血清MMP9蛋白ELISA检测方法的建立及在肝癌患者血清检测中的初步应用  

Establishment of a sandwich ELISA method for detection of matrix metalloproteinase 9 protein in sera samples and preliminary application for hepatocellular carcinoma patients

作　　者：高晨[1] 李锋[1] 周伟[1] 杨小洁[2] 石琦[1] 刘长利[3] 姜慧英[1] 毕新宇[2] 杨建国[2] 胡敬群[2] 韩俊[4] 董小平[4] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]中国医学科学院肿瘤医院肿瘤研究所 [3]北京市红十字血液中心 [4]中国疾病预防控制中心 病毒病预防控制所,北京100052

出　　处：《中华检验医学杂志》2007年第3期288-291,共4页Chinese Journal of Laboratory Medicine

基　　金：国家高科技研究发展计划项目(2002AA215021)

摘　　要：目的建立基质金属蛋白酶9(MMP9)的双抗体夹心 ELISA 检测方法,探讨血清中MMP9在正常人及肝癌患者中的差异。方法从人肝组织中经过反转录扩增出 MMP9基因721～1 156 bp 区段,插入到原核表达质粒 pQE30中,在大肠埃希菌 M15中诱导蛋白表达。以纯化的融合蛋白 HIS-MMP9为抗原免疫实验用兔及豚鼠,得到的 MMP9抗血清经过纯化后,建立双抗体夹心 ELISA法。对227份正常及193份肝癌患者血清中的 MMP9进行检测。结果 SDS-PAGE 显示所表达的HIS-MMP9融合蛋白相对分子质量约为17 000;以原核表达蛋白为抗原制备的 MMP9抗血清可有效地识别重组 MMP9和中性粒细胞和 HepG2细胞内源性 MMP9蛋白;利用建立的 MMP9 ELISA 检测技术发现肝细胞癌患者血清中 MMP9的含量高于正常人群,差异具有统计学意义,P<0.001。结论建立的双抗体夹心 ELISA 方法可用于血清 MMP9的检测,为建立以 MMP9为检测指标的肝细胞癌患者转移复发筛查方法提供了科学基础。Objective Expression of recombinant human matrix metalloproteinase 9 （MMP9） protein in E coli. To establish a sandwich ELISA method for detecting MMP9 in sera and analyses of the difference of serum MMP9 in health population and the patient with hepatoceilular carcinoma （HCC）. Methods A fragment MMP9 sequence from nt 721 - 1 156 was amplified with PCR from the synthesized cDNAs of human liver tissues and inserted into the prokaryotic-expression plasmid pQE30. HIS-MMP9 fusion protein was expressed and purified from E coli MIS ceils. The specific antibodies were elicited in rabbits and guinea pigs by immunization of the purified HIS-MMP9. After purifing antibodies with CNBr-activated Sepharose 4B, a sandwich ELISA technique was established. The serum MMP9 proteins were evaluated in 227 health adults and 193 HCC patients. Results SDS-PAGE displayed that the molecular-weight of the expressed fusion protein was about 17 000. The prepared antisera were able to recognize both recombinant and endogenously expressed MMP9 from human neutrophil and HepG2 ceils. It was found that the average level of MMP9 proteins in HCC patients was higher than that in health control, showing significantly statistic difference. Conclusions The established method could reflect the level of serum MMP9. The data in this study supply scientific basis of generating methodology for screening metastasis and reoccurrence of HCC, using serum MMP9 as index.

关 键 词：癌 肝细胞 基质金属蛋白酶类 酶联免疫吸附测定 

分 类 号：R735.7[医药卫生—肿瘤]