Cloning of a β-glucosidase Gene (bgl1) from Asperjillus niger and Expression in Chinese Hamster Ovary (CHO) Cells | 黑曲霉β-葡萄糖苷酶基因(bgl1)的克隆及其在中国仓鼠卵巢(CHO)细胞中的表达

Chinese. β-葡萄糖苷酶是纤维素酶系三大成员之一，被认为是纤维素糖化的限速酶。本研究克隆黑曲霉β-葡萄糖苷酶基因(bgl1)，并在中国仓鼠卵巢(CHO)细胞中进行表达分析。以GenBank上黑曲霉β-葡萄糖苷酶基因(bgl1)序列为模板，设计特异性引物，从黑曲霉(Asperjillus niger)基因组DNA中扩增目的片段，通过SOE-PCR的方法将猪腮腺分泌蛋白信号肽序列sp和bgl1相连，成功构建分泌型真核表达载体pc6-sp-bgl1，利用脂质体介导法瞬时转染哺乳动物CHO细胞，通过RT-PCR和Western blot检测，最后通过DNS(dinitrosalicylic acid)法测定表达产物酶学活性。PCR扩增得到的目的序列与文献报道序列的相似性为91%，预测的蛋白氨基酸序列相似性为99%。RT-PCR和Western blot检测证明，外源基因bgl1在CHO细胞中得到表达；分泌到细胞培养上清中的重组蛋白对水杨素的水解活性为1.74 U/mL，说明重组蛋白具有β-葡萄糖苷酶活性。本研究克隆得到黑曲霉bgl1基因并在哺乳动物CHO细胞中进行表达，为β-葡萄糖苷酶在单胃动物中的利用提供了基础研究资料。

English. β-glucosidase is one of the three members of the cellulase system and is considered to be the rate-limiting enzyme of cellulose saccharification. The objective of the study was to clone the β-glucosidase gene (bgl1) from Asperjillus niger, and analyze its expression in Chinese hamster ovary (CHO) cells. The DNA fragment was amplified by PCR from Asperjillus niger, and then cloned into pMD18-T vector. A DNA fragment containing the coding sequence of pig parotid secretory protein signal peptide (sp) and bgl1 was assembled by SOE-PCR and subcloned into the expression vector pcDNA6/His A using BamHⅠand XhoⅠrestriction sites to generate pc6-sp-bgl1. To evaluate transgene expression, plasmid DNA was purified and transiently transfected into CHO cells, then RT-PCR and Western blot analyses were used and the enzymatic activity was determined by DNS(dinitrosalicylic acid). Sequence analysis showed that the DNA sequence and the putative amino acid sequence shared 91% and 99% identity with the reported sequences, respectively. RT-PCR and Western blot analyses showed that the exogenous gene bgl1 was expressed and recombinant protein was secreted into cell culture medium. The enzymatic activity of culture supernatants from 48 h-transfected cells to D-(-)-salicin was 1.74 U/mL, indicating that the recombinant protein with the activity of β-glucosidase. We cloned the β-glucosidase gene bgl1 from Asperjillus niger and expressed the gene in CHO cells. The study paves the way for further research of β-glucosidase in monogastric animals.