Vascular endothelial growth factor 2
A technology of nucleic acid molecules and myocardial ischemia, applied in the fields of epidermal growth factor, growth factor/inducer, cardiovascular system diseases, etc.
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Embodiment 1
[0413] Expression patterns of VEGF-2 in human tissues and breast cancer cell lines
[0414] Northern blot analysis was used to detect the expression level of VEGF-2 in human tissues and human breast cancer cells. RNAzol TMTotal RNA samples from cells were isolated by System B (Biotecx Laboratories, Ltd.). Use 1% agarose gel to separate the total amount of about 10 mg of RNA isolated from each breast cancer tissue and specific cell line, and spot it on a nylon filter, [Sambrook et al., Molecular Cloning: A Laboratory Manual, p. Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989)]. Labeling reactions were performed with 50 ng of DNA fragments according to the Stratagene Prime-It kit. Labeled DNA was purified from 5Prime-3Prime using Select-G-50 columns, Ltd. [Boulder, CO]. The filter was hybridized with the full-length VEGF-2 gene labeled with a radioactivity of 1,000,000 cpm / ml in 0.5M NaPO4 and 7% SDS solution overnight at 65°C. Wash twice a...
Embodiment 2
[0417] Expression of a truncated form of VEGF-2 (SED ID NO: 4) by in vitro transcription and translation
[0418] VEGF-2 cDNA was transcribed and translated in vitro to determine the size of the truncated form of VEGF-2 and the translatable polypeptide partially encoded by VEGF-2 cDNA. Two VEGF-2 inserts in the pBluescript SK vector were PCR amplified with three pairs of primers, 1) M13 reverse and forward primers; 2) M13 reverse primer and VEGF primer F4; and 3) M13 reverse primer and VEGF primer F5. These primer sequences are shown below.
[0419] M13 reverse primer: 5'-ATGCTTCCGGCTCGTATG-3' (SEQ ID NO: 9). This sequence is positioned upstream of the 5' end of the VEGF-2 cDNA insert in the pBluescript vector, as the cDNA it is in the antisense start orientation. A T3 promoter sequence was positioned between the primer and the VEGF-2 cDNA. M13-2 forward primer: 5'GGGTTTTTCCCAGTCACGAC-3' (SEQ ID NO: 10). This sequence was positioned downstream of the 3' end of the VEGF-2 ...
Embodiment 3
[0424] Cloning and expression of VEGF-2 using baculovirus expression system
[0425] With reference to ATCC No.97149, use the PCR oligonucleotide primer corresponding to this gene 5' and 3' sequence to amplify the DNA sequence of the VEGF-2c albumen of 46 amino acids without N terminal:
[0426] The 5' primer sequence is TGT AAT ACG ACT CAC TAT AGG GAT CCCGCC ATG GAG GCC ACG GCT TAT GC (SEQ ID NO: 12) and contains a BamH1 endonuclease site (in bold) and VEGF-25' Sequence complementary 17 nucleotide sequence (nt.150-166).
[0427] The 3' primer has the sequence GATC TCT AGA TTA GCT CAT TTG TGGTCT (SEQ ID NO: 13) and contains a restriction enzyme cleavage site XbaI and 18 nucleotides complementary to the VEGF-23' sequence including a stop codon and a stop codon The 15nt sequence.
[0428] Amplified fragments were separated from 1% agarose gels using a commercially available kit ("Geneclean," BIO101, Inc., La Jolla, CA). This fragment was digested with endonucleases BamH1 and ...
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