【作者】 蒋昌龙；

【导师】 张红锋； 张庆华；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 本实验室应用荧光标记的mRNA差异显示技术分析了40例临床胃癌组织、癌旁组织之间表达基因的差异,确定了在基因的mRNA水平上在胃癌及癌旁组织中具有显著差异表达的基因S100P。S100P属于S100基因家族,位于染色体4p16处。近年来研究表明,S100P基因与肿瘤的发生进展关系密切,其表达在多种恶性肿瘤中明显增高,但对于S100P的功能却不清楚。 本研究采用了经典的分子生物学研究路线,即从基因表达蛋白,再制备抗体,从而获得抗S100P的高纯度和高特异性多克隆抗体,为临床胃癌检测提供重要依据和奠定基础。首先通过RT-PCR方法扩增S100P基因全长CDs序列,并将目的基因克隆至原核表达载体pET-22b（+）;并随后将阳性克隆导入大肠杆菌BL21（DE3）PlysS中进行诱导表达;利用pET-22b（+）载体在其多克隆位点末端含有多聚组氨酸的特点,通过Ni²⁺纯化柱进行亲和层析纯化;将纯化的S100P蛋白免疫BALB/C小鼠,制备抗S100P蛋白的多克隆抗体;进而通过Western印迹,在验证抗S100P蛋白多克隆抗体特异性的同时,即可为验证S100P基因在蛋白水平上是否与胃癌的发生、进展密切相关提供研究基础。 通过Western blot印迹实验结果显示,在抗S100P的多克隆抗体以1:16000稀释的情况下,免疫印迹显示了相当好的效果,证明了S100P蛋白的高纯度及多克隆抗体的高特异性。这为进一步研究S100P基因在胃肿瘤中的变化情况及其在胃肿瘤发生、进展过程中的作用打下了基础。更多还原

【Abstract】 S100P, the differentially expressed gene was screened directly by fluorescent differential display reverse transcription polymerase chain reaction (DDTR-PCR) analysis from human gastric cancer and corresponding paratumor tissues in our laboratory. S100P is a member of the S100 family of genes and is located at 4pl6. Recent researches have showed that The S100P gene is significantly related to the development and advance of some malignant tumors. However the function of S100P is not very clear.We adopted the classical molecular biologic means, that is from gene to protein and then to antibody. It makes a foundation for clinically gaistric cancer detection for the highly pure and specific antibody. We first clone its full CDs length into the prokaryotic expression vector pET-22b(+),and the recombinant protein was expressed in the E.coli BL21(DE3)PlysS. Making use of the polyHis tag in the C-deminal of the recombinant protein ,we purified S100P protein. Polyclonal antibody against S100P was generated by immunizing mice with purified S100P recombinant fusion protein. Western blotting indicated that the antiserum against S100P could bind with the expressed recombinant protein specifically and as well as make a foundation of validate the relationship between S100P and gastric cancer.The results showed when diluted to 1:16000, the prepared polyclonal antibody had better immunoreactivity. It strongly testified the high purification of S100P protein as well as high specificity of polyclonal antibody. The study made a foundation of further studying S100P biological function in the development and advance of gastric cancer.更多还原