Construction Of Human β2-glycoprotein-Ⅰ Domain Ⅰ Recombinant Plasmid And PSilencer3.1-H1/GFP Recombinant Plasmid

Human Beta2-glycoprotein I (β2GPⅠ), a plasma glycoprotein with amolecular mass of 50KD, is also known as apolipoprotein H (apoH) because thisprotein is also involved in lipid metabolism. The site of synthesis of β2GPⅠ ismain Liver. The plasma concentration of β2GPⅠ ranges from 10mg to 20 mgper 100ml. The complete amino acid sequence of the protein was determined bypeptide sequencing. Sequencing analysis showed the-protein is composed of 326amino acid. β2GPⅠ consists of five highly conserved subunits called sushidomain or complement control protein repeats, which are designated shortconsensus repeats. The fifth domain of β2GPⅠ has a unique structure andcontain a stretch of positively charged amino acids that is the majorphospholipid-binding site that mediates the binding to anionic phospholipids.There is a high structural homology among the human, bovine and mouse β2GPI molecules. Although pathophysiolgical role of β2GPI has not clearly beenelucidated, the protein is known to bind to negatively charged substances, such asphospholipids, lipoproteins and activated platelets. It has been known that β2GPI is involved in Lipid metabolism and significantly enchance the triglycerideclearance rate. β 2GP Ⅰ inhibits the intrinsic blood coagulation pathway,prothrombinase activity, and ADP-dependent platelets aggregation in vitro, and isa natural anticoagulant. β2GPⅠprobably inhibits the procoagulant role ofapoptotic cells, is involved in clearance of apoptotic cells and senescent cells, andthe immune clearance of "non-self" particles. The antiphospholipid antibody syndrome (APS), characterized by vascularthrombosis or recurrent pregnancy losses, is an autoimmune disorder whichinvolves multiple systems. Laboratory evidence for APA could be detected in thepatients. APA are a family of autoantibodies with heterogenicity that targetnegative phospholipid and protein cofactors. Many researchers focus on β2GPⅠsince it was thought to be an autoimmue antigen in 1990. The association ofplasma with autoimmune disease was extensively studied. High titers of anti-β2GPⅠ antibodies and β2GPⅠ-specific T lymphocytes have been found to beassociated with the APS of thrombotic events and/or recurrent fetal loss.Controversial results have been reported regarding the aPL-specific epitopesof β2GPI that are crucial in the development of APS. Many groups focused on itand reports about domain Ⅰ,Ⅳ,Ⅴ have been shown. It seems that most aPLantibodies recognize domain I of ?2GPI. So we postulated that β2GPⅠ-DⅠcould bind to pathogenic anti-β2GPⅠ .In this study , we obtained the cDNA of the first domain of hβ2GPI(hβ2GPI-DI), according to the isocaudarner character, constructing theconcatemer cDNA of hβ2GPI-DI (hβ2GPⅠ-DI2) .Inserting the cDNA of hβ2GPⅠ-DI and hβ2GPⅠ-DI2 into PQE30 prokaryotic expression vector, whichexpressed interest protein induced by IPTG. These recombinant protein werepurified and refolded by metal immobilization affinity chromatography, that is,one-step refold and purification. The biologic activities of recombinant andpurification protein were identified by CL-ELISA and Western blot.The proteins of hβ2GPⅠ-DI and hβ2GPⅠ-DI2 were highly effectiveexpressed by pQE30 expression vectors, and formed inclusion bodies. Theeffective of washing the inclusion bodies with 2% deoxycholic acid was very well.The highest level expression of hβ2GPⅠ was induced with 1mmol/L IPTG for5 hours in 37℃. Finally, the protein was purified and refolded by Ni-column andhas biologic function known well . through Western blot and ELISA assays,weidentified that proteins of hβ2GPⅠ-DI and hβ2GPⅠ-DI2 can specificly bind tomouse anti-hβ2GPⅠmonoclonal antibody the same as hβ2GPⅠ protein, thetwo recombination proteins have antigen activity of hβ2GPⅠ protein, and theactivity of they binding to the anti-hβ2GPⅠin APS patients serum have nosignificant difference to hβ2GPⅠ protein, which show that hβ2GPI specificantigen epi-position probably reside in its first domain. The study established afoundation for researching hβ2GPI specific antigen epi-position deeply, havingimportant meaning for studying the etiopathogenesis and treatment method ofautoimmune disease.