Claims for Patent: 9,550,983

Summary for Patent: 9,550,983

Title:	Nucleic acid sequence segment for enhancing protein expression
Abstract:	An isolated or a purified nucleic acid sequence for enhancing expression levels of a protein of interest, in 5\' to 3\' direction, comprises: a cytomegalovirus (CMV) promoter; an eEF-1 intron repeat (eEF-1 IR); and a regular sequence, which comprises: at least one tag element, a fixable linker sequence, wherein the fixable sequence is TEV sequence; and a multiple cloning site (MCS). By means of the array of the specific promoter and eEF-1 IR, the expression and purity of the recombinant protein could be enhanced; wherein eEF-1 IR can reduce the length of vector and assist RNA polymerase II transcription. Besides, TEV sequence of the fixable linker sequence of the regular sequence can remove a tag on recombinant protein; a specific target can be inserted into the multiple cloning site.
Inventor(s):	Ko; Ying-Chin (Taichung, TW), Lee; Chi-Pin (Taichung, TW), Chiang; Shang-Lun (Taichung, TW)
Assignee:	China Medical University (Taichung, TW)
Application Number:	14/820,836
Patent Claims:	1. An isolated or a purified nucleic acid sequence for enhancing expression levels of a protein of interest, in 5' to 3' direction, comprising: a cytomegalovirus (CMV) promoter as set forth in SEQ ID NO: 1; an eEF-1 intron repeat (eEF-1 IR) as set forth in SEQ ID NO: 2; and a regular sequence comprising: at least one tag element, a fixable linker sequence, wherein the fixable linker sequence is tobacco etch virus identification sequence (TEV sequence) as set forth in SEQ ID NO: 3; and a multiple cloning site (MCS). 2. The nucleic acid sequence as claimed in claim 1, wherein the at least one tag element is selected from the group consisting of histidine tag (His tag), hemagglutinin tag (HA tag), FLAG tag, green fluorescent protein (GFP), turbo GFP, red fluorescent protein (RFP) and glutathione-S-transferase (GST). 3. The nucleic acid sequence as claimed in claim 1, wherein the at least one tag element consists of a histidine tag and a hemagglutinin tag preceded by the histidine tag. 4. The nucleic acid sequence as claimed in claim 1, wherein the at least one tag element consists of a FLAG tag and a histidine tag preceded by a FLAG tag. 5. The nucleic acid sequence as claimed in claim 1, wherein the at least one tag element is a green fluorescent protein. 6. The nucleic acid sequence as claimed in claim 1, wherein the regular sequence further comprises an enhancing element following the multiple cloning site of the regular sequence. 7. The nucleic acid sequence as claimed in claim 6, wherein the enhancing element consists of an SV40 enhancer and a cytomegalovirus (CMV) enhancer preceded by the SV 40 enhancer. 8. An expression vector comprising: the nucleic acid sequence as claimed in claim 1, comprising: two tag elements consisting of the histidine tag and the hemagglutinin tag preceded by the histidine tag; and, at least one enhancing element following the regular sequence, wherein the at least one enhancing element comprises an SV40 enhancer and a cytomegalovirus (CMV) enhancer preceded by the SV40 enhancer. 9. The expression vector as claimed in claim 8, wherein the expression vector further comprises a selection antibiotic following the enhancer, wherein the selection antibiotic is selected from the group consisting of ampicillin, kanamycin, chloramphenicol, tetracyclin, hygromycin, neomycin or methotrexate. 10. The expression vector as claimed in claim 8, wherein a target gene ALPK1 is inserted into the multiple cloning site by restriction enzyme Asis I and pmer I. 11. An expression vector comprising: the nucleic acid sequence as claimed in claim 1 comprising: two tag elements consisting of a FLAG tag and a histidine tag preceded by the FLAG tag; and at least one enhancing element following the regular sequence, wherein the at least one enhancing element comprises an SV40 enhancer and a cytomegalovirus (CMV) enhancer preceded by the SV40 enhancer. 12. The expression vector as claimed in claim 11, wherein the expression vector further comprises a selection antibiotic following the enhancer, wherein the selection antibiotic is selected from the group consisting of ampicillin, kanamycin, chloramphenicol, tetracyclin, hygromycin, neomycin or methotrexate. 13. The expression vector as claimed in claim 11, wherein a target gene ALPK1 is inserted into the multiple cloning site by restriction enzyme Asis I and pmer I. 14. An expression vector comprising: the nucleic acid sequence as claimed in claim 1; the tag element being green fluorescent protein; and, at least one enhancing element following the regular sequence, wherein the at least one enhancing element comprises an SV40 enhancer and a cytomegalovirus (CMV) enhancer preceded by the SV40 enhancer. 15. The expression vector as claimed in claim 14, wherein the expression vector further comprises a selection antibiotic following the enhancer, and the selection antibiotic is selected from the group consisting of ampicillin, kanamycin, chloramphenicol, tetracyclin, hygromycin, neomycin or methotrexate. 16. The expression vector as claimed in claim 14, wherein a target gene ALPK1 is inserted into the multiple cloning site by restriction enzyme Asis I and pmer I. 17. A kit for detecting protein expression comprising: the vector of claim 8, at least one antibody for detecting the protein expression from the vector, wherein the antibody is selected from the group consisting of anti-histidine tag antibody, anti-hemagglutinin tag antibody, and combination thereof. 18. A kit for detecting protein expression comprising: the vector of claim 11, at least one antibody for detecting the protein expression from the vector, wherein the antibody is selected from the group consisting of anti-histidine tag antibody, anti-FLAG tag antibody, and combination thereof. 19. A kit for detecting protein expression comprising: the vector of claim 14, an antibody for detecting the protein expression from the vector, wherein the antibody is anti-green fluorescent protein antibody.

Details for Patent 9,550,983

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2035-03-27
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2035-03-27
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2035-03-27