Method for constructing peste des petits ruminant transgenic plant vaccine efficient expression vector

A plant expression vector and expression vector technology are applied in the field of recombinant vector construction and can solve problems such as the lack of effective treatment methods for epidemic diseases

Inactive Publication Date: 2015-04-08
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is currently no effective treatment for this disease, so it is

Method used

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  • Method for constructing peste des petits ruminant transgenic plant vaccine efficient expression vector
  • Method for constructing peste des petits ruminant transgenic plant vaccine efficient expression vector
  • Method for constructing peste des petits ruminant transgenic plant vaccine efficient expression vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Cloning of target gene and target sequence:

[0163] PCR amplification system (25 μL): 10× buffer 2.5 μL, dNTp (2.5 mM) 2 μL, upstream and downstream primers (20 μM) 0.5 μL each, Taq enzyme (5U / μL) 0.3 μL, template DNA 1 μL, ddH 2 O18.2 μL.

[0164] The PCR amplification program is:

[0165] Cloning of MAR sequence: using pMD-MAR plasmid as a template, use primers MAR12-F, MAR12-R and MAR34-F, MAR34-R to clone respectively; PCR amplification conditions: pre-denaturation at 94°C for 5min, denaturation at 94°C for 60s, Anneal at 55°C for 60s, extend at 72°C for 60s, after 35 cycles, extend at 72°C for 5min, and store the reaction product at 4°C.

[0166] GFP gene amplification: using pHL005 as a template, GFP-F and GFP-R as templates; PCR amplification conditions: pre-denaturation at 94°C for 4 minutes, denaturation at 94°C for 30s, annealing at 58°C for 45s, extension at 72°C for 45s, 30 cycles Afterwards, extend at 72°C for 7 minutes, and store the reaction product a...

Embodiment 2

[0171] The PCR product was connected to the pUCM-T vector by T / A cloning method, and the extracted plasmids were named: pGFP, pMAR12, pMAR34, pCsVMV, pF, pFKDEL. And the corresponding enzymes were digested and verified: pGFP (BamH Ⅰ, Xba Ⅰ) double digestion; 4: pCsVMV (Hind Ⅲ, Xba Ⅰ) double digestion; 2: pMAR12 (Hind Ⅲ) single digestion; 3: pMAR34 (Mun Ⅰ, EcoR Ⅰ) double enzyme digestion; 5: pF (Pvu Ⅱ, Bstx Ⅰ) step-by-step double enzyme digestion; 6: pFKDEL (Pvu Ⅱ, Bstx Ⅰ). For enzyme digestion results, see image 3 .

Embodiment 3

[0173] Construction of plant expression vectors:

[0174] 1. The replacement of the promoter is the construction of the expression vector pBI121-CsVMV ( Figure 4 , 5 )

[0175] The promoter CaMV35S on the expression vector pBI121 was replaced with the CsVMV on the cloning vector pCsVMV to improve the expression efficiency of the vector in plants.

[0176] (1) Respectively double-digest plasmid pBI121 and plasmid with restriction endonuclease HindIII and XbaI

[0177] pUCM-T-CsVMV

[0178] The enzyme digestion system was as follows: 10 μL plasmid, 2.5 μL 10×M buffer, 1 μL each of the two endonucleases, 11.5 μL ddH20, and the total reaction volume was 25 μL. Digest at 37°C for 2 hours, run 1% agarose gel electrophoresis for 35 minutes, purify and recover the large fragment of pBI121 with a DNA gel extraction kit, which is designated as fragment A, and recover the small fragment of pUCM-T-CsVMV, designated as fragment B.

[0179] (2) Connection of Fragment A and Fragment B ...

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Abstract

The invention relates to recombinant vector construction in the technical field of genetic engineering, in particular to construction used for recombining an efficient expression vector used for preventing and treating peste des petits ruminants. The expression vector comprises F protein genes of F proteins in PPRV and five kinds of recombinant plasmids connected with the genes, wherein the five recombinant plasmids include the pBI121-F, the 121-C-F-M12-M34, the 121-C-FKDEL-M12-M34, the 121-C-G-F-M12-M34 and the 121-C-G-FKDEL-M12-M34; and the gene sequence can be seen in a sequence table.

Description

technical field [0001] The invention relates to the construction of a recombinant vector in the technical field of genetic engineering, in particular to the construction of a recombinant plant high-efficiency expression vector for preventing Peste des petits ruminants. Background technique [0002] Peste des petits rμminants (PPR) is an acute and contagious disease caused by Peste des Petits Rμminants virμs (PPRV), which mainly infects small ruminants such as goats and sheep. The morbidity and mortality of the disease can be as high as 100% and 50%. There is currently no effective treatment for this disease, so it is necessary to find effective preventive methods to prevent this disease. With the development of biotechnology and genetic engineering technology, the research on PPRV is also more in-depth. The PPRV genome is a single-stranded negative-strand RNA, and six genes, N-P-M-F-H-L, are arranged sequentially from the 3' end to the 5' end of the RNA chain, encoding six...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66
Inventor 杨国锋苏昆龙宋智斌王薇王惠孙娟武海杰谭子恒李永臻王芳
Owner QINGDAO AGRI UNIV
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