本文選題：金黃色葡萄球菌腸毒素B　切入點：原核表達　出處：《西北農(nóng)林科技大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：金黃色葡萄球菌(Staphylococcus aureus, SA)是一種重要的人畜共患病原菌,革蘭氏染色陽性,可引起多種嚴重感染。由于抗生素的濫用,特別是耐甲氧西林菌株的迅速蔓延,使得依靠抗生素治療金黃色葡萄球菌引起的相關(guān)疾病變得越來越不可靠,疫苗是預(yù)防感染的有效手段,有望解決金黃色葡萄球菌感染這一棘手問題。 本研究根據(jù)金黃色葡萄球菌腸毒素B(SEB)的空間結(jié)構(gòu)模型,對其進行了三位點突變、密碼子優(yōu)化后導(dǎo)入原核表達系統(tǒng)進行表達、純化,并進行了動物免疫保護試驗,取得了以下結(jié)果: (1)于GenBank中查得SEB全序列,根據(jù)其空間結(jié)構(gòu)模型,進行三位點突變,經(jīng)密碼子優(yōu)化、序列合成后成功獲得rSEB基因序列。 (2)將合成后的基因序列經(jīng)Nde I和Hind III雙酶切,并與經(jīng)同樣酶切的pET-22b表達載體進行連接,通過PCR擴增、雙酶切鑒定及核苷酸測序證實原核表達載體構(gòu)建成功。 (3)為了促使目的蛋白以可溶性形式表達,對誘導(dǎo)條件進行了一系列摸索及優(yōu)化,如IPTG濃度、誘導(dǎo)時間、溫度。經(jīng)實驗結(jié)果比較最終確定1 mmol/L IPTG 37℃誘導(dǎo)8小時為最優(yōu)條件。對誘導(dǎo)的目的蛋白進行陰陽離子交換柱純化、BCA蛋白定量及透析袋濃縮后使得所得目的蛋白濃度為10 mg/mL。 (4)將所得目的蛋白免疫Balb/c小鼠,分注射劑型和滴鼻劑型。注射組以氫氧化鋁為佐劑,免疫兩次,滴鼻組以季銨化水凝膠為佐劑,免疫三次,每次免疫間隔兩周。于每次免疫后一周尾靜脈采血,分離血清并進行ELISA效價檢測以評價rSEB蛋白疫苗的免疫原性。注射組二免兩周后、滴鼻組三免兩周后分別進行注射及滴鼻攻毒,并對攻毒后的小鼠取肝、脾、肺、腎作病理組織觀察,以評價rSEB蛋白疫苗的保護效果。結(jié)果顯示,無論是注射途徑還是滴鼻途徑,rSEB均具有較強的較強的免疫原性及良好的保護力。
[Abstract]:Staphylococcus aureus (SA) is an important zoonotic pathogen, which is positive for Gram staining and can cause many serious infections. Due to the abuse of antibiotics, especially the rapid spread of methicillin-resistant strains, It is more and more unreliable to rely on antibiotics to treat Staphylococcus aureus related diseases. Vaccine is an effective means to prevent infection, which is expected to solve the thorny problem of Staphylococcus aureus infection. According to the spatial structure model of Staphylococcus aureus enterotoxin BGSEB, three locus mutation was carried out, the codon was optimized and introduced into prokaryotic expression system for expression, purification, and animal immunity protection test was carried out. The following results were achieved:. 1) the whole SEB sequence was found in GenBank. According to its spatial structure model, the three-site mutation was carried out. After codon optimization, the rSEB gene sequence was successfully synthesized. 2) the synthesized gene sequence was digested by Nde I and Hind III and ligated with the pET-22b expression vector digested by the same enzyme. The expression vector was successfully constructed by PCR amplification, double enzyme digestion identification and nucleotide sequencing. In order to promote the expression of the target protein in soluble form, a series of experiments were carried out to optimize the induction conditions, such as the concentration of IPTG and the induction time. The optimum conditions were determined by comparing the results of the experiment with 1 mmol/L IPTG at 37 鈩，

本文編號：1617363