μl.
[0041] The PCR reaction program was: denaturation at 94°C for 5min; 35 cycles of 94°C for 40s, 56°C for 40s, and 72°C for 40s; extension at 72°C for 5min. After the reaction, 5 μL of the PCR reaction product was added to 1 μL of a mixture of loading buffer (loading buffer) and Gene finder (1:9), and detected by electrophoresis on a 1.0% agarose gel containing Genefinder. The size of the PCR product is 631bp, and the result identification is shown in the description of the accompanying drawings and the accompanying drawings of the present invention. figure 1 . The amplified fragment was sequenced by Dalian Bao Biology Co., Ltd., and the...
Embodiment 2
[0042] Embodiment 2: Construction of recombinant vector
[0043] The PCR product was double-digested with EcoR I and Xho I, and the vector pYD1 was also double-digested with EcoR I and Xho I. After the results of the enzyme digestion were identified by electrophoresis, they were recovered and purified using the gel recovery kit of Bao Bio-Engineering Co., Ltd. Plasmid pYD1 and VP28 gene fragments, the recovered double enzyme digested and purified products (VP28 and pYD1) were treated with T 4 For DNA Ligase connection, the material ratio of the carrier to the target fragment is 1:8 to 1:10. Add 25 μl of the above ligation product to Top10 competent cells, heat shock in a water bath at 42°C for 45 seconds, and then quickly place it on ice for 1 minute, add a certain amount of LB medium (without Amp) to 1000 μl, and culture on a shaker at 37°C for 1 hours to restore resistance; apply 200 μl of the transformed bacteria solution to the solid LB medium containing Amp (50 μg / ml), a...
Embodiment 3
[0044] Example 3: Transformation of yeast competent cells by VP28-pYD1
[0045] Take 100 μl EBY100 competent cells (1×10 8 Cells / ml) and 5 μl pYD1-VP28 recombinant plasmid (0.1 μg / μl) were mixed in the EP tube, then added lithium acetate transformation solution (1 mmol / L liAc, 40% PEG3350, 1 × TE), shaken for 10 seconds, 30 ℃ water bath for 30 minutes; then add dimethyl sulfoxide, after 42 ℃ water bath for 7 minutes, centrifuge at 14000r / min for 5 seconds, discard the supernatant, and resuspend the cells with 0.5ml TE buffer. Take 100 μl of cell suspension and spread it on YNB (Leu) auxotroph selection plate lacking tryptophan (0.5-0.7% YNB, 1.0-2.0% glucose, 0.005-0.01% leucine, 1.5-2.0% agar) , cultured at 25-30° C. for 24-48 hours, and successfully transformed yeast colonies were obtained. In the same way, the competent cells were transformed with the empty vector pYD1 as a negative control for not expressing the target gene product.
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