Construction and Identification of Recombinant Expressing of Human Sjogren's Syndrome Antigen A / 医学研究杂志

ABSTRACT

Objective To clone human Sjogren's syndrome antigen A(SSA)for expressing of antigen SSA-52kD and establishing a new clinical detecting method.Methods According to the human SSA-52kD cDNA sequence reported in GenBank,primers of human SSA-52kD cDNA were designed and synthesized.Human SSA-52kD cDNA was amplified from RNA of cultured Hela cell by reverse transcriptase polymerase chain reaction(RT-PCR).The production of amplification was ligated to PET-30a vector and then transformed into the competent bacteria DH5?to construct the recombinant plasmid PET-30a-SSA-52kD.The recombinant plasmid was digested with Bgl Ⅱ and Hind Ⅲ,and positive clones were sequenced.Results The Human SSA-52kD cDNA fragment containing 1447bp was amplified by RT-PCR.Restriction endonuclease mapping using Bgl II and Hind III showed that the target gene was inserted into the recombinant plasmid.The complete coding sequence of Human SSA-52kD was consistent with that of GenBank through DNA sequencing.Conclusions The full length of human SSA-52kD cDNA was successfully cloned and the recombinant plasmid PET-30a-SSA-52kD was constructed.