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O type foot and mouth disease virus DNA vaccine and its preparing method

A foot-and-mouth disease virus and DNA sequence technology, applied in the direction of recombinant DNA technology, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of incomplete inactivation and escape of the virus

Active Publication Date: 2005-05-18
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the prevention and treatment of foot-and-mouth disease mainly uses the traditional vaccine immunization from live virus. Although this traditional vaccine is effective, in recent years, several outbreaks of foot-and-mouth disease, especially in Europe, may be related to the incomplete inactivation of the virus and the inactivation of the virus from the vaccine. Escaping from the place of production

Method used

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  • O type foot and mouth disease virus DNA vaccine and its preparing method
  • O type foot and mouth disease virus DNA vaccine and its preparing method
  • O type foot and mouth disease virus DNA vaccine and its preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 Construction of the first plasmid pcDNA3.1 / P12X3C

[0083] In order to introduce the XbaI site in the downstream primer as the restriction site for cloning when obtaining the full-length P12X3C gene, and at the same time utilize the stop codon in the XbaI site, an existing XbaI enzyme on the P1 gene fragment must be The cleavage site is mutated. Therefore, using upstream primer A: 5′-ACCTCCRACGGGTGGTACGC-3′ and downstream primer A1: 5′-CACTGCCACTTCAAGTTCTGCGAAG-3′, in which an altered (deactivated) XbaI site was introduced by base substitution in the downstream primer to bring The T vector pT / P1-2A with the P1-2A gene fragment [constructed in this laboratory, the product of connecting the P1-2A gene to pGEM-T Easy VectorSystem (purchased from Promega, Cat No: A3610)] as a template, Part of the P1 gene (fragment I) in which the 3'XbaI site was mutated was amplified by PCR. The reaction system (50 μl) for amplifying Fragment I is: 5 μl of 10× buffer, 4 μl o...

Embodiment 2

[0127] Embodiment 2 Construction of the second plasmid pcDNA3.1 / P12X3C3D

[0128] Using the T vector with 3D gene fragments [pGEM / 3D, which is the product of 3D gene ligation with pGEM-T EasyVector System (purchased from Promega, Cat No: A3610)] as a template, apply the upstream primer C (+) 5' -GG CACTCCGCAGGCGGCAACGGAGTTGG-3' and downstream primer Dg(-)5'-GG ATGCGTCACCGCACACGGCGTTACAA-3′ was amplified by PCR to obtain a 3D gene fragment, and KpnI and XbaI sites were introduced at the 5′ and 3′ ends of the 3D gene fragment, respectively. The reaction system (50 μl) for amplifying the 3D gene of the fragment is: 5 μl of 10× buffer, 4 μl of dNTPs, MgCl 2 3 μl, primer C(+) 0.5 μl, primer Dg(-) 0.5 μl, Taq DNA polymerase 0.5 μl, H 2O 36.5 μl. The reaction conditions are: 94°C for 5min, one cycle; 94°C for 1min, 56°C for 1min, 72°C for 1min30sec, cycle 35 times; finally, 72°C for 10min. A small amount of PCR products were separated by electrophoresis on 1% agarose gel and ...

Embodiment 3

[0187] Embodiment 3 expression of recombinant plasmid transfection animal cell and target gene

[0188] The JM109 Escherichia coli with two kinds of plasmid DNA was propagated in low-salt LB nutrient solution (NaCl 5g / L) containing Zeomycin (25 μg / ml), and extracted according to the operating instructions of Invitrogen’s SNAPMidiPrep Kit (Cat No: K1910-01) Plasmid DNA (concentration: 300 μg / ml), and its OD value was measured to determine the ratio of the transfected BHK-21 cells to the transfection reagent used. Transfect BHK-21 cells on a 35mm plate, dissolve 4 μg of plasmid DNA in 250 μg of DMEM medium (without fetal bovine serum and antibiotics), and then add 8 μL of lipofectamine transfection reagent LipofectaminePlus TM Reagent (purchased from Invitrogen, Cat No: 10964-013), after mixing, place it at room temperature for 20 min, and add 12 μL lipofectamine plus TM Reagent (purchased from Invitrogen, Cat No: 10964-013) was dissolved in 250 μL DMEM culture medium (without...

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Abstract

The present invention discloses type-One foot and mouth disease virus DNA vaccine and its preparation process, and is especially DNA vaccine including foot and mouth disease structure protein P1 gene and non-structure protein 2A, 2B and 3C gene as well as non-essential 3D gene and its preparation process.

Description

technical field [0001] The present invention mainly relates to O-type foot-and-mouth disease virus DNA vaccine and its preparation method, and specifically relates to the use of genetic recombination technology to construct two DNA vaccines containing O-type foot-and-mouth disease virus structural protein genes and a part of non-structural protein genes. The development method and its products ,use. Background technique [0002] DNA vaccines can induce protective cellular and humoral immune responses and have been used in recent years to develop vaccines against many different pathogens. Since currently used antiviral vaccines mainly induce humoral immunity, DNA immunization may be very useful for providing long-lasting protection against viral diseases. The large number of published articles in the field of DNA vaccines is mainly related to viral diseases, which reflects the strong need for novel, safe and effective immunization strategies in antivir...

Claims

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Application Information

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IPC IPC(8): A61K39/135A61K48/00C12N15/42C12N15/63C12N15/79C12N15/86
Inventor 谢庆阁刘在新郭慧琛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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