3-磷酸甘油酸脱氢酶及其抗体对自身免疫性肝炎的诊断意义

Cloning and expression of 3-phosphoglycerate dehydrogenase gene and its correlative antibodies in diagnosis of autoimmune hepatitis

目的克隆D-3-磷酸甘油酸脱氢酶（Phgdh）的cDNA，构建重组表达质粒，纯化重组蛋白，初步探讨Phgdh抗体在自身免疫性肝炎诊断中的意义。方法血清蛋白质组学方法鉴定差异蛋白，构建重组表达载体，在大肠杆菌中表达，融合蛋白经NiNTA树脂柱亲和层析纯化，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳及Western blot法进行免疫鉴定；应用表达蛋白建立间接酶联免疫吸附法，检测60名健康体检者、65例自身免疫性肝炎（AIH）、122例原发性胆汁性肝硬化、56例慢性乙型肝炎、117例慢性丙型肝炎患者血清中抗Phgdh抗体。组间率的比较用χ^2检验。结果经重组质粒测序和酶切鉴定结果证实，Phgdh目的基因已正确插入原核表达载体中，基因序列正确，符合表达框架。十二烷基硫酸钠聚丙烯酰胺凝胶电泳检测表达产物在相对分子质量约6．0×10^4附近有一明显的蛋白表达带，Westernblot分析结果显示重组蛋白具有Phgdh抗原反应性。酶联免疫吸附法检测血清标本结果显示，AIH、原发性胆汁性肝硬化、慢性乙型肝炎、慢性丙型肝炎及正常人抗Phgdh抗体阳性检出率分别为66．15％、21．42％、12．50％、6．83％和3．30％。AIH组Phgdh自身抗体阳性率与疾病对照组及正常对照组比较，差异均具有统计学意义（P〈0．01）。结论本研究成功克隆了Phgdh的cDNA，并将其在大肠杆菌中成功表达。该抗体主要在AIH患者中检出，此抗体的检测有助于提高AIH的临床诊断水平。

Objective To evaluate whether the D-3-phosphoglycerate dehydrogenase （Phgdh） correlative antibodies is crucial for AIH, we cloned Phgdh eDNA and constructed plasmid, then purified and identified the immunoreactivity of the recombinant protein, and established the enzyme linked immunosorbent assay （ELISA） to detect Phgdh autoantigen correlative antibodies in diagnosis of autoimmune hepatitis. Methods The constructed plasmid was transformed into E. coli. BL21（D3）. This fusion protein was purified by Ni-NTA chromatography and its immunoreactivity was identified by SDS-PAGE and Westem blot. The ELISA with the fusion protein was established first, then, the Phgdh autoantigen correlative antibodies in serum of patients with AIH （65） and patients with PBC （122） as well as chronic hepatitis B （CHB） （56）, chronic hepatitis C （CHC） （117）, and normal controls （60） were detected. Results The sequence of Phgdh autoantigen gene was the same as the sequence reported on the genebank. The fusion protein was found about 60kD strip on SDS-PAGE. Western blot analysis showed that the fusion protein had immunoreactivity. When analyzing the serum by ELISA, the immune reactivity to Phgdh was detected in 66.15% of patients with AIH, 21.42% of patients with PBC, 12.50% of patients with CHB, 6.83% of patients with CHC, and 3.30% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance （P 〈 0.01）. Conclusion The Phgdh cDNA is successfully cloned into E. coil BL21 （D3）. The frequency of antibodies to Phgdh is much higher in patients with AIH than in patients with PBC, CHB, CHC and normal control. The antibodies to Phgdh may have utility in improved diagnosis of AIH.