Objective: To construct pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and to investigate the interaction of chemokine receptor 4 (CXCR4) and viral macrophage inflammatory protein-II(vMIP-II) N terminal 21 peptides (NT21MP) in living cells. Methods: DNA fragment encoding NT21MP was chemically synthesized and inserted into BiFC eukaryotic expression vector pBIFC-VC155. The full length of CXCR4 DNA fragment was amplified by RT-PCR from SKBR (3) cells and inserted into BiFC eukaryotic expression plasmid pBIFC-VN173. Two recombinant vectors were identified by restriction enzyme digestion and DNA sequencing. The recombinant vectors were cotransfected into Africa green monkey kidney fibroblast COS-7 cells by using Lipofectamine 2000. The interaction of NT21MP and CXCR4 was detected by bimolecular fluorescence complementation (BiFC) assay. Results: The restriction enzyme digestion and DNA sequences and open read frames of two vectors were consistent with experiment design. The BiFC plasmids were successfully cotransfected into the target cells and expressed. The strong BiFC signals were detected in pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP cotransfected cells and the fluorescence signal was located in the cytoplasm. Conclusion: The eukaryotic expression plasmids for BiFC assay are successfully constructed. The interaction of NT21MP and CXCR4 in living cells can be detected by using this technology.
| 载体基本信息 | |||
| 载体名称 | pBiFC-VN173 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 5216 bp | 载体类型 | Bimolecular fluorescent complementation vectors (B |
| 筛选标记 | VN173 | 载体标签 | Flag |
pBiFC-VN173 载体载体图谱
pBiFC-VN173 载体载体序列
LOCUS Exported 5216 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5216)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5216
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 141..157
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
enhancer 318..697
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 698..901
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 935..958
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/note="FLAG"
/translation="DYKDDDDK"
polyA_signal 1557..2179
/note="hGH poly(A) signal"
/note="human growth hormone polyadenylation signal"
promoter 2208..2537
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2388..2523
/note="SV40 ori"
/note="SV40 origin of replication"
promoter complement(2576..2594)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2608..2624)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2632..2648
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(2656..2686)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 2701..2722
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3010..3598)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3769..4629)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4630..4734)
/gene="bla"
/note="AmpR promoter"
rep_origin 4761..5216
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
