Reteplase fusion protein：expression, purification and the effect of chaperones on its renaturation / 安徽医科大学学报

Objective To construct the prokaryotic expression system of reteplase fusion protein and research the effect of chaperones on its renaturation. Methods Inserted the reteplase gene into the prokaryotie expression vector PET32a and then expressed it by the induction of IPTG in E. coli BL21. Researched the effect of chaperones on the renaturation of fusion protein by adding different chaperones. Results The analysis of SDS-PAGE and Western blot indicated that reteplase fusion protein was expressed correctly. Chaperones DsbA,pKJE7,pTf16 had the conspicu-ous effect on the renaturation of fusion protein. The result of activity assay indicated that the refolded reteplase fu-sion protein had fibrinolytic activity. Conclusion Chaperones can promote renaturation of reteplase fusion protein.

Research progress of the anti-hepatitis virus mechanism of interferon-alpha and long-acting form of interferons / 中国药理学通报

Interferon-alpha is one of the most important drugs against hepatitis virus,and it can inhibit the replication of virus by inducing the expression of 2'-5'-OAS,PKR,MxA through Jak-STAT pathway in hepatocytes.The effects are also concerned with other signal pathways,such as MAPK,IRS-1/PI3K-p70S6 kinase pathways and so on.In order to prolong the half life of interferon-alpha and enhance its effectiveness,many other long-lasting interferons are developed,such as pegylated interferon,recombinant human serum albumin-interferon,and liposome interferon,which maybe take the place of interferon-alpha.The research and development of more long-acting and efficient interferons are becoming a more and more important way to the anti-virus treatment of hepatitis.

Progress and clinical application of TRAIL-induced apoptosis in cancer / 中国药理学通报

TNF related apoptosis inducing ligand (TRAIL) is a new identified member of TNF family, which selectively kills a broad spectrum of tumor cells, but nontoxic to most normal cells. TRAIL triggers tumor cell apoptosis via death receptor DR4 and DR5 anchored in the cell surface, which is mediated through intracellular induced apoptosis proteins. The major pathway of its action proceeds through the formation of DSIC and activation of caspase8. The apoptotic processes follow two signal pathways, namely the mitochondrial independent activation of caspase3, and mitochondrial dependent apoptosis through the cleavage of BID by caspase8, the release of cytochrome C, the formation of apoptosome, and activation of caspase9 and the downstream apoptosis. In previous researches, it is shown that nontagged TRAIL proved to be noncytotoxic to hepatocytes in monkeys and chimpanzees and to human normal hepatocytes. Thus, the nontagged TRAIL has attracted great attention in recent years as a promising anti cancer reagent.

Cloning expression purification and cytotoxic assay of sTRAIL in A549 cell line / 中国药理学通报

Aim To clone,express sTRAIL gene,then purify sTRAIL(114-281 amino acid) and assay its cytotoxic activity in human A549 cell line.Methods The intact full human TRAIL gene was amplified using PCR method from the human placenta and lung cDNA library.The full human TRAIL cDNA gene was inserted into pUC19 vector and sequenced.The extracellular DNA fragment was amplified using PCR,which was cloned to pET-11a vector and transformed into E.coli BL21.The denatured and refolded sTRAIL was purified and cytotoxic activity of sTRAIL was assayed using crystal violet staining and fluorescence-activated cell sort(FACS) in A549 cell line.Results The full length TRAIL cDNA gene was amplified from the human placenta and lung cDNA library,which was identical to the published TRAIL sequence.The extracellular DNA fragment was cloned to pET-11a.The expression level reached 50% of the total protein of BL21.The purity of sTRAIL was about 98%,while IC_(50) was about(24?5.2) ?g?L~(1) in TRAIL-treated A549 cells with crystal violet staining method.The time-dependent relationship of sTRAIL-induced apoptotic death in A549 cells was significant with FASC analysis.Conclusion sTRAIL gene has been cloned and successfully expressed.The process of refolding and purification of sTRAIL has been established.sTRAIL demonstrated cytotoxicity in A549 cell line.