Abstract:

Objective To compare the prokaryotic expression system and eukaryotic expression system for the expression of cysteine protease of Clonorchis sinensis，and the diagnostic efficiency of their objective products. Methods According to the se? quence of cysteine protease of C. sinensis，two pairs of primers were designed to amplify the genes from the total cDNA of C. sinen? sis. The genes were cloned into plasmid pET28a（+）and pPIC9K， respectively，and these recombinant plasmids were transformed into E. coli BL21 and GS115 separately after they were identified through double digests and sequencing. The cysteine protease of C. sinensis was expressed and purified，and then the sero?diagnostic effects of the purified proteins for clonorchiasis by ELISA were compared. Results The cysteine protease of C. sinensis was expressed as inclusion bodies in BL21，and its yield was 6.8 mg/L， while it was expressed as a kind of soluble protein in GS115，and its yield reached to 65.00 mg/L. Their sensitivities for serodiagno? sis of clonorchiasis were 95.00% and 93.30%，respectively，and their specifities were 91.67% and 94.10%，respectively，with no statistically significant differences between them（all P values were above 0.05） . Conclusion The application value on cysteine protease of C. sinensis expressed through eukaryotic expression system is higher than that expressed through prokaryotic expression system.

Key words: Clonorchis sinensis；Cysteine protease；Prokaryotic expression system；Eukaryotic expression system；Immuno? diagnosis

摘要：

目的 比较原核和真核2种表达系统重组表达华支睾吸虫半胱氨酸蛋白酶方法的优劣和目的产物的血清免疫学检测效果。方法 根据已知的华支睾吸虫半胱氨酸蛋白酶序列分别设计引物扩增目的基因， 与相应的表达载体相连分别构建原核表达质粒pET28a?CP和真核表达质粒pPIC9K?CP， 双酶切及测序鉴定后， 将正确的重组质粒分别转入大肠埃希菌（E. coli） BL21 （DE3） 和毕赤酵母GS115中诱导表达、 纯化； 将获得的纯化蛋白采用ELISA方法检测华支睾吸虫病人血清和正常人血清， 比较诊断效果。结果 原核表达系统表达的华支睾吸虫半胱氨酸蛋白酶以包涵体形式存在， 表达量为6.84 mg/L； 真核表达系统表达量达到65.00 mg/L， 且为可溶性蛋白； 两者检测华支睾吸虫病的敏感性分别为95.00%（57/60） 和 93.30% （56/60）， 特异性分别为91.67% （77/84） 和94.10% （79/84）， 差异均无统计学意义 （P均 > 0.05）。结论 真核表达华支睾吸虫半胱氨酸蛋白酶较原核表达系统具有更高的应用价值。

关键词: 华支睾吸虫； 半胱氨酸蛋白酶； 原核表达系统； 真核表达系统； 免疫诊断