摘要
目的获取可溶性SmDl重组蛋白，并建立抗SmDl抗体ELISA检测方法。方法采用PCR方法扩增SmDl基因，构建表达质粒pET-21a-SmDl，转化到大肠杆菌BL21，在16℃条件下，1mMIPTG诱导表达10h，SDS-PAGE电泳分析表达产物，镍柱亲和层析纯化，Bradford法测定蛋白浓度，建立间接ELISA法，检测215例常见自身免疫性疾病血清样本。结果通过双酶切和测序结果表明成功构建表达质粒pET-21a-SmDl，SDS-PAGE电泳得知目的蛋白以可溶性表达为主，经镍柱纯化获得单一目的蛋白，浓度为0.62mg／mL。46．1％SLE患者呈抗StuDl抗体阳性，其次是MCTD（20％），其他疾病的检出率均小于15％，SLE组与非SLE组的灵敏度差异有统计学意义（P〈0．05）。抗StuDl抗体对SLE特异性为93％，高于非SLE组，其他疾病组均小于80〉。结论可溶性表达的StuDl蛋白具有较好的抗原性，抗StuDl抗体有助于临床辅助诊断SLE。
Objective To obtain soluble StuD1 recombinant protein and establish indirect ELISA for Anti- SmD1. Methods StuD1 gene was amplified by PCR and cloned into pET-21a, pET-21a-SmD1 constructed succefully was transfected into E. coli BL21 which expressed protein 10 hour inducing by 1ram IPTG on the condition of 16℃. Recombinant protein was identified by SDS- PAGE electrophoresis and purified by HIS-tag Ni affinity chromatogra- phy. Bradford method was used to determinate protein concentration. Indirect ELISA was used to detect 215 serum in several autoimmunitic patients. Results The result of double digestion and sequencing showed that expression plas- mid pET-21a-SmD1 was successfully constructed. Most of soluble SmD1 protein stayed in supernantant, which showed by SDS-PAGE electrophoresis and purified by HIS-tag Ni affinity chromatography. 46. 1% SLE patients showed anti-StuD1 antibodies positive, followed by MCTD（20%）, anti-StuD1 in any other diseases were less than 15%. The difference between sensitivity of anti-StuD1 to SLE and non-SLE showed significant statistically. 93% spe- cificity of anti-SmD1 to SLE was higher than other diseases, which were less than 80%. Conclusion Soluble recombi- nant StuD1 has good antigenicity,and anti-StuD1 could be helpful for clinical diagnosis of SLE.
出处
《检验医学与临床》
CAS
2014年第A02期26-29,共4页
Laboratory Medicine and Clinic
关键词
SmDl
系统性红斑狼疮
原核表达
StuD1
systemic lupus erythematosus
prokaryotic expression
