Carrier for in vitro transcription mRNA, and construction method of carrier, method for obtaining mRNA by carrier transcription and application of carrier

A technology of in vitro transcription and construction methods, applied in the direction of using vectors to introduce foreign genetic material, vectors, recombinant DNA technology, etc., which can solve the problems of reduced transcriptional activity

Inactive Publication Date: 2020-02-07
青岛宁逸生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a considerable portion of the m7GpppG analogs are incorporated into the reverse structure of the mRNA and are not recognized by the transcription-translation machinery, resulting in a decrease in transcriptional activity

Method used

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  • Carrier for in vitro transcription mRNA, and construction method of carrier, method for obtaining mRNA by carrier transcription and application of carrier
  • Carrier for in vitro transcription mRNA, and construction method of carrier, method for obtaining mRNA by carrier transcription and application of carrier
  • Carrier for in vitro transcription mRNA, and construction method of carrier, method for obtaining mRNA by carrier transcription and application of carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Construction of plasmid vectors for in vitro transcription of mRNA by molecular cloning

[0098] 1 method

[0099] 1.1 Synthesis of target fragments

[0100] The 5'UTR plasmid, EGFP plasmid, 3'UTR plasmid and polyA plasmid with restriction sites were synthesized by BGI.

[0101] 1.2 Digestion of the target fragment

[0102] Table 1 5'UTR plasmid digestion system

[0103] Element concentration Volume (μl) CutsmartBuffer 10× 2 wxya 1 BamHI-HF 1 5'UTR plasmid template 200ng / μl 10 water 6 total capacity 20

[0104] Digestion conditions: 37°C for 4 hours. 1% agarose gel electrophoresis (120V, 30min), separate the 250bp (5'UTR sequence) fragment after digestion, and perform gel recovery and purification.

[0105] Table 2 Enzyme digestion system of GFP plasmid

[0106] Element concentration Volume (μl) Cutsmart Buffer 10× 2 BamHI-HF 1 AscI 1 EGFP plasmid template 20...

Embodiment 2

[0163] In vitro transcription of mRNA and expression

[0164] 2.1 Preparation of templates for in vitro transcription

[0165] Polymerase chain reaction (PCR)

[0166] Use PfuPCRMasterMix (KP201) to PCR amplify the target fragment, the system is as follows:

[0167] Table 7 Amplification system

[0168] PCR system volume 2×Pfu PCR MasterMix 10 Upstream primer M13F (10uM) 1 Downstream primer M13R (10uM) 1 water 7 Vector for in vitro transcription of mRNA (50ng) 1 total capacity 20

[0169] Amplification conditions: 94°C for 5min; 30 cycles of 94°C for 30s, 57°C for 30s, 72°C for 2min; 72°C for 10min.

[0170] 2.2 Agarose gel electrophoresis

[0171] 1) 1% agarose gel: Weigh 1g of agarose, add 100ml 1×TAE, heat in a microwave until the agarose is completely melted, add 6ul Gel-Red, pour into a mold and cool for later use.

[0172] 2) After the PCR reaction by agarose gel electrophoresis, add 4 μl of 6× loading buffer to th...

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Abstract

The invention provides a carrier for in vitro transcription mRNA, and a construction method of the carrier, a method for obtaining mRNA by carrier transcription, and belongs to the technical fields ofmolecular cloning and in vitro transcription. A 5' UTR sequence, a target gene sequence, a 3' UTR sequence and a polyA sequence are connected to the carrier sequentially, and the carrier for in vitrotranscription of the mRNA is obtained. The carrier provided by the invention contains the 5' UTR sequence, the target gene sequence, the 3' UTR sequence and the polyA sequence, so that in vitro transcription of the mRNA can be effectively performed, addition of cap structure analogs is avoided, and the 3' UTR sequence can prolong the half life of translation protein, and the translation efficiency can be improved.

Description

technical field [0001] The invention belongs to the technical field of molecular cloning and in vitro transcription, and in particular relates to a vector for transcribing mRNA in vitro, a construction method thereof, a method for obtaining mRNA by transcribing the vector, and an application thereof. Background technique [0002] Vaccines are an important means to prevent and control the occurrence and spread of infectious diseases. Classical vaccines include inactivated vaccines and live attenuated vaccines, but they have certain limitations in terms of effectiveness and speed of development. [0003] After the 1970s, people began to use genetic engineering technology to produce vaccines. In 1990, Wolff first discovered that mice were injected intramuscularly with plasmid DNA, and the plasmid and the genes it carried could be taken up and expressed by cells. At that time, limited to the low stability of mRNA, researches mostly focused on plasmid DNA and viral DNA. But mR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/67C12N15/66
CPCC12N15/63C12N15/66C12N15/67C12N2830/50
Inventor 王芳
Owner 青岛宁逸生物科技有限公司
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