ABSTRACT
Atherosclerosis is a chronic inflammatory disease of the vascular walls. As the essential mediators of inflammation, cytokines play important roles in atherosclerosis. Through widespread and penetrating studies, many cytokines, such as TNF-, IL-1, IL-6 and IFN-, are found involved in the development of atherosclerosis, as well as the related signal transduction pathways such as the NF- B pathway, the JNK / AP-1 pathway, and the JAK / STAT pathway. This implies that cytokines have a favorable perspective of clinical application. A few cytokines are identified as biomarkers, which help the early diagnosis of the disease and estimation of the risk of the clinical vascular events. Meanwhile, great progress has been made on the cytokine-targeted treatment of atherosclerosis, providing a novel therapy in the treatment of the disease.
ABSTRACT
AIM: To construct prokaryotic expression vector of His-tagged human IP-10 for further study of its biological function in the inflammatory response. METHODS: The coding sequence of IP-10 lacking signal peptide was amplified from human lung cDNA library by polymerase chain reaction (PCR) and the fragment was cloned into pET-14b plasmid for the construction of His-tagged fusion protein expressing vector, pET-14b/IP-10. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli, BL21 (DE_3). The expression of His-tagged fusion protein was induced with IPTG and purified with Ni+-NTA affinity chromatography. Then the chemotactic activity of IP-10 was determined by transwell migration assay on THP-1 cells. RESULTS: The construction of pET-14b/IP-10 recombinant vector was proved by enzyme digestion and sequencing. The fusion protein IP-10, which was purified by a routine Ni+ affinity method, had an activity on the induction of cell migration of THP-1. CONCLUSION: We successfully construct IP-10 fusion protein expressing vector and get the fusion protein with high bioactivity, which provides essential materials for the future studies on IP-10.
ABSTRACT
Objective To clone the 5′ non-coding region (NCR) of human interferon-?-inducible protein 10(IP-10), and to identify the transcriptional activity of IP-10 promoter induced by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). Methods Genomic DNA of lymphocytes was isolated from the human blood. With above DNA as the template, the 5'NCR of human IP-10 was amplified by nest polymerase chain reaction (PCR) method. Then, the IP-10 promoter was cloned into luciferase reporter vector, pGL3. The recombined vector was transfected into HUVEC, and then the activity of the luciferase was determined after the cells were stimulated by LPS. Results Human IP-10 promoter was obtained and the pGL3/IP-10 was successfully constructed. Moreover, the activity of luciferase driven by human IP-10 promoter was observed to obviously increase in the HUVEC stimulated by LPS. Conclusion We successfully cloned human IP-10 promoter, constructed luciferase reporter vector driven by the human IP-10 promoter, and confirmed that high transcriptional activity of human IP-10 promoter was induced by LPS in HUVEC. The results supplied an experimental base for the further study of the transcriptional regulation of human IP-10.