红色荧光蛋白mRuby2的优化表达
毕业论文关键词：红色荧光蛋白；mRuby2；接种培养方法；优化表达。
Optimal expression of red fluorescent protein mRuby2
Abstract： Using the red fluorescent protein mRuby2 (recombinant expression plasmid pBMR in host cell KRX)，the effects of culturing temperature and inoculation method on the expression of recombinant mRuby2 were investigated by scanning the excitation spectrum and emission spectrum, and measuring their peak fluorescent brightness. The results showed that the excitation peak of the recombined fluorescent protein mRuby2 was 561 nm, while the emission peak was 598 nm, slightly different from their values of pure protein. Its excitation spectrum and emission spectrum, similar to those of pure protein, were single peak spectrum. After comparing its expressions at 24℃ and 37℃, detailed comparison of three inoculation methods was carried out with 37℃ culturing temperature. Generally, single colony inoculation expression method was significantly better than both ordinary two-step inoculation expression method and one-ring inoculation expression method. The fluorescent brightness could be significantly increased by storing the expression cells in 4℃ refrigerator.
Key words: red fluorescent protein; mRuby2; inoculation method; optimized expression.
目 录
摘要1
关键词1
Abstract1
Key words1
引言（或绪论）1
1 材料与方法2
1.1 材料 2
1.1.1 载体和细胞2
1.1.2 主要试剂2
1.1.3 主要仪器2
1.2方法 2
1.2.1荧光蛋白质表达克隆pBMR(pRSETB/mRuby2)的构建2
1.2.2两个培养温度、三种诱导表达培养方法2
1.2.3诱导培养后的低温处理3
1.2.4红色荧光蛋白mRuby2波峰值的确定3
1.2.5数据分析3
2结果与分析3
2.1重组表达载体的构建3
2.2红色荧光蛋白mRuby2光化学特性的初步测定分析3
2.3三种接种方法荧光强度比较4
2. 3. 1激发和发射波谱扫描4
2. 3. 2单点荧光强度4
2.4 37℃下不同接种培养方式单点荧光强度统计分析5
2. 4. 1不同接种培养方式单点荧光强度方差分析5
2. 4. 2图表5
3 讨论 6
致谢7
参考文献7
红色荧光蛋白mRuby2的优化表达
较早的红色荧光蛋白(red fluorescent protein，RFP)是从珊瑚纲生物Anthozoa corals中分离出来的一类与绿色荧光蛋白（green fluorescent protein，GFP）相似、在激发光的照射下可以发射出红色的荧光[1]。在多种颜色的成像实验中需要红色探针，在活细胞及动物全身成像中也需要表现较好的红色荧光蛋白；加上基于较长的激发波长产生的光毒性较小，可以用来探测较深的生物组织，红色荧光蛋白质有其独有的应用需要[13]。自 1999年第一个红色荧光蛋白质DsRed被开发应用以来，红色荧光蛋白以其激发和发射波长较长、细胞内成像背景低、细胞毒性小而备受关注。迄今为止，RFP自开发应用后为改良光化学特性，产生了多种突变体如mCherry、mApple等。另一个光化学性能更好红色荧光蛋白质是从奶嘴海葵（Entacmaea quadricolor)开发得到的mRuby2[2]。它是mRuby的单亚基突变体，它有亮度高等特点。与最初的红色荧光蛋白相比，改良的荧光蛋白质有成熟时间短、荧光强度强、发射波长更长、单体等优点[3]。红色荧光蛋白质的性能的改良使它们更多地应用于分子标记以及蛋白相互作用等研究[8]。
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