Cloning, Expression, And Characterization Of Soluble Human Vascular Endothelial Cell Growth Inhibitor

Vascular endothelial cell growth inhibitor (VEGI) was first discovered by Tan and his colleagues through the selection of cDNA library of human umbilival vein endothelial cells (HUVEC). The full length of VEGI genome is about 17kb, which consists of exons and three introns. It can produce three kinds of mRNA splicing into three VEGI isoforms consisting of 251 192 and 174 amino acid residues. Each isoform shows endothelial cell-specific expression. The VEGI251 and VEGI174 share a C-terminal 151-residue segment. The protein VEGI174 has been well studied so far. The extracellular domain consisted of 151 amino acids of the C-terminal comprises the soluble VEGI174 (sVEGI). It can inhibit the growth of endothelial cells but not other types of cells examined, and also can inhibit the formation of capillary-like structures by endothelial cells in vitro. In vivo, the sVEGI174 significantly inhibited the growth of colon carcinoma in mice. However, there are few reports on the most abundant isoform, VEGI251.VEGI251 (also named TL1A), the most abundant isoform, belongs to the TNF superfamily and exhibits 24.6% overall sequence homology with the TNF superfamily. The VEGI251 is a type II member protein with a brief intracellular N-terminal segment and most of the protein (residues 72-251) comprising the extracellular domain. The results on biological activities of VEGI251 showed that it is the TNF-like ligand of DR3 and TR6/DcR3. Additionally, it can induce proliferation of T cells and apoptosis of erythroleukemia and it can't inhibit the growth of endothelial cells and formation of capillary-like structures by endothelial cells in vitro. Although the three isoforms share the same C terminal 151-residues segment, the biological activities of them shows significant difference. In this study, we cloned and expressed the extracellular domain of VEGI (amino acid residues 72-251), and investigated the biological functions of the soluble sVEGI174.[Objectives](1) To clone the extracellular domain of VEG 174.251 from human umbilival vein endothelial cells (HUVEC).(2) To express the extracellular domain in E.coli , induce its expression and purify it.(3) To study the biological activity and possible mechanism of VEGI72-251.(4) To compare the biological activity between VEGI72-251 and[Methods](1) The extracellular region of VEGI251 was amplified by RT-PCR. The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E.coli DH5 a by lithium chloride method, screened and identified by restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) VEGI72-251 gene was subcloned into expression vector pBV220 and transformed into E.coli DH5 a . The recombinant vector was screened and identified by enzymatic digestion. The recombinant protein was induced to express by heating, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.(3) The VEGI72-251 protein was purified.(4) The content of LPS in the purified recombinant protein was detected.(5) The biological activity was assayed on tumor cells by microplate photometer.(6) The inhibitory effect of soluble VEGI72-251 on chick embryonic chorioallantic membrane angiogenesis was detected.(7) The contents of IL-2 and IFN in the supernatants of the PBMCs medium were determined.(8) A B16 xenograft tumor model was established. The effect of VEGI72-251 on B16 xenograft tumor model was determined.(9) Compared the biological activities between soluble VEGl72-251 and sVEGI174 and analyzed the possible mechanisms.[Results](1) The extracellular region gene of VEGI72-251 was successfully cloned from HUVEC. The DNA sequences were identical to that recorded in GenBank.(2) The extracellular region of VEGI72-251 was highly expressed in E.coli DH5 a , the expression rate was 25.6%. The molecular weight of...