去唾液酸糖蛋白受体H1亚单位基因片段的克隆表达及其检测自身免疫性肝炎的价值

Cloning and expression of the human asialoglycoprotein receptor H1 subunit and preliminary application

目的 克隆去唾液酸糖蛋白受体（asialoglycoprotein receptor，ASGPR）H1亚单位显性表位435bp基因片段，构建表达重组质粒，进行表达、纯化，以获得具有免疫活性的ASGPR蛋白，建立间接ELISA法，并探讨其在检测自身免疫性肝炎（AIH）中抗ASGPR抗体的价值。方法将ASGPRH1亚单位糖类识别区（CRDH1）435bp的基因片段定向克隆至真核表达载体PEGH，经D-半乳糖诱导表达，表达产物用谷胱甘肽凝胶珠（Glutathione Sepharose4B）亲和纯化，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹法（WB）及蛋白质谱（MALDI—TOF）进行免疫活性鉴定，应用表达蛋白建立ELISA法。同时用该方法检测45例AIH、30例SLE、30例类风湿关节炎（RA）、10例原发性干燥综合征（SS）患者、30名健康献血者的抗ASGPR抗体阳性率。结果经重组质粒测序结果证实，CRDH1／PEGH目的基因已正确插入真核表达载体中，基因序列正确，符合表达框架；SDS—PAGE检测表达产物为1条相对分子质量42500的蛋白表达条带。质谱蛋白肽指纹数据通过Mascot在人类蛋白质数据库中分析比对，与ASGPRH1亚单位蛋白同源性为98．34％，WB分析表明，重组蛋白具有人ASGPR抗原反应性；间接ELISA检测血清结果显示，AIH组中抗ASGPR抗体的阳性率为35．6％（16／45），非AIH组均为阴性，差异有统计学意义（X^2=31．85，P〈0．01）。结论成功克隆的ASGPRH1基因可在啤酒酵母菌中成功表达，且重组的自身抗原具有较好的抗原性和特异性。应用纯化蛋白建立的间接ELISA检测AIH抗ASGPR抗体具有较好的特异性。

Objective To clone and express the human asialoglycoprotein receptor（ASGPR） H1 subunit, purify and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay （ELISA） to detect anti-ASGPR antibodies in diagnosis of autoimmune hepatitis. Methods The CRDH1 eDNA （435 bp） was subcloned into eukaryotic vector PEGH, and the recombinant protein expression was induced by D （ ＋ ）-Galactose. The recombinant CRDH1 was purified with Glutathione Sepharose 4B, and its immunoreactivity was identified by SDS-PAGE and western blot as well as MALDI-TOF. ELISA was established to detect the anti-ASGPR antibodies in serum samples of 45 patients with AIH, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequencing of recombinant plasmid showed the CRDH1 gene was successfully inserted to the eukaryotic expression vector with correct sequence and open reading frame. The fusion protein showed a molecular weight of 42 500 Da on SDS-PAGE gel and confirmed to be the human ASGPR by MALDI-MS through peptide mass fingerprint analysis with Mascot in human protein database. It shared 98.34% homology with ASGPR HI subunit. Western blot analysis showed that the fusion protein had the same immunoreactivity as human ASGPR. The results of ELISA indicated that the positive rate of anti- ASGPR was 35.6% （ 16/45 ）, but the ELISA was negative in other control. There was significant difference of positivity of the autoantibodies between AIH and non-AIH controls （X^2 = 31.85 ,P 〈 0. 01 ）. Conclusions The human plasmid containing ASGPR is successfully clone into Saccharomyces cerevisiae Y258. The recombinant autoantigen owns good antigenieity and specificity. ELISA established with the purified protein shows good specificity for diagnosis of AIH.