ABSTRACT
The NS1 gene of the H5N1 subtype avian influenza virus was amplified by RT-PCR, and the am-plified product was cloned into the eukaryotic expression vector pCMV-Myc, then it was transfected into A549 cells. After 48 h, the expression of NS1 was detected by Western blot. Fluorescence and electron microscopy and flow cytometry showed that the NS1 gene of H5N1 avian influenza virus could induce apop-tosis in human pulmonary carcinoma cell line A549.
Subject(s)
Humans , Annexin A5 , Apoptosis , Cell Line, Tumor , Cloning, Molecular , Influenza A Virus, H5N1 Subtype , Genetics , Virulence , Lung Neoplasms , Pathology , Viral Nonstructural Proteins , Genetics , PhysiologyABSTRACT
Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Subject(s)
Animals , Female , Humans , Escherichia coli , Genetics , Metabolism , Interleukin 1 Receptor Antagonist Protein , Genetics , Pharmacokinetics , Mutagenesis, Site-Directed , Methods , Mutant Proteins , Pharmacokinetics , Recombinant Proteins , Genetics , PharmacokineticsABSTRACT
The single-chain antibody gene (ox26-scFv) to transferrin receptor (TfR) was synthesized and amplified by three-step PCR. After sequencing, the gene was cloned into prokaryotic expression vector pTIG-Trx which carried thioredoxin (Trx) gene and a C-terminal His.tag. The Ox26-scFv proteins achieved 31% yields of total bacteria proteins at 20 degrees C, after 0.02mM IPTG induction using the strain E. coli BL21 (DE3). The soluble scFv proteins in cytoplasm suspension were about 35% and the inclusion bodies were about 65%. The soluble products were purified by immobilized metal chelation affinity chromatography (Ni-NTA), a single band with molecular weight 29 kD appeared on SDS-PAGE gel. Rat GH3 cell immunocytochemistry staining showed that Ox26-scFv protein could recognize and bind to transferrin receptor. Injected SD rats with Ox26-scFv proteins by tail veins, the antibodies were detected from brain tissues specially on the brain capillaries 4 h later which indicate that Ox26-scFv proteins have a good target function to brain capillaries and can permeate the blood-brain barrier mediated by the transferrin receptors.