双孢蘑菇2796凝集素基因的扩增与原核表达

林勇; 肖冬来; 刘俊锋; 唐小峦

doi:10.19303/j.issn.1008-0384.2020.04.011

双孢蘑菇2796凝集素基因的扩增与原核表达

Amplification and Expression of a Lectin Gene from Agaricus bisporus AS2796

摘要

摘要:
目的获得双孢蘑菇凝集素基因，了解双孢蘑菇凝集素家族蛋白的功能。
方法根据已报道的双孢蘑菇凝集素基因序列设计引物，以总DNA为模板扩增双孢蘑菇凝集素基因。将长432 bp的基因亚型克隆后连接至携带His标签的原核表达载体pET-28a，转化大肠杆菌BL21（DE3），经IPTG诱导表达，表达产物经亲和层析纯化后，进行Western blot印迹分析。
结果扩增的凝集素基因大小为432 bp，重组表达质粒pET-Lectin构建正确，重组凝集素蛋白在大肠杆菌BL21（DE3）中的表达产物约为18 kDa，重组蛋白纯化后纯度达95%以上。
结论本研究首次扩增双孢蘑菇凝集素基因，并获得纯度较高的双孢蘑菇凝集素蛋白，可利用凝集试验检测其生物学活性，为目的基因的深入研究提供参考依据。

Abstract:
Objective A lectin gene from Agaricus bisporus AS2796 was isolated to study the functions of the agglutinin protein in mushrooms.
Method Based upon the reported sequences, a primer was designed to amplify the lectin gene with the total DNA as templates. A 432 bp PCR product was purified and connected to the prokaryotic expression vector pET-28a carrying the His label, then transformed into E. coli BL21 (DE3). The IPTG-induced expression product was subsequently purified by affinity chromatography and identified by Western blot.
Result The amplified lectin gene was 432 bp with correctly constructed recombinant expression plasmid, pET-lectin. The recombinant agglutinin protein was about 18 kDa and more than 95% pure after purification,
Conclusion For the first time, a lectin gene was successfully isolated from A. bisporus and amplified. The agglutinin protein obtained could be used to further study its bioactivity and biosynthesis.

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