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Plasmid vector for expressing humoral immune vaccine mRNA as well as construction method and application thereof

A technology of humoral immunity and plasmid vectors, which is applied in the fields of cell culture, molecular biology techniques, immunobiology and image analysis, and can solve the problems of lack of academic or commercial plasmid vectors

Inactive Publication Date: 2020-09-01
深圳市新合生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, in the field of molecular biology, there is a lack of appropriate academic or commercial plasmid vectors that can express secretory B epitopes in mammalian cells

Method used

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  • Plasmid vector for expressing humoral immune vaccine mRNA as well as construction method and application thereof
  • Plasmid vector for expressing humoral immune vaccine mRNA as well as construction method and application thereof
  • Plasmid vector for expressing humoral immune vaccine mRNA as well as construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] (1) Construction of in vitro transcription expression vector pNeoCura-BVac

[0044] In order to overcome the current lack of vectors for in vitro transcription that can obtain high stability and high translation expression in the mammalian body or in vitro cultured cells expressing the mRNA of the humoral immune vaccine antigenic site (abbreviated as B antigenic epitope) located in the extracellular secretion, it is difficult The shortcomings of large-scale development of B antigen epitope immune response and vaccine screening are solved by replacing the replaceable segment in the pNeoCura-Exp060 vector with a specific segment that assists B antigen expression and subcellular localization.

[0045] Specifically, the present invention uses restriction endonucleases XbaI and XhoI to remove the replacement segment with a length of 30 in the pNeoCura-Exp060 vector, and connects and inserts a segment with XbaI site sticky end, T7 RNA polymerase recognition fragment , 5'-UTR ...

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Abstract

The invention discloses a plasmid vector for expressing humoral immune vaccine mRNA as well as a construction method and application of the plasmid vector. The plasmid vector comprises a 5'-untranslated region of a hemoglobin beta subunit gene (HBB) and an immunoglobulin heavy chain epsilon (IGHE) protein signal peptide sequence which can be arranged at the 5'-end of the front part of a humoral immune vaccine site (B antigen epitope) peptide fragment gene to be inserted and expressed; and a 3'-untranslated region of the HBB gene and a Poly (dA) tail sequence with the length of 60 arranged at the 3'-end of the rear part of the gene. The plasmid vector for in-vitro expression of mRNA can directly make B antigen epitope site inserted into an expression target protein gene, expressed mRNA carries the poly (A) tail sequence with the length of 60, and the expressed mRNA can be directly and efficiently translated into a B antigen epitope capable of being expressed and secreted outside a cellin a mammal body or in-vitro culture cells.

Description

technical field [0001] The invention relates to the technical fields of molecular biology technology, cell culture, immunobiology, image analysis and the like. Specifically, it includes replacing the original replaceable segment in the existing expression plasmid with the T7 RNA polymerase binding site, the key elements required for translation expression, and the key elements required for extracellular secretion localization by enzyme digestion, ligation, etc. Constructed as a plasmid vector pNeoCura-BVac that can express mRNA in mammalian in vivo or in vitro cultured cells; then a specific humoral immune vaccine antigenic site (abbreviated as B epitope) is constructed as a gene fragment and inserted into this vector to constitute an example Plasmid, transform this sample plasmid into an appropriate E. coli strain, extract the plasmid after cultivation, carry out restriction restriction linearization in vitro, and transcribe to obtain sample mRNA; and detect the concentration...

Claims

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Application Information

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IPC IPC(8): C12N15/85
CPCC12N15/85C12N2800/107C12N2830/50
Inventor 潘有东宋麒王奕肖安万季刘刚文颖
Owner 深圳市新合生物医疗科技有限公司