苦瓜蛋白MAP30的原核表达及活性研究

发布时间：2018-05-05 10:21

本文选题：苦瓜 + 原核表达　；参考：《陕西科技大学》2017年硕士论文

【摘要】：苦瓜,在明代《滇南本草》中第一次被记录入药,作为一种古老的药食同源植物,传统中药功效为除邪热,解劳乏,清心明目。随着现代提取分离技术的发展,如今已经从苦瓜中提取得到了多种具有药理活性的物质,包括皂苷、多糖、蛋白及萜类化合物。其中,从苦瓜中分离得到的核糖体失活蛋白MAP30(Momordica anti-HIV protein of 30 KD)具有广谱的抑菌性、抗病毒和抗肿瘤活性,其对未感染的靶细胞及其他正常细胞也表现出极小细胞毒性或细胞抑制效应,因而成为本次研究的具体对象。利用传统提取分离方法去提取MAP30不仅步骤繁琐,而且提取效率低,提取量无法满足后期研究需要。本研究将传统中药学与现代分子技术结合起来,利用基因工程手段去实现MAP30的异源生产,提高MAP30产量,并对异源表达得到的MAP30进行抑菌研究,主要结果如下:(1)MAP30生物信息学分析:MAP30分子质量约为32.02 kDa,氨基酸残基数为286个,理论等电点为9.08,不稳定系数为28.33,在体外哺乳动物网织红细胞中的半衰期为30 h,预测前23位氨基酸MVKCLLLSFLIIAIFIGVPTAKG为信号肽,属于稳定蛋白。(2)MAP30的TA克隆:根据MAP30基因序列设计出一对特异性引物,采用PCR技术扩增到与预期大小相符约900 bp的片段,该片段经回收与克隆载体pMD18-T相连后导入Escherichia coli DH5α,经逐级筛选最终的测序结果表明,克隆得到的基因序列与GenBank中已报道的MAP30序列Blast比对,二者一致性高达99.77%,表明已成功克隆MAP30基因。(3)MAP30原核表达工程菌的构建及诱导表达:用EcoRⅠ及SalⅠ分别双酶切pMD18-T-MAP30、pET28a及pET32a三种质粒,MAP30基因与表达载体pET28a及pET32a分别连接,获得的重组表达质粒pET28a-MAP30及p ET32a-MAP30分别转化至大肠杆菌BL21(DE)及Rosetta(DE3)pLysS中,最终成功筛选出两株阳性重组原核表达工程菌pET32a-MAP30/Rosetta及p ET28a-MAP30/BL21;其中,pET28a-MAP30/BL21工程菌在诱导后,经SDS-PAGE电泳显示未发现与理论值大小相一致的目的蛋白条带,表明该菌株不能成功表达MAP30;p ET32a-MAP30/Rosetta工程菌在诱导后,经SDS-PAGE电泳及Western blot分析均发现与理论值大小相一致的目的蛋白条带,表明该菌株能成功表达MAP30蛋白;pET32a-MAP30/Rosetta工程菌放大培养后收集上清,并经Ni-NTA亲和层析得到纯化后的MAP30蛋白溶液,最终经脱盐冷冻干燥得到蛋白样品,发酵产率能达242 mg/L。(4)MAP30抑菌活性研究:借助牛津杯法得到MAP30融合蛋白的抗菌谱,为革兰氏阳性菌的金黄色葡萄球菌及枯草芽孢杆菌,革兰氏阴性菌的大肠埃希菌及真菌的酿酒酵母;MAP30融合蛋白对金黄色葡萄球菌、大肠埃希菌、酿酒酵母及枯草芽孢杆菌的最低抑菌浓度分别为0.35 mg/mL、0.65mg/mL、0.55 mg/mL及0.75 mg/mL;生长抑制试验表明苦瓜融合蛋白MAP30对金黄色葡萄球菌、枯草芽孢杆菌、大肠埃希菌及酿酒酵母的正常生长都有抑制作用。综上所述,本研究构建了一株能成功表达融合蛋白MAP30的原核表达工程菌,并对该融合蛋白的活性进行了研究,为异源生产MAP30蛋白及该蛋白活性研究奠定基础。
[Abstract]:Bitter gourd is first recorded in the Ming Dynasty "South Yunnan native grass". As an ancient medicine and food homologous plant, traditional Chinese medicine has the effect of eliminating evil heat, removing fatigue, and clearing heart and eyes. With the development of modern extraction and separation technology, many kinds of substances with pharmacological activity have been extracted from bitter melon, including saponins, polysaccharides, proteins and terpenes. Among them, the ribosome inactivating protein MAP30 (Momordica anti-HIV protein of 30 KD) isolated from bitter gourd has broad-spectrum bacteriostasis, antiviral and antitumor activity, which also showed minimal cytotoxicity or cell inhibition to uninfected target cells and other normal cells. The traditional extraction and separation method to extract MAP30 is not only tedious, but also the extraction efficiency is low, and the extraction amount can not meet the needs of late research. This study combines traditional Chinese medicine with modern molecular technology, uses genetic engineering to realize the MAP30 production, raises the output of MAP30, and advances the MAP30 into the heterologous expression. The main results are as follows: (1) MAP30 bioinformatics analysis: the molecular weight of MAP30 is about 32.02 kDa, the amino acid residue is 286, the theoretical isoelectric point is 9.08, the