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Preparation method for bluetongue virus core sample grain

A technology of bluetongue virus and core-like particles, which is applied in the fields of molecular biology and biology, can solve the problems of baculovirus pollution of public health and safety, the small number of virus core-like particles, and the inability to use large-scale production, so as to facilitate industrialization Effects of production application, uniformity of virus and core-like particles, and low production cost

Active Publication Date: 2019-08-02
SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, attenuated vaccines and inactivated vaccines for bluetongue have been developed in my country, but their protective effects are not good and there are some disadvantages. Therefore, researchers have shifted their attention to the development of new genetically engineered vaccines, mainly focusing on bluetongue virus. In the study of bluetongue virus-like particles, bluetongue virus core-like particles were obtained by co-expressing bluetongue virus VP3 and VP7 proteins in sf9 cells with recombinant baculovirus
However, this method cannot be used for large-scale production, and there is also the risk of baculovirus contamination causing public health safety hazards
In the prior art, some prokaryotic expression systems are used to express VP2-VP7, etc., and then mix and assemble virus core-like particles, but the actual process of this method is low in efficiency, cumbersome to operate, and the number of virus core-like particles produced is small, and the particles Poor uniformity, relatively low practical application value
[0004] At present, there are few reports at home and abroad to obtain bluetongue virus core-like particles through the co-expression of prokaryotic expression systems. Based on this, the present invention is proposed

Method used

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  • Preparation method for bluetongue virus core sample grain
  • Preparation method for bluetongue virus core sample grain
  • Preparation method for bluetongue virus core sample grain

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0053] Example 1. Sequence optimization of target gene and construction of pETDuet-VP7 plasmid

[0054] (1) Considering that the natural genes of VP3 and VP7 of bluetongue virus are difficult to co-express in the prokaryotic system, this application analyzed and optimized the sequences of VP3 and VP7, designed a series of optimized sequences through single factor experiments, and established the recombinant expression pre-tests Optimized sequence suitable for this application. Specifically, the optimized sequence of VP3 is shown in SEQ ID NO.1, and the optimized sequence of VP7 is shown in SEQ ID NO.2.

[0055] (2) According to the optimized VP7 sequence, refer to SEQ ID NO.2, synthesize the entire open reading frame of BTV-VP7 (introduce NdeI restriction site at the 5' end of VP7 sequence, and introduce AvrII restriction site at the 3' end), Thus obtained recombinant plasmid pUC-VP7.

[0056] (3) Plasmid pUC-VP7 and vector pETDuet-1 were digested with restriction endonuclea...

example 2

[0064] Construction of example 2.pETDuet-VP3-VP7 plasmid

[0065] (1) According to the optimized VP3 sequence, see SEQ ID NO.1, synthesize the entire open reading frame of BTV-VP3 (introduce a NotI restriction site at the 5' end of the VP3 sequence, and introduce an NcoI restriction site at the 3' end), Thus obtained recombinant plasmid pUC-VP3.

[0066] (2) Plasmid pUC-VP3 and plasmid pETDue-VP7 were double-digested with restriction enzymes NotI and NcoI, respectively, to obtain VP3 and pETDuet-VP7 with cohesive ends. The enzyme digestion system is as follows:

[0067]

[0068] 37°C, react for 1.5h. The digested product was subjected to 1% agarose gel electrophoresis, and was recovered and purified using the agarose gel DNA purification kit from Takara Company.

[0069] (3) Ligation Use T4 DNA ligase to ligate VP3 with cohesive ends and pETDuet-VP7, the molar ratio of VP3 and pETDuet-VP7 is about 5:1, and connect at 16°C for 3 hours. The connection system is as follows...

example 3

[0073] Example 3. Obtaining of expression bacteria containing recombinant expression plasmid pETDuet-VP3-VP7

[0074] (1) Transformation The recombinant expression plasmid pETDuet-VP3-VP7 was transformed into expressive Escherichia coli competent cells BL21(DE3) by means of heat shock transformation. Take 50ul competent cells BL21(DE3) and melt on ice, add 10ng recombinant expression plasmid pETDuet-VP3-VP7. Place on ice for 30min. Subsequently, heat shock was performed in a water bath at 42°C for 1 min, and then placed on ice for 2 min. Add 500 μL of SOC medium, and culture with shaking at 37° C. for 1 h (160-225 rpm). Appropriate amount was spread on LB plates containing Amp, and cultured overnight at 37°C.

[0075] (2) Identification of positive bacteria The positive bacteria were identified by colony PCR, that is, the expression bacteria containing the recombinant expression plasmid pETDuet-VP3-VP7 were obtained.

[0076] Colony PCR was carried out with VP7-specific pr...

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Abstract

The invention discloses a preparation method for a bluetongue virus core sample grain and escherichia coli for simultaneously expressing bluetongue virus vp3 and vp7 proteins. The method comprises thefollowing steps: inserting bluetongue virus vp3 and vp7 genes into a dual-expression vector pETDuet-1, thereby constructing a pronucleus dual-expression plasmid pETDuet-VP3-VP7; converting into expression type escherichia coli BL21(DE3), thereby acquiring an expression bacterium containing the pronucleus dual-expression plasmid pETDuet-VP3-VP7; performing ultrasonic breaking and sucrose gradientcentrifugation after self-induced expression, thereby acquiring the bluetongue virus core sample grain. According to the invention, high-volume high-purity expression for the bluetongue virus core sample grain with excellent immunological competence is realized in the manner of prokaryotic expression.

Description

technical field [0001] The application relates to the fields of molecular biology and biotechnology, in particular to a prokaryotic expression system for simultaneously expressing bluetongue virus VP3 and VP7 proteins. Background technique [0002] Bluetongue (Bluetongue, BT) is a non-contact viral infectious disease of ruminants transmitted mainly by Culicoides, caused by Bluetongue virus (BTV) of the family Reoviridae and Orbiviridae. Bluetongue has been recognized by the World Organization for Animal Health (OIE) as one of the most important diseases affecting ruminants. Bluetongue was discovered in South Africa as early as the 19th century and has spread to tropical, subtropical and temperate regions. In 1979, after the occurrence of bluetongue disease was identified in Yunnan Province for the first time in China, the disease broke out in Hubei Province, Anhui Province, Sichuan Province and Shanxi Province one after another. It has been confirmed that there are 26 sero...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12N15/66C07K14/005C12R1/19
CPCC07K14/005C12N15/66C12N15/70C12N2720/12123
Inventor 黄超华曹琛福花群义史卫军杨俊兴林彦星阮周曦曾少灵王潇
Owner SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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