抗人羧酸酯酶-II多克隆抗体的制备、表征及应用

[Preparation, characterization and application of polyclonal antibody against human carboxylesterases-II].

作者信息

Wu Chun-ling, Bao Hong-xia, Tang Xiao-bo, Zhu Da-ling

机构信息

Biopharmaceutical Department, College of Pharmacy, Key Lab of Biopharmaceutical Engineering of Heilongjiang Province, Key Laboratory of Drug and Endogenous Substance Metabolism of Higher Learning Institution of Heilongjiang Province, Harbin, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Feb;25(2):158-60, 163.

PMID:19174014

Abstract

AIM

To construct a prokaryotic plasmid expressing human carboxyles-terases-II (hCE-II ), purify the recombinant protein and investigate the rabbit polyclonal antibody against hCE-II .

METHODS

A recombinant plasmid expressing pGEX-4T-1-hCE-II (hCE-II -GST) and pET-32a- hCE-II (hCE-II -His) was constructed and then it was expressed in E.coli BL21 induced by IPTG. The polyclonal antibody was prepared by immunizing the rabbits with the purified recombinant protein. The sensitivity and specificity of the antibody was detected using enzyme -linked immunosorbent assay, immunohistochem ical staining and Western blot analysis.

RESULTS

The polyclonal antibody against hCE-II was successfully prepared. Western blot analysis showed that the antibody specifically reacted with the recombinant protein and natural human liver microsomal proteins. Immunohistochemis result demonstrated that the pAb could combine with liver cytoplasm protein but not vascular smooth muscle cell protein.

CONCLUSION

The successful preparation of the pAb against hCE-II with high titer and specificity lays a foundation for the investigation of diagnosis kit of liver cancer.

摘要

目的

构建表达人羧酸酯酶-II（hCE-II）的原核质粒，纯化重组蛋白并研究抗hCE-II的兔多克隆抗体。

方法

构建表达pGEX-4T-1-hCE-II（hCE-II -GST）和pET-32a-hCE-II（hCE-II -His）的重组质粒，然后在IPTG诱导下于大肠杆菌BL21中表达。用纯化的重组蛋白免疫兔子制备多克隆抗体。使用酶联免疫吸附测定、免疫组织化学染色和蛋白质印迹分析检测抗体的敏感性和特异性。