摘要温度作为重要的环境因子，对变温动物的生理特征和生存状态具有重要的影响。热激蛋白是变温动物适应环境中的温度变化的重要因子。热激蛋白70（Hsp70）作为热激蛋白家族的重要成员，与免疫密切相关。本论文将通过构建重组质粒pIRESPsHsp70实现Hsp70在大肠杆菌和食用甲鱼及其胚胎中的表达，从分子生物学水平分析食用甲鱼的诱导性热激蛋白70（PsHsp70）。发现在比较严苛的孵化温度34℃下，携带有Hsp70基因的重组质粒pIRES2-EGFP诱导Hsp70在胚胎中的过量表达并没有显著影响新生食用甲鱼和2月龄的幼鳖的生存率，但是其血清中的溶菌酶活性在病原菌注射前后均有显著的改变。由此可见，食用甲鱼Hsp70可不仅在应对环境和生物学压力方面发挥作用，同时有可能参与免疫过程。71268

As an important environmental factor, temperature affects the physiological characteristics and survival of ectotherm significantly. Heat shock proteins(Hsps) are the main molecular factors accioating with ectotherm termal adaption. Heat shock protein 70(Hsp70) is an important family member of Hsps involving in immune response. In this study, according to constructing the recombinant plasmid pIRESPsHsp70 to achieve the Hsp70’ expression in e. coli , soft-shelled turtles and its’ embryo, an inducible Hsp70 homologue (PsHsp70) of Chinese soft-shelled turtle soft-shelled turtles was analyzed at molecular level. The recombinant PsHsp70 was purified from Escherichia coli and showed apparent ATPase activity by spectrophotometric assay. Under harsh temperature 34 °C, overexpression of PsHsp70 carried by pIRES2-EGFP in the turtle embryos did not significantly affect the survival rate of both hatchlings and two months old turtles, but the lysozyme activity of both hatchlings and two months old turtles changed significantly either before or after bacterial challege. Taken together, these results suggest that PsHsp70 is likely to play a role in coping with environmental and biological stresses and may involve in immunity.

毕业论文关键词：食用甲鱼，热激蛋白70，温度，表达水平，死亡率，酶活力

Keyword: soft-shelled turtles; Heat shock protein 70; temperature; expression level; mortality; lysozyme activity

目录

1.引言 4

2.材料与方法 4

2.1菌株和培养条件 4

2.2食用甲鱼的孵化 5

2.3 基因的序列鉴定和分析 5

2.3.1基因的扩增 5

2.3.2 TA克隆 5

2.3.3测序及基因的鉴定 7

2.4质粒的构建 7

2.4.1质粒pEASYPsHsp70的提取 7

2.4.2 DNA片段的获得 7

2.4.3载体的处理 7

2.4.4载体和插入片段的连接转化 8

2.4.5阳性克隆的鉴定：载体引物和片段引物PCR检测 8

2.4.6质粒的提取 8

2.5在卵中实现PsHsp70的过量表达 8

2.5.1将过量表达质粒导入胚胎中 8

2.5.2检测过量表达质粒在食用甲鱼各个组织中的分布 9

2.5.3使用反转录PCR检测质粒的表达 9

2.5.4免疫能力测定 9

2.6数据分析 Hsp70过量表达对食用甲鱼细菌感染死亡率的影响:http://www.youerw.com/shiping/lunwen_80891.html