Recombination baculovirus for expressing Africa swine fever CD2V protein in SF9 cell

A technology of recombinant baculovirus and African swine fever virus, which is applied in the direction of double-stranded DNA virus, virus, viral peptide, etc., can solve the problem of vaccine without African swine fever, etc., and achieve the effect of high protein expression and easy purification

Inactive Publication Date: 2019-08-23
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is currently no vaccine against African swine fever. If it can block the spread of the virus in domestic pig

Method used

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  • Recombination baculovirus for expressing Africa swine fever CD2V protein in SF9 cell
  • Recombination baculovirus for expressing Africa swine fever CD2V protein in SF9 cell
  • Recombination baculovirus for expressing Africa swine fever CD2V protein in SF9 cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: Construction of bacmid expressing CD2V gene

[0015] 1.1 Cutting and optimization of CD2V gene

[0016] After analyzing the structural domain of the CD2V gene whose nucleotide sequence is SEQ ID NO: 1 (the amino acid sequence of the encoded protein is SEQ ID NO: 2), the structural domain of the CD2V gene is cut, and the C-terminal 150aa cytoplasmic The internal structure is cut off, which helps the protein to better fold into a tertiary structure and exposes its antigenic site better. At the same time, its codons are optimized; thus, the protein can be expressed more efficiently in sf9 cells. The amino acid sequence after optimization and modification is SEQ ID NO: 3, which can well express the antigenic site. The nucleotide sequence of the optimized gene is SEQ ID NO:4.

[0017] 1.2 Construction of bacmid expressing CD2V gene

[0018] 1.2.1 Enzyme digestion reaction

[0019] 1.2.1.1 Mark the 1.5mL EP tube to be used, and add and mix the sample in the...

Embodiment 2

[0085] Example 2 SF9 cell transfection

[0086] (1) Preparation: UV sterilization in a biosafety cabinet for 30 minutes; TNM-FH culture solution was placed in a 27°C water bath and preheated to 27°C.

[0087] (2) Add 2 μg of recombinant DNA to 100 μl of TNM-FH medium without serum and double antibody, and mix well. Add 9 μl Cellfectin Reagent to 100 μl TNM-FH medium without serum and double antibody, and mix well. The liposomes were mixed with the recombinant DNA and allowed to stand at room temperature for 40 min.

[0088] (3) Take out the 6-well plate cells from the incubator at 27°C, discard the supernatant medium, wash the cells three times with pre-warmed TNM-FH culture medium, and discard the TNM-FH culture medium.

[0089] (4) Add 2 ml of 10% fetal bovine serum TNM-FH culture solution to each cell well.

[0090] (5) Gently add the mixture of recombinant DNA and liposomes into each well of cells, mix gently, and culture statically at 27°C for 5-6 hours.

[0091] (6) ...

Embodiment 3

[0094] Embodiment 3 protein purification and detection

[0095] 4.1 Expression and Identification of Recombinant CD2V Protein in Sf9 Insect Cells Infect insect cells Sf9 with recombinant baculovirus CD2V-P1 and culture at 27°C for 72 hours. Meanwhile, normal insect cells Sf9 without virus infection were cultured at 27°C for 48 hours as a control, and the cells were harvested , and the culture supernatant was frozen for future use. After the cells were washed with PBS at pH 7.4, 1×SDS-PAGE loading buffer [50mM Tris-HCl (pH6.8), 100mM Dithiothreitol (DTT), 2% SDS, 0.05% Bromophemolblue, 10% Glycerol] was added, Boil for 5 minutes, use 12% separating gel and 5% stacking gel for polyacrylamide gel electrophoresis, 100 volts for about 2.5 hours, stain with Coomassie brilliant blue R250, and find that the recombinant baculovirus of the unoptimized CD2V gene has no expression in insect cells , while the optimized CD2V recombinant baculovirus-infected insect cell Sf9 lysate has CD2V ...

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Abstract

The invention provides a recombination baculovirus for expressing Africa swine fever CD2V protein in an SF9 cell. The CD2V protein can be recombined and expressed in the SF9 cell; the amino acid sequence of the CD2V protein is shown in SEQ ID NO:4; the sequence of the nucleotide fragment is shown in SEQ ID NO:3. The recombination baculovirus is used for preparing the Africa swine fever CD2V protein in an insect cell. The insect cell Sf9 is used for recombining and expressing the Africa swine fever CD2V protein, the protein expression amount is high, purification is easy, the recombination baculovirus is used for preparing and identifying a diagnosis product, and a solid foundation is laid for producing Africa swine fever subunit vaccines and diagnosis reagents.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, in particular to a recombinant baculovirus expressing African swine fever CD2V protein in SF9 cells. Background technique [0002] African swine fever (African Swine fever, East African Swine fever, ASF) is an acute, febrile and highly contagious viral disease caused by a viral virus, characterized by a short course of disease, but a mortality rate of up to 100%. , clinical manifestations of fever, skin cyanosis, lymph nodes, kidneys, gastrointestinal mucosal bleeding. The disease was first reported in Kenya in 1909 and has always existed in sub-Saharan African countries. It spread to Western Europe and Latin American countries in 1957, and most of them were extinguished in time. However, there are still cases in Portugal, southwestern Spain and Sardinia in Italy. Popularity. Since 2007, African swine fever has occurred, spread, and spread in many countries around the wor...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N5/10C07K14/18A61K39/187A61P31/20
CPCA61K39/12A61P31/20C07K14/005C12N15/86C12N2710/12022C12N2710/12034
Inventor 郭伟伟向银辉陈俭梅刘大卫范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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