【作者】 陈晓燕；

【导师】 汪志平；

【作者基本信息】 浙江大学 ， 生物物理学， 2006， 硕士

【摘要】 一、SMR1的分离与纯化 通过对3株节旋藻原螺旋形藻丝、由螺旋形变直的藻丝,以及直线形回变成螺旋形的藻丝所作的蛋白质SDS-PAGE分析表明,螺旋形与直线形藻丝间至少存有5个差异蛋白,分别位于53.1、52.0、31.8、21.9和20.3 kDa处。以钝顶节旋藻Sp-S为材料,对其中差异较明显、位于21.9 kDa处的蛋白(SMR1)进行分离与纯化,主要步骤、技术参数与结果如下:(1)采用冻融法破碎节旋藻细胞,SMR1提取液的最佳pH为6.8;(2)硫酸铵分级沉降可有效去除杂蛋白,SMR1主要在硫酸铵饱和度为40～50%的级分中;(3)用Tris-HCl缓冲液溶解硫酸铵沉降级分并依次经Sephacryl S-200凝胶过滤层析、Q-Sepharose FF强阴离子交换层析后,制得了电泳纯的SMR1,提取率约为粗提液总蛋白的0.065%。 二、SMR1的分子特征 纯化后SMR1的双向电泳分析显示,其纯度大于90%,等电点(pI)为4.98;圆二色谱分析结果表明,SMR1的二级结构中α-螺旋、β-折叠和无规卷曲的组成依次为31%、12%和57%。同时,ESI-MS鉴定结果显示SMR1中有5条肽段、共53个氨基酸的序列与藻蓝蛋白连接多肽CpcI的高度一致,且分布于CpcI第30～101位氨基酸残基内;N-端测序结果显示SMR1的N-端序列与CpcI的第30～44位氨基酸序列完全一致;MALDI-TOF MS测得SMR1的精确分子量为16712 Da。由上述结果推测SMR1很可能是一种由CpcI基因表达并经修饰后形成、氨基酸序列与CpcI中第30～171位一致的同源蛋白。考虑到不同藻株间CpcI基因的差异,我们应用PCR等技术从Sp-S中克隆出cpcHID的操纵子并进行测序,进而根据Sp-S中SMR1的核苷酸序列推导得其氨基酸序列。生物信息学分析结果显示,根据SMR1氨基酸序列预测出来的分子量、等电点、二级结构等理论值与测得的实验值非常吻合,并可能由7个α-螺旋形成结构域,在节旋藻形态建成中行使其生物学功能。 三、SMR1的原核表达及抗体制备 由于从节旋藻中提取并分离纯化SMR1的得率低,难以满足抗体制备等实更多还原

【Abstract】 1. The purification of SMR1The proteins SDS-PAGE analyses of the wild-type Arthrospira platensis strains Sp-S, Sp-B and Sp-T with regular helical morphology, and their corresponding linear varieties and the revertant varieties were performed. The results indicated that there were 5 significantly different bands in common between the helical and linear filaments, which were located at 53.1, 52.0, 31.8, 21.9 and 20.3 kDa, respectively. Furthermore, using the follow process and conditions, the most significant band at 21.9 kDa was purified from Sp-S. Firstly, the soluble protein extracts was extracted with Tris-HCl （pH 6.8）, Secondly, in order to remove part of other proteins effectively, the crude extract was precipitated with ammonium sulfate in 40-50% saturation. Finally, the redissolved precipitate was applied to Sephacryl S-200 gel-filtration and Q-Sepharose FF anion-exchange chromatography in turn, and the electrophoresis puritied SMR1 was obtained and its yield was about 0.065%.2. The molecular characteristics of SMR1The result of 2-DE analysis showed that the pI of SMR1 was 4.98 and the purity of prepared SMR1 exceeded 90%. The secondary structure of SMR1 was investigated by Circlar Dichroism analysis, and the result revealed that the content of a-helix, β-sheet and random coil of was 31%, 12% and 57%, respectively. Furthermore, the ESI-MS analysis showed that the detected 5 polypeptides （contain 53 amino acids） belonged to CpcI, and the matched sequences were distributed in the 30^th-101^st amino acid. Moreover, the N-terminal sequence of SMR1 was completely matched with a section of CpcI, and the exact molecular weight of SMR1 was 16712 Da, which was determined by MALDI-TOF MS. According to the results above, it was speculated that SMR1 was a homologue of CpcI with identical amino acid sequence, and was formed by cleavage and modification of CpcI.It was reported that the nucleotide sequences of CpcI were differed withArthrospira strain. Thus the cpcHID operon in Arthrospira Sp-S was cloned and sequenced, and then the amino acid of SMRl was deduced according to the corresponding nucleiotide sequence. Than the theoretical data of molecular weight, pi and secondary structure was predicted and be compared with the experimental one. The result indicated that the two kinds of data were consistent. Moreover, it was revealed from the predicted secondary structure that SMRl domain was composed of 7 a-helix sections, whose function was involved in morphogenesis of Arthrospira, probably.3. Expression and its antibody preparation of SMRlIt was so difficult to prepare sufficient SMRl from Arthrospira for preparing antibody that we turned to express SMRl in E. coli. After studied the expression conditions, such as induced temperature and induced time, it could be concluded that the feasible temperature and time for expression of SMRl were 25-30 °C and 6-12 h respectively. Finally, the expressed product was applied to Ni-affinity chromatography for obtaining SMRl with high purity.Furthermore, the antibody against expressed SMRl was prepared by immunizing the pure-blood New Zealand rabbits for 4 times. The quantity of SMRl for every time was 1. 0.5, 0.5 and 0.5 mg, in turn. Than the antibody （antiserum） was collected in 2 weeks after the last immunization, and was determined its titer and specificity with indirect ELISA and western blot. The results indicated that the antibody against SMR1 was with a titer of 1:51200, and bound specifically to expressed SMRl and Arthrospira SMRl either. Thereby, the function of SMRl in Arthrospira morphogenesis could be revealed by some immunological methods, such as coimmunoprecipation, and protein microarry.Key words: Arthrospira platensis;morphogenesis;protein;purification;molecular characteristic;prokaryotic expression;antibody更多还原