| In recent years, it is an important breakthrough in endocrinology thatadipose tissue has endocrine function. The adipose tissue itself servesas the site of triglyceride storage and free fatty acid release inresponse to changing energy demands. Adipose tissue also participatesin the regulation of energy homeostasis as an important endocrine organthat secretes a number of biologically active adipokines such as leptin,TNF-α, IL-6, adiponectin(ADPN),and so on.Adiponectin is one such adipokine that has significant physiologicalfunctions. Adiponectin can improve glucose metabolism andlipometabolism, insulin resistance. Adiponectin has been reported to havedirect antiatherosclerotic effects. Adiponectin was demonstrated tostrongly inhibit the expression of adhesion molecules, includingintracellular adhesion molecule, vascular cellular adhesion molecule.Adiponectin can induce weight loss, too. Recent studies suggest thatadiponectin may play a role in the modulation of inflammatory vascularresponse by suppressing macrophage function. So adiponectin has a closerelation with insulin resistance, type 2 diabetes, obesity, andcardiovascular disease.Now some researchers apply many kinds of protein expression systemsto express recombinant adiponectin. In spite of low cost, ProkaryoticExpression System express exogenous proteins in E.coli as inclusion bodyand the process of purification is complicated, ln addition ,Prokaryoticcells can not carry out protein modification after transcription.Eukaryotic expression system is accordingly more valuable. Thebaculovirus-insect cell expression system is extensively applied toexpress exogenous proteins. Through this expression system, recombinantprotein yield is well. As eukaryotic expression system, it can carry out protein modification after transcription. Recombinant protein has highbioactivity and immunogenicity. Baculovirus is safe for people. Thereforwe decide to express recombinant adiponectin in the baculovirus-insectcell expression system.Objectives:1. To clone human adiponectin gene, construct the baculovirus-insectcell expression vector. 2. To transfect cultured Sf9 insect cell line.Obtained recombinant Baculovirus infect Sf9 insect cells to expressrecombinant protein. Identify the expression product.Methods:1. To extract the total RNA from human adipose tissue. We got theadiponectin cDNA with RT-PCR method. Design a pair of primers to amplifythe encoding fragment of human ADPN gene by PCR. Construct recombinantclone vector pMD18-T/ADPN. Using BamHI , EcoRI enzymes to digestplasmids, the encoding fragment of human ADPN gene was ligated intotransfer vector pFastBacl, and recombinant vector pFastBacl-ADPN wasextracted.2. After transformation, recombinant transfer vector pFastBacl-ADPNwas introduced into E. coli DHIOBac Competent cells which included ashuttle vector, Bacmid. By site-specific transposition, ADPN gene wasintegrated into Bacmid, and a recombinant shuttle vector wasconstructed, named Bacmid-iDPN.3. The cultured Sf9 cells were transfected with Bacmid-iDPN, and therecombinant baculovirus was obtained. The expression product in celllysate and culture supernatant was identified by SDS-PAGE, Western Blot.4. Optimize expression condition and express recombinant adiponectinprotein.Results:1. Recombinant transfer vector pFastBacl-ADPN was constructed successfully. ADPN gene was inserted in correct location.2. Recombinant shuttle vector Bacmid-ADPN transfected Sf9cells. Recombinant baculovirus DNA contained ADPN gene by PCRidentification.3. Recombinant protein with itself signal peptide was secreted intoculture medium. The MW of recombinant protein was about 30kD. Western blotproved that expressed protein could combine with ADPN polyclonalantibody.Conclusions:Recombinant baculovirus vector was successfully constructed toexpress solubility and secreted human adiponectin protein in insect cells.The baculovirus-insect cell expression system is efficient and suitablefor adiponectin expression.
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