Isolated nucleic acid molecule and application thereof
A liver-specific expressional regulatory element, including, in order from 51 to 31: a promoter of a dog serpinAl gene or a functional fragment thereof, a promoter of a Xenopus laevis vitellogenin A2 gene or a functional fragment thereof, a promoter of a Xenopus laevis albumin gene or a functional fragment thereof, and a promoter of a human serpinAl gene or a functional fragment thereof.
1 . An isolated nucleic acid molecule, comprising, in a 5′-to-3′ direction, a liver-specific expression regulatory element and a gene of interest operably linked to the liver-specific expression regulatory element,
wherein the liver-specific expression regulatory element comprises, in order in the 5′-to-3′ direction: a promoter derived from the dog serpinAl gene or functional fragment thereof, a promoter derived from the xenopus laevis vitellogenin A2 gene or functional fragment thereof, a promoter derived from the xenopus laevis albumin gene or the functional fragment thereof, and a promoter derived from the human serpinAl gene or functional fragment thereof, wherein, the promoter derived from the dog serpinAl gene or functional fragment thereof comprises the nucleotide sequence of SEQ ID NO: 2;
the promoter derived from the xenopus laevis vitellogenin A 2 gene or functional fragment thereof comprises the nucleotide sequence of SEQ ID NO: 3;
the promoter derived from the xenopus laevis albumin gene or functional fragment thereof comprises the nucleotide sequence of SEQ ID NO: 4; and
the promoter derived from the human serpinAl gene or functional fragment thereof comprises the nucleotide sequence of SEQ ID NO: 5.
2 . The isolated nucleic acid molecule according to claim 1 , further comprising an intron located between the liver-specific expression regulatory element and the gene of interest.
3 . The isolated nucleic acid molecule according to claim 2 , wherein the intron is an SV40 intron or an MVM intron.
4 . The isolated nucleic acid molecule according to claim 1 , further comprising a polyadenylation signal sequence located at a 3′-terminal of the gene of interest.
5 . The isolated nucleic acid molecule according to claim 4 , wherein the polyadenylation signal sequence is a BGH-polyA signal sequence or an SV40 polyA signal sequence.
6 . The isolated nucleic acid molecule according to claim 1 , further comprising a Kozac sequence located between the liver-specific expression regulatory element and the gene of interest.
7 . The isolated nucleic acid molecule according to claim 1 , comprising, in order in the 5′-to-3′ direction: the liver-specific expression regulatory element, an intron, a Kozac sequence, the gene of interest, and a polyadenylation signal sequence.
8 . The isolated nucleic acid molecule according to claim 1 , wherein the gene of interest encodes a protein of interest, and the protein of interest comprises a reporter protein, and/or a therapeutic protein.
9 . The isolated nucleic acid molecule according to claim 8 , wherein the protein of interest comprises sfGFP and firefly luciferase.
10 . The isolated nucleic acid molecule according to claim 1 , further comprising AAV inverted terminal repeats ITRs located at a 5′-terminal of the liver-specific expression regulatory element and at a 3′-terminal of the gene of interest.
11 . The isolated nucleic acid molecule according to claim 10 , wherein the AAV inverted terminal repeat ITR is derived from an AAV serotype which is AAV 2 .
12 . The isolated nucleic acid molecule according to claim 10 , wherein the AAV ITR comprises the nucleotide sequence SEQ ID NO: 12 or SEQ ID NO: 13.
13 . A vector, comprising the isolated nucleic acid molecule of claim 1 .
14 . The vector according to claim 13 , which is a viral vector or a polynucleotide vector.
15 . The vector according to claim 13 , which is a plasmid, a cosmid or a transposon.
16 . The vector according to claim 13 , which is a viral vector, wherein the viral vector comprises an AAV vector.
17 . A method for expressing a gene of interest, comprising introducing the isolated nucleic acid molecule of claim 1 into a host cell, and allowing the gene of interest to be expressed in the host cell.