摘要
目的克隆人干燥综合征A抗原基因(SSA-52kD),为SSA抗原的表达和使用重组抗原用于自身抗体的临床检测奠定基础。方法根据GenBank中检索到的人SSA-52kDcDNA序列,在5′非编码区和3′非编码区设计特异性引物,提取人源Hela细胞总RNA作为模板,反转录RT-PCR扩增人干燥综合征SSA-52kD抗原cDNA。PCR产物纯化后连接至载体PET-30a,导入大肠杆菌DH5α,构建重组质粒PET-30a-SSA-52kD。对重组质粒进行酶切鉴定,选择阳性克隆测序;对DNA测序结果进行鉴定分析。结果RT-PCR扩增产物为1447bp。重组质粒PET-30a-SSA-52kD经BglⅡ和HindⅢ双酶切证实含目的基因片段。序列分析提示与GenBank中检索到的一致。结论成功克隆人SSA-52kD基因,并构建重组质粒PET-30a-SSA-52kD。
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 DHSctto construct the recombinant plasmid PET- 30a - SSA -52kD. The recombinant plasmid was digested with BglⅡ 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.
出处
《医学研究杂志》
2008年第2期47-49,共3页
Journal of Medical Research
基金
国家863高技术研究发展计划(20024032)
