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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 To establish a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of matrix metalloproteinase 9 (MMP9) in human serum and explore the difference of serum MMP9 between normal and hepatocellular carcinoma patients. Methods The 721 ~ 1 156 bp fragment of MMP9 gene was amplified by reverse transcription from human liver tissue and inserted into the prokaryotic expression plasmid pQE30 to induce the expression of the protein in Escherichia coli M15. The purified fusion protein HIS-MMP9 was used as antigen to immunize rabbits and guinea pigs. The obtained anti-MMP9 antiserum was purified and then double-antibody sandwich ELISA was established. The serum levels of MMP9 in 227 normal and 193 hepatocellular carcinoma patients were determined. Results SDS-PAGE showed that the relative molecular mass of the expressed HIS-MMP9 fusion protein was about 17 000. The MMP9 antisera prepared by using the prokaryotic expressed protein as an antigen could effectively recognize the recombinant MMP9 and the endogenous MMP9 of neutrophils and HepG2 cells MMP9 ELISA was used to detect serum MMP9 levels in patients with hepatocellular carcinoma than in normal people, the difference was statistically significant (P <0.001). Conclusion The established double antibody sandwich ELISA method can be used for the detection of serum MMP9, which provides a scientific basis for establishing a screening method for metastasis and recurrence of hepatocellular carcinoma with MMP9 as a detection index.