本文選題：苦瓜 + 原核表達(dá)　； 參考：《陜西科技大學(xué)》2017年碩士論文

【摘要】：苦瓜,在明代《滇南本草》中第一次被記錄入藥,作為一種古老的藥食同源植物,傳統(tǒng)中藥功效為除邪熱,解勞乏,清心明目。隨著現(xiàn)代提取分離技術(shù)的發(fā)展,如今已經(jīng)從苦瓜中提取得到了多種具有藥理活性的物質(zhì),包括皂苷、多糖、蛋白及萜類(lèi)化合物。其中,從苦瓜中分離得到的核糖體失活蛋白MAP30(Momordica anti-HIV protein of 30 KD)具有廣譜的抑菌性、抗病毒和抗腫瘤活性,其對(duì)未感染的靶細(xì)胞及其他正常細(xì)胞也表現(xiàn)出極小細(xì)胞毒性或細(xì)胞抑制效應(yīng),因而成為本次研究的具體對(duì)象。利用傳統(tǒng)提取分離方法去提取MAP30不僅步驟繁瑣,而且提取效率低,提取量無(wú)法滿(mǎn)足后期研究需要。本研究將傳統(tǒng)中藥學(xué)與現(xiàn)代分子技術(shù)結(jié)合起來(lái),利用基因工程手段去實(shí)現(xiàn)MAP30的異源生產(chǎn),提高M(jìn)AP30產(chǎn)量,并對(duì)異源表達(dá)得到的MAP30進(jìn)行抑菌研究,主要結(jié)果如下:(1)MAP30生物信息學(xué)分析:MAP30分子質(zhì)量約為32.02 kDa,氨基酸殘基數(shù)為286個(gè),理論等電點(diǎn)為9.08,不穩(wěn)定系數(shù)為28.33,在體外哺乳動(dòng)物網(wǎng)織紅細(xì)胞中的半衰期為30 h,預(yù)測(cè)前23位氨基酸MVKCLLLSFLIIAIFIGVPTAKG為信號(hào)肽,屬于穩(wěn)定蛋白。(2)MAP30的TA克隆:根據(jù)MAP30基因序列設(shè)計(jì)出一對(duì)特異性引物,采用PCR技術(shù)擴(kuò)增到與預(yù)期大小相符約900 bp的片段,該片段經(jīng)回收與克隆載體pMD18-T相連后導(dǎo)入Escherichia coli DH5α,經(jīng)逐級(jí)篩選最終的測(cè)序結(jié)果表明,克隆得到的基因序列與GenBank中已報(bào)道的MAP30序列Blast比對(duì),二者一致性高達(dá)99.77%,表明已成功克隆MAP30基因。(3)MAP30原核表達(dá)工程菌的構(gòu)建及誘導(dǎo)表達(dá):用EcoRⅠ及SalⅠ分別雙酶切pMD18-T-MAP30、pET28a及pET32a三種質(zhì)粒,MAP30基因與表達(dá)載體pET28a及pET32a分別連接,獲得的重組表達(dá)質(zhì)粒pET28a-MAP30及p ET32a-MAP30分別轉(zhuǎn)化至大腸桿菌BL21(DE)及Rosetta(DE3)pLysS中,最終成功篩選出兩株陽(yáng)性重組原核表達(dá)工程菌pET32a-MAP30/Rosetta及p ET28a-MAP30/BL21;其中,pET28a-MAP30/BL21工程菌在誘導(dǎo)后,經(jīng)SDS-PAGE電泳顯示未發(fā)現(xiàn)與理論值大小相一致的目的蛋白條帶,表明該菌株不能成功表達(dá)MAP30;p ET32a-MAP30/Rosetta工程菌在誘導(dǎo)后,經(jīng)SDS-PAGE電泳及Western blot分析均發(fā)現(xiàn)與理論值大小相一致的目的蛋白條帶,表明該菌株能成功表達(dá)MAP30蛋白;pET32a-MAP30/Rosetta工程菌放大培養(yǎng)后收集上清,并經(jīng)Ni-NTA親和層析得到純化后的MAP30蛋白溶液,最終經(jīng)脫鹽冷凍干燥得到蛋白樣品,發(fā)酵產(chǎn)率能達(dá)242 mg/L。(4)MAP30抑菌活性研究:借助牛津杯法得到MAP30融合蛋白的抗菌譜,為革蘭氏陽(yáng)性菌的金黃色葡萄球菌及枯草芽孢桿菌,革蘭氏陰性菌的大腸埃希菌及真菌的釀酒酵母;MAP30融合蛋白對(duì)金黃色葡萄球菌、大腸埃希菌、釀酒酵母及枯草芽孢桿菌的最低抑菌濃度分別為0.35 mg/mL、0.65mg/mL、0.55 mg/mL及0.75 mg/mL;生長(zhǎng)抑制試驗(yàn)表明苦瓜融合蛋白MAP30對(duì)金黃色葡萄球菌、枯草芽孢桿菌、大腸埃希菌及釀酒酵母的正常生長(zhǎng)都有抑制作用。綜上所述,本研究構(gòu)建了一株能成功表達(dá)融合蛋白MAP30的原核表達(dá)工程菌,并對(duì)該融合蛋白的活性進(jìn)行了研究,為異源生產(chǎn)MAP30蛋白及該蛋白活性研究奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：陜西科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S567.219;Q943.2

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