[Cloning of expression vector for VEGF121 and VEGF165 genes encoding human vascular endothelial growth factor].
OBJECTIVE
To clone and construct the expression vector for human vascular endothelial growth factor (VEGF) genes.


METHODS
Total RNAs were extracted from human lung tissue of a 4-month-old fetus and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) with the amplified products cloned into pMD18-T vector. Sequence analysis was performed before the amplified products were cloned into the expression plasmid pcDNA3.1-, the recombinant of which was verified by endonuclease digestion.


RESULTS
After RT-PCR using a pair of primers (sense -21/7 bp and antisense 554/576 bp), two bands were identified. The band (487 bp) shorter in length was confirmed as VEGF121 (with the full length of VEGF121 being 444 bp) while the longer band (619 bp) was normal VEGF165 (with the full length of VEGF165 being 576 bp). Interestingly, another slightly longer VEGF165 nucleotide sequence was identified by sequencing analysis, which featured an unique 20 bp insertion precisely between exon 3 and exon 4 from the first ATG of human VEGF165 cDNA. The 20 bp insert was identified as the retaining intron 3 terminal nucleotides containing the splicing signal, which caused frame shift mutation in the reading frame and could probably give rise to a short polypeptides consisting of only 97 amino acid residuals due to the early appearance of stop code UAG in the middle of exon 4.


CONCLUSIONS
We have successfully constructed the expression vector for VEGF121 and VEGF165 genes, and a new possible alternative splicing isoform of VEGF is identified in normal fetus whose molecular mechanism and physiological function needs further investigation.
Zhou
Zhong-Jiang
ZJ
Department of Cardiology, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China.
Liu
Yi-Li
YL
Wu
Ping-Sheng
PS
Li
Xiao-Wei
XW
Zha
Dao-Gang
DG
Zhou
Ying
Y
D003001:Cloning, Molecular; D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D005333:Fetus / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors