论文部分内容阅读
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 alliinase gene from garlic bulbs and construct alliinase expression vector and express in Pichia pastoris system to further explore the biological activity of recombinant alliinase. Methods The alliinase gene of garlic was cloned by RT-PCR and the recombinant plasmid pPIczαC-alliinase was constructed by pPIczαC vector. The recombinant plasmid was introduced into Pichia pastoris X-33 by electroporation, and the positive clones were screened. After induced by methanol The supernatant was analyzed by SDS-PAGE and Western blotting. The activities of recombinant alliinase and extracted alliin were detected by pyruvic acid method. The contents of two alliinase proteins were determined by Lowry’s method. The activity of two enzymes was compared by specific activity. Results The alliinase gene was cloned from garlic with a size of 1500 bp. The relative molecular mass of recombinant alliinase was about 5.5 × 104, which was present in the supernatant of Pichia pastoris. The specific activity of alliinase was (82.09 ± 3.89) U / mg, and the alliinase activity was (176.49 ± 5.06) U / mg. CONCLUSION Alliinase gene can be expressed in Pichia pastoris expression system. Recombinant alliinase has enzyme activity but its activity is lower than that of natural alliinase extracted.