胰腺癌相关自身抗原47原核表达、纯化及其抗体检测胰腺癌的价值初探

目的 验证胰腺癌相关性自身抗原47(PAA47)的质谱鉴定结果，初探抗PAA47检测在诊断胰腺癌中的意义。方法 构建pQE—30—PAA47重组表达载体；在大肠杆菌M15中诱导表达KIAA0111编码蛋白质；经纯化后用点印迹、免疫印证法验证表达产物与相应胰腺癌相关性自身抗体阳性血清的抗原反应性；建立间接酶联免疫吸附实验(ELISA)，检测60名健康体检者、60例胰腺癌、20例慢性胰腺炎及120例其他肿瘤(大肠癌、肝癌、胃癌及肺癌各30例)患者血清中的抗PAA47，并与回顾性糖链抗原19—9(CAl9—9)检测结果相比较。结果 表达产物的序列与KIAA0111编码序列一致；点印迹法和免疫印迹法显示纯化表达产物与抗PAA47血清具有抗原反应性。ELISA结果显示，胰腺癌患者中，该抗体阳性率为31．67％(19／60)，大肠癌、肝癌、胃癌分别为10．00％、3．33％、6．67％，正常人、慢性胰腺炎及肺癌患者中未见阳性。抗PAA47对胰腺癌的灵敏度为31．67％，特异性为95．89％。与慢性胰腺炎患者比较，PAA47的灵敏度为31．67％，特异性为100％，CAl9—9的灵敏度为75．00％，特异性为86．96％。平行法联合检测2项指标，既保持了相同的特异性，又提高了检测的灵敏度(90％，P＜0．05)。采用顺序法联合检测，灵敏度和特异性分别为70％和100％。结论 本研究成功克隆了人KIAA0111编码基因，并将其在大肠杆菌中成功表达；新鉴定的PAA47，其抗体血清学检测与CAl9—9等肿瘤标志物联合，对提高胰腺癌诊断的灵敏度和特异性具有一定的意义。

cDNA cloning, expression of PAA47 and its significance in pancreatic cancer

Objective To clone, express and purify the protein, KIAA0111 gene product, which was formerly identified as the target antigen of pancreatic cancer associated autoantibody (PAA47) and to testify the truth of the former identification with mass spectrometry after separation with two dimensional electrophoresis, also to further investigate the clinical significance of anti-PAA47 in the diagnosis of pancreatic cancer.Methods Construct expression vector pQE30-PAA47. The 6-His tagged protein was induced via E.coli M15 and purified with affinity chromatography following SDS-PAGE. The expressed product were examined with anti-PAA47 positive sera of pancreatic patients with dot blotting and western blotting. ELISA for anti-PAA47 has been established with the purified fusion protein PAA47 as antigen and detected 260 sera samples, including 60 from normal controls, 60 from pancreatic cancer patients, 20 from chronic panreatitis patients and each 30 from patients with colonrectal cancer, hepatocelluar cancer, gastric cancer or lung cancer. Results The recombinant pQE30-KIAA0111 was confirmed by sequencing. Dot blotting and western blotting results confirmed the KIAA0111 gene product to be the specific antigen of anti-PAA47. The positive rate of anti-PAA47 in pancreatic cancer is 31.67%(19/60), while 10.00% in colonrectal cancer, 3.33% in hepatocellular cancer, 6.67% in gastric cancer. None of the normal people, chronic pancreatitis and lung cancer patients was positive. 19 cases of pancreatic cancer were correctly predicted diagnosis using anti-PAA47, with a sensitivity of 31.67% and a specificity of 95.89%. Compared with chronic pancreatitis group, anti-PAA47 had a sensitivity as 31.67% and specificity as 100% for the diagnosis for pancreatic cancer. Retrospective analysis of CA19-9 level of pancreatic cancer and chronic pancreatitis patients showed that, it had a higher sensitivity as 75.00% and a lower specificity as 86.96%, compared with anti - PAA47( P<0.05). Analysis of the two indexes with parallel testing showed a better sensitivity as 90% than CA19-9 alone, while the same specificity. Analysis the two index with series testng showed a better specificity as 100% than CA19-9 alone, although the sensitivity was lower as 70%( P<0.05). Conclusion The formerly identified novel autoantigen, KIAA0111 gene product, named PAA47, is a prospectively serological marker of pancreatic cancer. Associated with CA19-9, the detection of anti - PAA47 may be helpful to improve the sensitivity and specificity of the diagnosis of pancreatic cancer.