重组金黄色葡萄球菌肠毒素B蛋白疫苗的制备及免疫效果评价

发布时间：2018-03-15 23:33

  本文选题：金黄色葡萄球菌肠毒素B　切入点：原核表达　出处：《西北农林科技大学》2011年硕士论文　论文类型：学位论文

【摘要】：金黄色葡萄球菌(Staphylococcus aureus, SA)是一种重要的人畜共患病原菌,革兰氏染色阳性,可引起多种严重感染。由于抗生素的滥用,特别是耐甲氧西林菌株的迅速蔓延,使得依靠抗生素治疗金黄色葡萄球菌引起的相关疾病变得越来越不可靠,疫苗是预防感染的有效手段,有望解决金黄色葡萄球菌感染这一棘手问题。 本研究根据金黄色葡萄球菌肠毒素B(SEB)的空间结构模型,对其进行了三位点突变、密码子优化后导入原核表达系统进行表达、纯化,并进行了动物免疫保护试验,取得了以下结果: (1)于GenBank中查得SEB全序列,根据其空间结构模型,进行三位点突变,经密码子优化、序列合成后成功获得rSEB基因序列。 (2)将合成后的基因序列经Nde I和Hind III双酶切,并与经同样酶切的pET-22b表达载体进行连接,通过PCR扩增、双酶切鉴定及核苷酸测序证实原核表达载体构建成功。 (3)为了促使目的蛋白以可溶性形式表达,对诱导条件进行了一系列摸索及优化,如IPTG浓度、诱导时间、温度。经实验结果比较最终确定1 mmol/L IPTG 37℃诱导8小时为最优条件。对诱导的目的蛋白进行阴阳离子交换柱纯化、BCA蛋白定量及透析袋浓缩后使得所得目的蛋白浓度为10 mg/mL。 (4)将所得目的蛋白免疫Balb/c小鼠,分注射剂型和滴鼻剂型。注射组以氢氧化铝为佐剂,免疫两次,滴鼻组以季铵化水凝胶为佐剂,免疫三次,每次免疫间隔两周。于每次免疫后一周尾静脉采血,分离血清并进行ELISA效价检测以评价rSEB蛋白疫苗的免疫原性。注射组二免两周后、滴鼻组三免两周后分别进行注射及滴鼻攻毒,并对攻毒后的小鼠取肝、脾、肺、肾作病理组织观察,以评价rSEB蛋白疫苗的保护效果。结果显示,无论是注射途径还是滴鼻途径,rSEB均具有较强的较强的免疫原性及良好的保护力。
[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，

本文编号：1617363