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
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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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