Recombinant nucleic acid molecule for transcribing circular RNA and application of recombinant nucleic acid molecule in protein expression

A technology for recombining nucleic acids and molecules, applied in the fields of molecular biology and bioengineering, can solve problems such as insufficient protein expression and short expression duration

Active Publication Date: 2021-03-12
SUZHOU CUREMED BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, although linear mRNA can achieve mRNA-mediated protein expression, its expression duration is short and the protein expression is insufficient. Therefore, it is necessary to develop new mRNA technologies with the ability to express proteins persistently

Method used

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  • Recombinant nucleic acid molecule for transcribing circular RNA and application of recombinant nucleic acid molecule in protein expression
  • Recombinant nucleic acid molecule for transcribing circular RNA and application of recombinant nucleic acid molecule in protein expression
  • Recombinant nucleic acid molecule for transcribing circular RNA and application of recombinant nucleic acid molecule in protein expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0263] Example 1: Exploring the expression of circular mRNAs mediated by different IRESs in 293T cells

[0264] 1.1 Experimental method and steps

[0265] (1) Plasmid construction

[0266] To construct the EGFP target gene containing different elements, this step entrusts Suzhou Jinweizhi Biotechnology Co., Ltd. to perform gene synthesis and cloning. The DNA vector for constructing circular RNA used here includes T7 promoter, 5' homology arm, 3' intron, second exon E2, 5' spacer, IRES element, EGFP coding region, downstream spacer region, the 5' intron, the first exon E1, the 3' homology arm, and the enzyme cutting site XbaI that can be used for plasmid linearization. The resulting gene fragment was ligated into pUC57 vector.

[0267] The IRES component information is as follows:

[0268]

[0269] Wherein, the definition of the IRES element V domain can be found in references (Proc Natl Acad Sci U SA. 2009 Jun 9; 106(23):9197-9202.)

[0270] The circular RNA sequences ...

Embodiment 2

[0352] Example 2: Expression of circular mRNA obtained by combining EV29 IRES with different homology arms and spacer sequences in 293T cells

[0353] 2.1 Experimental method and steps

[0354] On the basis of the above example 1, using the same intron and exon elements as in the above example, with EV29IRES (nucleotide sequence shown in SEQ ID NO: 10), a novel 5' homology arm 1 (nucleotide sequence shown in SEQ ID NO: 2), 3' homology arm 1 (nucleotide sequence shown in SEQ ID NO: 17) and 5' spacer 1 sequence (shown in SEQ ID NO: 6 Nucleotide sequence) and 3' spacer 1 sequence (nucleotide sequence shown in SEQ ID NO: 52) as the basic elements of circular mRNA, construct the circular mRNA (Circ- RNA EV29 H1S1).

[0355] Using the same intron and exon elements as the above-mentioned circular mRNA, with EV29 IRES, 5' homology arm 2 (nucleotide sequence shown in SEQ ID NO: 3), 3' homology arm 2 (SEQ ID The nucleotide sequence shown in NO: 18) and the 5' spacer 2 sequence (the n...

Embodiment 3

[0371] Example 3: Circular mRNA encoding novel coronavirus Spike antigen RBD protein realizes protein expression in 293T cells

[0372] 3.1 Experimental method and steps

[0373] On the basis of the above-mentioned Example 2, using EV29 IRES, 5' homology arm 1, 3' homology arm 1 and 5' spacer 1, 3' spacer 1 sequences as the basic elements of circular mRNA to construct a novel Circular mRNA of the RBD domain (receptor binding domain) of the coronavirus Spike antigen. The RBD protein sequence is shown in SEQ ID NO:32, and the DNA sequence encoding RBD is shown in SEQ ID NO:31. DNA synthesis was entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd.

[0374] Finally, the complete DNA fragment containing the T7 promoter, class I PIE element, 5' homology arm 1, 3' homology arm 1, 5' spacer 1, 3' spacer 1, EV29 IRES, RBD coding region was cloned into pUC57 plasmid. Linearize the plasmid DNA, transcribe the linear mRNA in vitro, purify the linear mRNA, circularize the mRNA, purify...

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Abstract

The invention relates to a recombinant nucleic acid molecule for transcribing circular RNA and application of the recombinant nucleic acid molecule in protein expression. Specifically, the invention relates to the recombinant nucleic acid molecule for transcribing circular RNA, a recombinant expression vector, linear RNA, circular RNA, a recombinant host cell, a pharmaceutical composition and a method for preparing protein. The recombinant nucleic acid molecule is transcribed to form circular RNA containing a specific IRES element, the IRES element can improve the protein expression level of the circular RNA in eukaryotic cells, efficient and durable expression of protein is achieved, and the recombinant nucleic acid molecule has important application values for preparing mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines and mRNA-based dendritic cell tumor vaccines, and in the fields of gene therapy based on mRNA, chimeric antigen receptor T cell therapy based on mRNA,protein supplement therapy and the like.

Description

technical field [0001] The disclosure belongs to the technical fields of molecular biology and bioengineering, in particular, the disclosure relates to a recombinant nucleic acid molecule for transcribing circular RNA and its application in protein expression. More specifically, the present disclosure relates to a recombinant nucleic acid molecule transcribing circular RNA, a recombinant expression vector, linear RNA, circular RNA, a recombinant host cell, a pharmaceutical composition, and a method for preparing a protein. Background technique [0002] Messenger Ribonucleic Acid (mRNA) is transcribed from DNA and provides the required genetic information for the next step of protein translation. When the mRNA encoding the antigenic protein is injected into the human body, it can synthesize the antigenic protein in the body, thereby inducing a strong cellular and humoral immune response, and showing the characteristics of an autoimmune adjuvant, which is an excellent vaccine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/85C12N15/11C12N5/10
CPCC12N15/79C12N15/85C07K14/00C12N2840/203C12N2310/532A61K38/00C12N2840/206C12N2770/20022C12N2770/20034Y02A50/30C07K14/005C07K14/4748C07K14/505C07K14/5443C07K14/70535C07K16/2818
Inventor 孙振华左炽健朱佳凤
Owner SUZHOU CUREMED BIOMEDICAL TECH CO LTD
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