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Recombinant classical swine fever virus NS3 protein, ELISA kit and application of kit

A technology of swine fever virus and recombinant adenovirus, applied in the field of genetic engineering, can solve the problems of incapable of large-scale production and small purification of NS3 protein

Inactive Publication Date: 2014-10-15
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Another object of the present invention is to provide a method for preparing the recombinant CSFV NS3 protein, aiming to solve the problem that the existing NS3 protein has a small amount of protein purification and cannot be produced on a large scale

Method used

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  • Recombinant classical swine fever virus NS3 protein, ELISA kit and application of kit
  • Recombinant classical swine fever virus NS3 protein, ELISA kit and application of kit
  • Recombinant classical swine fever virus NS3 protein, ELISA kit and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Construction of pET32a-NS3-Intein-ELK16 expression plasmid

[0065] 1. Construction of plasmid pET32a-Intein-ELK16

[0066] First, the Escherichia coli expression vector pTXB1 containing the Mxe Gy rA (Intein in the carrier) intein of the Mycobacterium xenopi gyrA gene was double-digested with EcoR I and Sma I to obtain the Mxe Gy rA intein. The enzyme digestion system was as follows:

[0067]

[0068] Incubate overnight in a constant temperature water bath at 30°C.

[0069] Then send the amino acid sequence to the company to synthesize 16-amino acid short peptide ELK16-(LELELKLK)2 and PT linker, that is, PTlinker-ELK16, and then fill in the end of the intein. The filling system is as follows:

[0070]

[0071]

[0072] After mixing, act at 37°C for 3 hours, then act at 65°C for 5 minutes.

[0073] Connect with PTlinker-ELK16, the enzyme digestion system is as follows:

[0074]

[0075]After mixing, connect overnight at 16°C. That is, the ligat...

Embodiment 2

[0130] 1. Induced expression and purification of NS3 gene in Escherichia coli

[0131] The identified positive recombinant prokaryotic expression plasmid pET32a-NS3-Intein-ELK16 and the empty vector pET32a were respectively transformed into competent cells of the host expression strain Rosetta (DE3), and the transformation steps were the same as before to transform DH5α. Randomly pick a single colony and inoculate into 5mL LB (Amp + , 100μg / mL) liquid medium, at 37°C, 200rpm shaking culture for 12h ~ 16h, and then transfer the original bacterial solution to 10mLLB (Amp + , 100μg / mL) liquid medium, cultured at 37°C with shaking at 200rpm until OD 600 = 0.6 ~ 0.8, take out 2mL of bacterial liquid as the negative control before induction, then add isopropylthio-β-D-galactoside (IPTG) with a final concentration of 1mmol / L, and induce at 16°C and 150rpm After expressing overnight, the bacterial liquid was collected for SDS-PAGE and Western blot analysis.

[0132] According to th...

Embodiment 3

[0150] 1. Establishment of indirect ELISA detection method for CSFV NS3 antibody

[0151] 1.1. Indirect ELISA operating procedure

[0152] (1) Coating: Coat the antigen-NS3 recombinant protein on the ELISA plate with carbonate buffer solution, 100 μL / well, mix well on a shaker, and then put it in a refrigerator at 4°C for overnight coating.

[0153] (2) Plate washing: Wash 3 times with PBST and pat dry.

[0154] (3) Blocking: block with PBS containing 5% skim milk, 200 μL / well, and act at 37° C. for 1 hour.

[0155] (4) Plate washing: Wash 3 times with PBST and pat dry.

[0156] (5) Add the serum to be tested, dilute it with PBSTM (PBST containing 5% skim milk), 100 μL / well, and set a blank control at the same time, and act at 37° C. for 1 hour.

[0157] (6) Plate washing: Wash 5 times with PBST and pat dry.

[0158] (7) Add horseradish peroxidase-labeled rabbit anti-pig secondary antibody (rabbit anti-pig-IgG-HRP), 100 μL / well, and react at 37° C. for 50 minutes.

[0159] ...

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Abstract

The invention provides a recombinant classical swine fever virus (CSFV) NS3 protein, an ELISA kit and the application of the kit. The recombinant CSFV NS3 protein is prepared by the following steps: firstly, cloning the NS3 coding sequence in a complete genome sequence of CSFV into a pET32a-Intein-ELK16 plasmid vector, expressing, then performing inductive cutting, and obtaining an recombinant CSFV NS3 protein by collecting supernatant in a centrifugation manner, wherein the amino acid sequence of the recombinant CSFV NS3 protein is shown in SEQ ID NO: 1; secondly, establishing an ELISA kit based on the recombinant CSFV NS3 protein so as to realize the application of identification of a CSFV vaccine and a recombinant adenovirus vaccine. The recombinant CSFV NS3 protein is high in protein purification efficiency, good in reactivity, low in cost, and simple in operating process, can realize large-scale production, is matched with recombinant adenovirus for use, so as to distinguish antibodies generated by totivirus and a recombinant adenovirus stimulated body, which has an important significance of rapid detection of NS3 antibodies.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant swine fever virus NS3 protein, an ELISA kit and applications thereof. Background technique [0002] The production of recombinant proteins is very important no matter in the industrial field or laboratory scale, and the cost of separation and purification of recombinant proteins accounts for about 60% to 80% of the total cost (references: Chen Hao, Chen Yuhong, Zhu Dexu, Liu Jianning, Recombinant protein purification technology, China Biotechnology Journal, 2002, 22(5):p.87-92.). [0003] Current protein purification methods mainly include traditional ion-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and expression of fusion tags with self-cleavage and self-aggregation functions. Ion exchange chromatography and hydrophobic interaction chromatography have certain requirements on the initial conditio...

Claims

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Application Information

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IPC IPC(8): C07K14/18C12N15/40C12N15/70G01N33/68G01N33/569
CPCC07K14/005C12N15/70C12N2770/24322G01N33/56983G01N2333/183
Inventor 孙永科杨义艾
Owner YUNNAN AGRICULTURAL UNIVERSITY
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