结缔组织生长因子的分泌表达及ELISA检测法的建立

Secretive expression and establishment of ELISA determination of connective tissue growth factor

目的 制备结缔组织生长因子 (CTGF)抗原 ,建立ELISA检测方法。方法 从血管内皮细胞中提取总RNA ,逆转录合成cDNA ,设计特异性引物 ,通过PCR方法扩增CTGFC区 2 4 2～ 349位氨基酸基因 ,插入噬菌体包膜蛋白基因Ⅲ的上游 ,制备CTGF融合蛋白。用纯化后的融合蛋白免疫家兔 ,获得抗体 ,抗体纯化后标记HRP ,建立ELISA。结果 成功克隆CTGFC区基因 ,插入表达载体经IPTG诱导后获得分子量为 2 5 0 0 0的融合蛋白 ,免疫学检测证实为CTGF。所得抗体用HRP标记。建立的ELISA法检测灵敏度为 2ng/ml,批内及批间平均变异系数为 5 .4 %和 7.5 % ,平均回收率为 10 4 .2 %。血红蛋白浓度在 0～ 5 0mmol/L ,总胆红素在 0～ 15 0 μmol/L ,三酰甘油在 0～ 2 .0mmol/L对测定结果无影响。 2例慢性乙型肝炎病人血标本 ,5例糖尿病肾病、3例肾功能衰竭、3例慢性肾炎尿标本为阳性。结论 CTGF可以作为判断纤维化发生、进展及预后的一个新指标。CTGFELISA检测方法的成功建立 。

Objective To produce connective tissue growth factor (CTGF) standard material and establish the method for determination of CTGF.Method Total RNA was extracted from human endothelial cells and was reverse-transcripted into cDNA.The gene encoded CTGF C region 242-349 residues was obtained by PCR with designed primers and inserted to upstream of the gene of phage coat protein Ⅲ.The CTGF fused protein was expressed and rabbits were immunized to produce antibody after purification.The purified antibody was conjugated with horseperoxidase (HRP),and used to establish an ELISA method.The established method was evaluated according to its sensitivity,precision,recovery,interference and etc.For the investigation of relevance between CTGF and diseases 162 clinical samples were tested.Result The gene of CTGF C region was successfully cloned and inserted into expressive vector to obtain the fused protein of 25 kD by induced IPTG.The required protein was identified by immunological method.The antibody was effectively labeled with HRP.The sensitivity of this method was 2 ng/ml.The average intra-assay and inter-assay precision were 5.38% and 7.50% respectively.The average recovery was 104.23%.The concentrations of hemoglobin (0-50mmol/L),total bilirubin (0-150 μmol/L),triglyceride (0-2.0mmol/L) did not interfere the test result.The result of clinical application showed that two blood samples from chronic hepatitis and urine samples from 5 diabetic nephropathy patients,3 renal failure patients and 2 chronic nephritis patients were CTGF-positive.Conclusion CTGF was a new marker and is closely related to the formation,development and prognosis of fibrosis.Successful establishment of ELISA method for detection of CTGF paved a way for its clinical application.