桦褐孔菌纤维素酶基因的原核表达与纯化

发布时间：2018-10-12 12:01

【摘要】：桦褐孔菌Inonotus obliquus(Ach.exPers.)Pilat,是一种非常珍贵的药用菌,菌核为主要药用价值部位,其在抗肿瘤、抗高血压、降血糖、提高免疫力等方面有显著疗效,一直深受人们的喜爱。然而桦褐孔菌野生资源十分稀少,且野生的桦褐孔菌在桦树上要经过10-15年才具有良好的药用价值,因此桦褐孔菌人工栽培工作的探索迫在眉睫。目前桦褐孔菌菌核的栽培已取得成功,但产量较低,研究证明,胞外酶活性变化与菌核生长关系密切,继而本试验以桦褐孔菌JL01为试材,继续在与菌核发育有关的胞外酶——纤维素酶的分子水平上展开研究,为进一步了解胞外酶的蛋白质功能和分泌表达变化规律,以及建立高效的外源表达载体提供理论依据,具体实验结果如下:1.克隆并获得了桦褐孔菌JL01菌株中内切葡聚糖酶(endoglucanase,IO-EG)和β-葡萄糖苷酶(beta-glucosidase,IO-BGL)的ORF序列,β-葡萄糖苷酶的ORF序列与Gen bank中公布的ORF序列完全吻合,而内切葡聚糖酶的ORF序列与Gen bank中公布的ORF序列比对,发现1092个碱基T缺失,导致ORF序列改变,得到了新的内切葡聚糖酶ORF序列,新基因命名为eg I,eg I的ORF序列全长为1149 bp,编码382个氨基酸,推测的蛋白分子量为40.1 kDa,理论等电点为4.78,序列比对和功能分析鉴定后,确定其为纤维素酶糖苷水解酶第5家族;2.构建了桦褐孔菌内切葡聚糖酶基因(eg I)和β-葡萄糖苷酶基因(bgl)原核表达载体,并命名为pET-30a-eg I和pET-30a-bgl;3.IO-EG和IO-BGL原核表达载体pET-30a-eg I和pET-30a-bgl正确导入大肠杆菌BL21中,并获得稳定表达的蛋白,SDS结果显示蛋白均以包涵体形式存在,IO-EG和IO-BGL重组蛋白分子质量分别约为40 kDa和90 kDa;4.采用Ni-IDA蛋白纯化试剂盒在变性条件下对目的蛋白进行纯化,纯化后的IO-EG蛋白纯度为88%,IO-BGL蛋白纯度为75%,透析浓缩后的IO-EG和IO-BGL蛋白浓度分别为0.5 mg·L-1和0.65 mg·mL-1,得到的蛋白质量分别为3.0 mg和3.6 mg。5.将纯化得到的蛋白进行Western Blotting分析,IO-EG蛋白条带单一,和理论分子量相符,IO-BGL蛋白条带和理论分子量相符,但35 KDa附近出现杂带,推测原因是IO-BGL蛋白降解。
[Abstract]:Betula obliquus Inonotus obliquus (Ach.exPers.) Pilat, is one of the most precious medicinal bacteria. Sclerotia is the main medicinal value. It has remarkable effect in anti-tumor, anti-hypertension, hypoglycemia and improving immunity, and has been loved by people all the time. However, the wild resources of Betula platyphylla are very rare, and the wild Betula platyphylla has a good medicinal value in birch trees after 10-15 years, so it is urgent to explore the artificial cultivation of Betula platyphylla. At present, the sclerotia of Betula platyphylla has been successfully cultivated, but the yield is relatively low. It is proved that the change of extracellular enzyme activity is closely related to the growth of sclerotia, and the JL01 of Betula platyphylla is used as the test material in this experiment. To continue to study on the molecular level of cellulase, an extracellular enzyme related to sclerotia development, in order to further understand the changes of protein function and secretion expression of extracellular enzymes, and to establish efficient exogenous expression vectors. The experimental results are as follows: 1. The ORF sequences of endoglucanase (endoglucanase,IO-EG) and 尾 -glucosidase (beta-glucosidase,IO-BGL) in JL01 strain of Betula obliquus were cloned and obtained. The ORF sequence of 尾 -glucosidase was in good agreement with the ORF sequence published in Gen bank. However, the ORF sequence of endoglucanase was aligned with the ORF sequence published in Gen bank, and 1092 base T deletions were found, which led to the change of ORF sequence, resulting in a new ORF sequence of endoglucanase. The total length of the ORF sequence named eg Igneg I is 382 amino acids, and the deduced molecular weight of the protein is 40.1 kDa, the theoretical isoelectric point is 4.78. After sequence alignment and functional analysis, it is identified as the fifth family of cellulase glycoside hydrolase; 2. The prokaryotic expression vectors of (eg I) and 尾 -glucosidase (bgl) were constructed and named as pET-30a-eg I, pET-30a-bgl;3.IO-EG and IO-BGL prokaryotic expression vectors pET-30a-eg I and pET-30a-bgl into E. coli BL21. The stable expression protein was obtained. The SDS results showed that the proteins existed in the form of inclusion bodies. The molecular weight of IO-EG and IO-BGL recombinant proteins were about 40 kDa and 90 kDa;4., respectively. Ni-IDA protein purification kit was used to purify the target protein under denaturation conditions. The purity of purified IO-EG protein was 88% and the purity of IO-BGL protein was 75.The concentration of IO-EG and IO-BGL protein after dialysis was 0.5 mg L-1 and 0.65 mg mL-1, respectively, and the protein contents were 3.0 mg and 3.6 mg.5., respectively. The purified protein was analyzed by Western Blotting. The band of IO-EG protein was single, which was consistent with the theoretical molecular weight, and the band of IO-BGL protein was consistent with the theoretical molecular weight, but the heterocyclic bands appeared near 35 KDa, presumably due to the degradation of IO-BGL protein.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.3

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