instability coefficient is 28.33, the half-life of the mammalian reticulocyte in vitro is 30 h, and the pre test 23 amino acid MVKCLLLSFLIIAIFIGVPTAKG is a signal peptide. Stable protein. (2) TA cloning of MAP30: a pair of specific primers were designed according to the sequence of MAP30 gene, and PCR technique was used to amplify the fragments corresponding to the expected size about 900 BP. The fragment was introduced into Escherichia coli DH5 alpha after being reclaimed and cloned pMD18-T, and the final sequencing results showed that the sequence of the cloned gene sequence was obtained. Compared with the MAP30 sequence Blast reported in GenBank, the consistency of the two was up to 99.77%, indicating that the MAP30 gene had been cloned successfully. (3) the construction and induced expression of MAP30 prokaryotic expression engineering bacteria: EcoR I and Sal I were used to cut pMD18-T-MAP30, pET28a and pET32a three plasmids, and MAP30 gene was connected with the expression vector pET28a and purified respectively. The recombinant expression plasmid pET28a-MAP30 and P ET32a-MAP30 were transformed into Escherichia coli BL21 (DE) and Rosetta (DE3) pLysS, and two positive Recombinant Prokaryotic expression engineering bacteria pET32a-MAP30/Rosetta and P ET28a-MAP30/BL21 were successfully screened. The target protein strip with the same value and size shows that the strain can not express MAP30 successfully; after the induction of P ET32a-MAP30/Rosetta engineering bacteria, the target protein bands consistent with the theoretical value are found by SDS-PAGE electrophoresis and Western blot analysis, indicating that the strain can successfully reach MAP30 protein and pET32a-MAP30/Rosetta engineering bacteria magnifying culture. After collecting the supernatant, the purified MAP30 protein solution was obtained by Ni-NTA affinity chromatography, and the protein samples were obtained by desalting and freeze drying. The fermentation yield could reach 242 mg/L. (4) MAP30 bacteriostasis: the antibacterial spectrum of MAP30 fusion protein was obtained by the Oxford cup method, which was the Staphylococcus aureus and Bacillus subtilis of Gram-positive bacteria. The minimum inhibitory concentrations of MAP30 fusion proteins to Staphylococcus aureus, Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis were 0.35 mg/mL, 0.65mg/mL, 0.55 mg/mL and 0.75 mg/mL, respectively, and the growth inhibition test showed that the bitter melon fusion protein MAP30 was to Staphylococcus aureus. The normal growth of Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae had inhibitory effects. In summary, a prokaryotic expression engineering strain that could successfully express the fusion protein MAP30 was constructed, and the activity of the fusion protein was studied, which laid the foundation for the study of the production of MAP30 protein and the activity of the protein.

【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.219;Q943.2

【参考文献】