Recombination And Expression Of Huamn Smith D1 Antigen And Establishing A Method For Detecting Its Antibody

SLE is a heterogeneous,idiopathic autoimmune disorder unified by the presence of a host of autoantibodies, many of which recognize antigens from the cell nucleus and cell membrane phospholipid components. Among the different autoantigenic candidates that are recognized by autoantibodies in SLE, there are only two nuclear antigens that are considered pathognomonic of SLE: double-stranded DNA (dsDNA) and the Sm antigens of the U-1 small nuclear ribonucleoprotein complex. Antibodies to these autoantigens are sufficiently discriminating to be part of the American College of Rheumatology (ACR) classification criteria for SLE.Although anti-dsDNA antibodies play an important clinical significance in diagnosing of SLE and estimating the activity of SLE,SLE patients may be low positive for anti-dsDNA antibodies after curing.Even though anti-Sm antibodies are not correlate with the activity of SLE, SLE patients may keep positive for anti-Sm antibodies after curing.So anti-Sm antibodies are known as retrospective antibodies and are characteristic hallmarks of SLE.Among the six core proteins B/B',D,E,F,G and N,which are the Sm antigens, only the B/B'and D proteins can react with anti-Sm antibodies.The specificity of anti-B/B'antibodies for SLE is lower than anti-Sm D antibodies,because anti- B/B'antibodies can cross-react with anti-RNP antibodies. The predominant immuno- reactive protein of Sm D,which are specific for SLE,is Sm D1.Thus the detecting of anti-Sm D1 antibodies are of importance in diagnosing of SLE.The coding sequence of human Sm D1 was amplified from HL-60 cells cDNA library of human leukemia with specific primers, and cloned into pGEX-5T after the restriction enzyme digestion.The recombinant vector was introduced into E. coli BL21(DE3 plysS+).The recombinant proteins were expressed at a high level and good antigen after induction with IPTG , and analyzed by 12% SDS-PAGE and Western blot. The inclusion body was treated by denaturation and renaturation. The fusion proteins were purified from GST columns, which was a prerequisite for preparation and detection of anti-Sm D1 antibodies.Because the production of prokaryotic expression was inclusion body and the purification was complicated, we tried to induce the recombinant proteins in eukaryon expression. The coding sequence of human Sm D1 was also amplified from HL-60 cells cDNA library of human leukemia with specific primers, but cloned into pPIC9K after the restriction enzyme digestion. The recombinant vector was introduced into SMD1168.The recombinant proteins were extracellular secretion and expressed at a high level after induction with methanol, and analyzed by Tricine-SDS-PAGE and Western blot after concentration by ammonium sulfate.We established DIGFA for detecting anti-Sm D1 antibodies using the preliminary purification of eukaryon expression.The sensitivity of DIFFA was similar to IB,but the specificity of DIGFA was lower than IB.However,DIGFA was a simple, rapid and reliable assay. Further studies are required before using it in clinic duing the limitation of clinic specimen.Thus we isolate human Sm D1 antigens through molecular cloning technique,and establish DIGFA for detecting anti-Sm D1 antibodies.