Modified Nucleotide Sequence Encoding Glucagon-Like Peptide-1 (GLP-1), Nucleic Acid Construct Comprising Same for Production of Glucagon-Like Peptide-1 (GLP-1), Human Cells Comprising Said Construct and Insulin-Producing Constructs, and Method of Use Thereof
a technology of glucagon-like peptide and nucleotide sequence, which is applied in the field of nucleic acid constructs, can solve the problems of short half life, inability to rapidly degrade, and limited potential as a new therapeutic agent, and achieve the effects of reducing increasing the glucose blood level, and reducing the weight of a subj
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example 1
GLP-1 Constructs
[0117]In order to obtain a biologically active form of secreted GLP-1 from genetically engineered cells, the following strategy is used to generate the GLP-1 nucleic acid constructs. The active GLP-1(7-37) peptide is comprised of 31 residues, accordingly the nucleotide sequence corresponding to these residues is conveniently split in half to generate two separate fragments viz., N-terminal and C-terminal fragments as shown in FIG. 3-B. Complementary oligonucleotides corresponding to these amino acids are synthesized with the modifications listed below. Sequences of the oligonucleotides are listed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
[0118]An XhoI restriction site is added at the split site, without altering the amino acid sequence. A furin cleavage site (RQK′R) and the Kozak sequence and the bases consistent with the overhang of NheI restriction site are added to the 5′ end of the N-terminal fragment as shown in FIGS. 3-B and C. Bases consistent ...
example 2
Assessment of GLP-1 Secreted in Transiently Transfected Cells
[0124]Various human cell lines (293T, HeLa, SHP77, U2OS) are transiently transfected with GLP-1 constructs to assess production of GLP-1 in the culture medium. The transfections are performed as follows.
[0125]Cells are plated at 2.5×105 cells / 60 mm dish in 4.0 ml culture medium. The cells are allowed to adhere overnight. The cells are 40-60% confluent after the overnight culture. The medium is removed from the cells so that there is 1.5 ml remaining in the plate.
[0126]In a sterile microtube 1 μg DNA is added into a total of 150 μl DNA-condensation buffer (EC buffer, Qiagen Effectene transfection kit). Enhancer solution (8 μl l) is added into the DNA / EC buffer, vortexed for 1 second and incubated for 2-5 minutes at room temperature. 25 μl Effectene is added into each tube and vortexed for 10 seconds. The tubes are allowed to sit at room temperature for 5-10 min. The transfection mixture is diluted with 1.0 ml media and adde...
example 3
Engineering of Human U2OS Cells to Produce GLP-1
[0129]Human osteosarcoma cell line U2OS, available from the ATCC, is stably transfected with GLP-1 cDNA using the following protocol. The vector is linearized with BglII. The cells are co-transfected with pCDNA-huFurin expressing human furin. pCDNA-huFurin is linearized using MfeI. U2OS cells are seeded in 10-cm plates the day before at 2.5×105 per plate. The cells are transfected using calcium phosphate (Promega).
[0130]Three days post-transfection, medium is supplemented with 1000 μg / ml Geneticin. Once colonies are evident, cells are expanded in 6-well plates and tested for GLP-1 expression by active GLP-1 ELISA kit (Linco Diagnostic Services, Inc.). Cell populations producing high levels of GLP-1 are further cloned by limiting dilution at 1 cell / well. The clones are tested for their ability to produce GLP-1 in the presence and absence of DPPIV inhibitor.
[0131]The production of active GLP-1 in U2OS cells transiently transfected with n...
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