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Surface display and application of yeast of white spot syndrome virus (WSSV) VP37

A technology for vitiligo syndrome and VP37, applied in the field of genetic engineering, can solve problems such as unreported, and achieve the effects of good stability, simple and easy cultivation method, and convenient large-scale production.

Inactive Publication Date: 2011-04-20
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Genetically engineered live vector vaccines based on yeast cell surface display technology have not been reported in the study of shrimp vaccines

Method used

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  • Surface display and application of yeast of white spot syndrome virus (WSSV) VP37
  • Surface display and application of yeast of white spot syndrome virus (WSSV) VP37
  • Surface display and application of yeast of white spot syndrome virus (WSSV) VP37

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: VP37 gene amplification

[0038] The PCR primers were synthesized by Shanghai Bioengineering Co., Ltd., and the following reaction system was established in a PCR tube:

[0039] 10×Buffer 2.5μl; dNTP 2.0μl; WSSVDNA template 1.0μl; 37-a (10pmol / μl) 1.0μl; 37-s (10pmol / μl) 1.0μl; rTaq enzyme (5U / μl) 0.5μl; ddH 2 O 17 μl.

[0040] 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 800bp, 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

[0041] Embodiment 2: Construction of recombinant vector

[0042] 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 VP37 gene fragments, the recovered double enzyme digested and purified products (VP37 and pYD1) were treated with T 4 For DNA Ligase connection, the material ratio of the carrier to the target fragment is 1:8-1:10. 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), and ...

Embodiment 3

[0043] Example 3: Transformation of yeast competent cells by VP37-pYD1

[0044] Take 100 μl EBY100 competent cells (1×10 8 Cells / ml) and 5 μl pYD1-VP37 recombinant plasmid (0.1 μg / μl) were mixed in the EP tube, then added lithium acetate conversion 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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Abstract

The invention discloses a preparation method of recombinant yeast for the surface display of white spot syndrome virus (WSSV) protein VP37, comprising the steps of: constructing a fused expression recombinant plasmid by using a surface display carrier pYD1 of the yeast and the gene recombination of the WSSV protein VP37, and transforming the brewing yeast EBY100 to obtain the recombinant yeast for the surface display of the VP37. The preparation method is characterized in that the recombinant yeast EBY100 containing the WSSV protein VP37 gene has VP37 on the surface. The invention also provides the application of a recombinant yeast live vaccine for the surface display of the WSSV VP37 in the cultivation of crustaceans. The prepared recombinant yeast can be used as an oral vaccine for immunizing the shrimps and protecting the shrimps against the WSSV. The genetic engineering live vaccine in the invention has the advantages that the brewing yeast is safe, and the cultivation method is simple and easy, and convenient for mass production. Relative to other vaccines, the displayed VP37 protein is easy to be identified by an immune system and has good stability.

Description

technical field [0001] The present invention relates to a genetic engineering technology, specifically, the present invention relates to a yeast surface display method of shrimp white spot syndrome virus structural protein VP37, and the present invention also relates to a production method and application of the displayed shrimp white spot syndrome virus VP37. Background technique [0002] Shrimp white spot rod virus (WSSV) is one of the main virus origins that have harmed artificially cultured prawns in my country and the Asia-Pacific region in recent years. It can infect most types of prawns with high lethality, and can also infect freshwater and marine A variety of crustaceans such as crabs, lobsters, amphipods, and water flies in the ecosystem have a wide range of hosts, causing serious economic losses to the aquaculture industry. Therefore, the development of drugs against shrimp white spot syndrome virus has received extensive attention. [0003] Shrimp is an invertebr...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81A61K39/12A61P31/20C12N15/70C12N1/21C12R1/93C12R1/865C12R1/19
Inventor 刘庆慧黄捷李新新张秀丽
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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