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摘要：螺原体粘附到宿主细胞是其侵染的第一步，当其粘附到宿主细胞时可能会引起宿主的免疫反应。本文探究蜜蜂螺原体黏附相关蛋白 ALP609 对宿主意蜂免疫的影响。为了在原核表达系统中表达全长的ALP609基因我们将TGA定点突变成TGG。外源表达的ALP609蛋白免疫意蜂后，利用间接免疫荧光技术检测黏附蛋白在意蜂中肠细胞中的定位，用分光光度计法测定酚氧化酶活性，通过荧光定量 PCR 技术测定免疫相关基因酚氧化酶原基因（proPO）和防卫素抗菌肽基因（Def2）的相对表达量。结果表明，ALP609蛋白具有粘附性；外源表达的ALP609 蛋白免疫意蜂后，宿主酚氧化酶活性略有升高，两个免疫相关基因 proPO 和Def2的相对表达量上升。以上研究证明，蜜蜂螺原体的黏附相关蛋白 ALP609 能够被宿主意蜂的受体识别并引起意蜂的免疫反应，为后续研究其致病机理提供了重要信息。 25796
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毕业论文关键词：螺原体；黏附相关蛋白；蜜蜂；免疫应答
Adhesin-like protein ALP609 of Spiroplasma melliferum induce immune response of Apis mellifera
Abstract ： Adhering to host cells is the first step of spiroplasma infection, which may
cause host immune reactions. In this research, we investigated adhesin-like protein ALP609 of Spiroplasma
melliferum induce immune response of Apis mellifera. The ALP609 gene was site-mutated from TGA to
TGG and transcribed in prokaryotic expression system to full expression. Application of indirect
immunofluorescence technique to detect the presence of adhesion proteins at the midgut cells of Apis
mellifera, using spectrophotometer to measure phenoloxidase activity, qRT-PCR was used for detection of
expression levels of the immune genes proPO and Def2 after protein ALP609 infection. Results show that
protein ALP609 is a kind of adhesion protein, phenoloxidase activity increased slightly in Apis mellifera,
expression levels of the immune genes proPO and Def2 up-regulated. Findings indicate that adhesin-like
protein ALP609 of Spiroplasma melliferum can be recognized by its host Apis mellifera, and cause
host immune reaction, which provides important information for the following researches on the
pathogenic mechanism of Spiroplasma melliferum..
Key words: spiroplasma; adhesin-like protein; Apis mellifera; immune response
目 录
摘要„2
关键词„2
Abstract„2
Key words„„2
引言„2
1 材料与方法„„3
1.1 材料 „3
1.2 实验方法4
1.2.1 黏附蛋白基因定点突变4
1.2.1.1 螺原体总 DNA 提取4
1.2.1.2 螺原体黏附蛋白相关基因的扩增„4
1.2.1.3 表达载体的构建„„5
1.2.2 螺原体黏附相关蛋白的原核表达与纯化„5
1.2.2.1 重组质粒在大肠杆菌中的原核表达„„5
1.2.2.2 重组蛋白纯化„„6
1.2.3 间接免疫荧光验证黏附相关蛋白的黏附性„„6
1.2.3.1 组织细胞分离培养6
1.2.3.2 间接免疫荧光技术7
1.2.4 酚氧化酶活力测定„7
1 . 2. 5 荧光定量 PCR 法基因表达量测定7
2 结果与分析8
2.1 黏附相关蛋白基因定点突变结果„„8
2.2 黏附相关蛋白表达与纯化„„9
2.3 间接免疫荧光验证黏附相关蛋白的黏附性10
2.4 黏附相关蛋白对蜜蜂免疫调节的影响„„11
2.4.1 酚氧化酶活力变化„11
2.4.2 proPO 和 De f2 基因的相对表达量„„11