Prokaryoti Expression Of Human Adiponectin And Its Bioactivity Determination

In recent years,adipose tissue is considered not only as an energy storage but also as a very important endocrine organ.Adipose tissue secretes a number of biologically active adipokines such as leptin,tumor necrosis factor-a(TNF-a),resistin, interleukin-6(IL-6),adiponectin(ADPN) and so on.These hormones participate in the nervous,endocrine and immune regulation,and especially play an important role in the energy metabolism.Adiponectin as one such adipokine has significantly physiological functions.It can improve glucose metabolism,lipid metabolism and insulin resistance. Adiponectin has been reported to have direct effects of antiatherosclerosis through inhibiting the expression of adhesion molecules,including intracellular adhesion molecule and vascular cellular adhesion molecule.Adiponectin could induce weight loss too.Recent studies suggest that adiponectin may play a role in the modulation of inflammatory vascular response by suppressing macrophage function.Therefore adiponectin has a close relation with insulin resistance,type 2 diabetes,obesity and cardiovascular disease.Lately it has been reported that adiponectin may directly or indirectly influence the occurrence and development of tumor.The hypoadiponectinemia was correlated with insulin resistance and type 2 diabetes.We can find the plasma adiponectin level reduced in the condition of CVD, hypertension,and dyslipidemia.So the determination of plasma adiponectin will be a good marker for diagnosis and prognosis of diabetes mellitus,and addition of recombinant adiponectin or increasion of endogenous adiponectin secretion provides a new way for prevention and treatment of disease related to insulin resistance.Till now researchers have applied many kinds of protein expression systems such as prokaryotic and eukaryotic systems to get recombinant adiponectin.Prokaryotic expression system is widely used as the advantages of low cost,simple operation and high output.In our study,the expression vector of pQE-30 Xa we used is one kind of prokaryotic expression system from Qiagen company which has high efficiency in expressing and convenience for purifying.At present there is no domestic reporting about using this vector to get adiponectin protein.Objectives:1.To clone human adiponectin gene,and construct the prokaryotic expression vector-pQE-30 Xa/ADPN.2.To transfect E.coli Ml 5 competent cells,obtain the expression of recombinant adiponection protein,and identify the expression product.3.To optimize the expression conditions,and establish an efficient technological process.Methods:1.To extract the total RNA from human adipose tissue.We got the adiponectin cDNA with RT-PCR method.Design a pair of primers to amplify the encoding fragment of human ADPN gene by PCR.Construct recombinant clone vector pMD18-T/ADPN. Using BamHI,HindⅢenzymes to digest plasmids,the encoding fragment of human ADPN gene was ligated into expression vector pQE-30 Xa,and then recombinant vector pQE-30 Xa/ADPN was extracted.2.The expression vector then was transformed into the M15 competent cells,and protein was expressed induced with IPTG.The protein in the inclusion body was extracted and identified by the methods of SDS-PAGE and Western Blot.The optimum of expression including induce time,temperature and concentration of induce reagent was tested.3.The recombinant protein was largely expressed and purified by His Band Kit in the denatured condition.Protein was refolded and identified by SDS-PAGE.The concentration and bioactivity of the protein were determined.Results:1.Recombinant expression vector pQE-30 Xa/ADPN was successfully constructed. ADPN gene was inserted in correct location.2.Recombinant protein was obtained by the expression of E.coli M15.The optimum of induce time was 6 hours,the temperature was 37℃and concentration IPTG was 0.6mM.3.The protein was fairly purified by His Band Kit.The yield was 330μg/100ml LB and accounted for 24.8%of total protein in E.coli M15[pREP4].The result of MTS experiment showed that the protein had certain bioactivity.Conclusions:Recombinant prokaryotic expression vector pQE-30 Xa/ADPN was successfully constructed to express adiponectin protein with certain bioactivity in E.coli M15.The pQE-30 Xa expression system is efficient and suitable for adiponectin.