目的 从大蒜鳞茎中克隆蒜氨酸酶基因，构建蒜氨酸酶真核表达载体，并在毕赤酵母系统中表达，进一步探讨重组蒜氨酸酶的生物学活性。方法 利用RT－PCR方法克隆大蒜鳞茎蒜氨酸酶基因，通过pPIczαC载体构建pPIczαC－蒜氨酸酶真核表达质粒，电转化法导入毕赤酵母X－33，筛选阳性克隆，经甲醇诱导后取表达上清进行SDS－PAGE和Western blotting分析。利用丙酮酸法检测重组蒜氨酸酶和提取的天然蒜氨酸酶的活性，Lowry法测2种蒜氨酸酶蛋白质量，通过比活力比较2种酶活性大小。结果 从大蒜内克隆出蒜氨酸酶基因，大小为1 500 bp，重组蒜氨酸酶相对分子质量约为5．5×10⁴，存在于毕赤酵母表达上清液中。重组蒜氨酸酶比活力为(82．09±3．89)U／mg，天然蒜氨酸酶比活力为(176．49±5．06)U／mg。结论 蒜氨酸酶基因可以在毕赤酵母表达系统中获得表达，重组蒜氨酸酶具有酶活性，但是其活性低于提取的天然蒜氨酸酶。

Objective To clone the alliinase gene from the garlic bulb and construct the eukaryote expression plasmid for expressing the recombinant alliinase in Pichia pastoris system and analyzing its bioactivity. Metheds The alliinase gene was cloned from the Zhejiang garlic bulb by RT-PCR and the eukaryote expression plasmid of alliinase was constructed with the pPIczαC vector. The recombinant plasmid was transformed into Pichia pastoris X-33 by eletroporation. The positive clones were screened and were induced by methanol. Supernatants after induction were analyzed by SDS-PAGE and Western blotting. The activities of the recombinant protein and the extracted alliinase were detected by the pyruvic acid method and compared by specific activity. The contents of the two kinds of alliinase were detected by Lowry method. Results The alliinase gene was successfully cloned from the garlic bulb, the length of alliinase gene was 1 500 bp, the molecule of the recombinant alliinase was about 5.5 × 10⁴, existed in the supernatant of Pichia pastoris. The specific activity of the recombinant protein was (82.09 ± 3.89) U/mg and the nature alliinase was (176.49 ± 5.06) U/mg. Conlusion The alliinase gene is successfully expressed in Pichia pastoris system. The recombinant alliinase has the activity of enzyme, but is lower than that of the extracted alliinase.

浙江省教育厅课题(Y200805242)；浙江省中医药管理局资助课题(2010ZB026)；浙江中医药大学重点课题(2009ZZ05)；浙江省科技厅新苗人才计划项目(67401219)；浙江中医药大学校级课题(17108015)