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Construction of carriers for prokaryotic expression and eukaryotic expression of candidate gene of porcine rota virus vaccines

A porcine rotavirus, prokaryotic expression technology, applied in the direction of virus antigen components, antiviral agents, microbial-based methods, etc., can solve the problems of rotavirus nano-vaccine development without reports

Inactive Publication Date: 2011-05-04
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, many scholars at home and abroad have carried out in-depth research on new rotavirus vaccines and made new progress, but the development of rotavirus nano-vaccine has not been reported yet.

Method used

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  • Construction of carriers for prokaryotic expression and eukaryotic expression of candidate gene of porcine rota virus vaccines
  • Construction of carriers for prokaryotic expression and eukaryotic expression of candidate gene of porcine rota virus vaccines
  • Construction of carriers for prokaryotic expression and eukaryotic expression of candidate gene of porcine rota virus vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cloning and prokaryotic expression of porcine rotavirus VP4 gene of embodiment 1

[0045] 1. Materials

[0046] 1.1 Disease material, strain and plasmid

[0047] Disease material: PRV lung tissue, preserved in our laboratory.

[0048] Strains: competent cells TOP10, BL21(DE3) (geneplow Ltd).

[0049] Plasmid: The plasmid vector pMD 18-T Vector was purchased from Treasure Bioengineering (Dalian) Co., Ltd., and the prokaryotic expression vector pGEX-4T-1 plasmid was donated by the Animal Genetics Laboratory of Shandong Agricultural University.

[0050] 1.2 Enzymes and main reagents

[0051] Enzymes: AMV reverse transcriptase and rTaq DNA polymerase were purchased from Bao Biological Engineering (Dalian) Co., Ltd.; restriction endonucleases BamHI and Xhol were purchased from NEB Company.

[0052] Main reagents: TRNZOL-A+ was purchased from Tiangen Company; RNase inhibitor RNase, DNAMarker DL2000, DNA Marker DL10000, small amount of gel recovery kit (Agarose GelDNA Puri...

Embodiment 2

[0202] Example 2 Construction and transfection of rotavirus gene and insect cell sf9 expression vector PFastbacl recombinant vector

[0203] 1. Materials

[0204] 1.1 Disease material, strain and plasmid

[0205] Disease material: porcine lung tissue (PRV)

[0206] Bacterial strain: with embodiment 1

[0207] Cells: Insect cells sf9 were maintained by our laboratory (Invitrogen).

[0208] Plasmid: The insect expression vector PFastbacl is preserved by our laboratory, and the rest are the same as in Example 1.

[0209] 1.2 Enzymes and main reagents

[0210] Enzyme: KOD-Plus enzyme was purchased from Shuomeng Bio, and the rest were the same as in Example 1.

[0211] Main reagents: with embodiment 1.

[0212] 1.3 Medium and reagent preparation

[0213] SFX-insect medium was purchased from Dalian Bao Biological Company. All the other are with embodiment 1.

[0214] 1.4 Main instruments and equipment

[0215] With embodiment 1.

[0216] 2. Method

[0217] 2.1 Extraction...

Embodiment 3

[0399] Example 3 Construction of Rotavirus Gene and Yeast Cell GS115 Expression Vector PPIC9K Recombinant Vector

[0400] 1. Materials

[0401] 1.1 Disease material, strain and plasmid

[0402] Disease data: PRRSV lung group

[0403] Bacterial strain: with embodiment 1

[0404] Cell: Yeast GS115 preserved by our laboratory

[0405] Plasmid: yeast expression vector PPIC9K (invitrogen), and the rest are the same as in Example 1.

[0406] 1.2 Enzymes and main reagents

[0407] Enzyme: KOD Plus enzyme was purchased from Shuomeng Bio, and the rest were the same as in Example 1.

[0408] Main reagents: with embodiment 1.

[0409] 1.3 Medium and reagent preparation

[0410] With embodiment 1.

[0411] 1.4 Main instruments and equipment

[0412] With embodiment 1.

[0413] 2. Method

[0414] 2.1 Extraction of RNA in PRV lung tissue

[0415] With embodiment 1.

[0416] 2.2 PRV-VP4, VP7 gene RT-RCR amplification

[0417] 2.2.1 Design and synthesis of primers

[0418] Desi...

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Abstract

The invention discloses a construction method for carriers for prokaryotic expression and eukaryotic expression of candidate gene of porcine rotavirus vaccines VP4 and VP7, comprising: cloning and prokaryotic expression of genes of porcine rotavirus VP4, construction and transfection of the recombinant vector of genes of rotavirus VP4 and VP7 and the expression carriers of insect cell sf9, and the construction of a recombinant vector of genes of rotavirus VP4 and VP7 and the expression vector of yeast cells GS115.

Description

technical field [0001] The invention relates to the field of biological genes, in particular to the prokaryotic expression of porcine rotavirus VP4 and VP7 vaccine candidate genes and the construction of eukaryotic expression vectors. Background technique [0002] Porcine rotavirus (Porcine Rotavirus, PRV) is a member of the genus Rotavirus in the Viroviridae family and is one of the main pathogens causing viral diarrhea in piglets. [0003] PRV mainly exists in the digestive tract of diseased pigs and infected pigs. After being excreted into the external environment with feces, PRV pollutes feed, clean water, bedding and soil, etc., and infects susceptible pigs through the digestive tract. The detoxification time can last for several days, which seriously pollutes the environment. In addition, the virus has a strong resistance to the external environment, so that rotavirus (Rotavirus, RV) is repeatedly infected among pigs, middle pigs and piglets, and takes root in pig farm...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/85C12N15/81C12P21/02A61K39/15A61P31/14C12R1/84C12R1/19
Inventor 易建中肖长峰
Owner SHANGHAI ACAD OF AGRI SCI
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