用双重识别基团分子印迹聚合物富集天然微量蛋白质的研究

【作者】 韩瑞芳；

【导师】 宓怀风；

【作者基本信息】 南开大学 ， 化学生物学， 2009， 博士

【摘要】 本文分为两个部分:第一部分是用蛋白质印迹聚合物分离富集天然微量蛋白质的研究;第二部分是用nested PCR方法阐明编码猪内质网体中FKBP23的cDNA及克隆表达的研究。第一部分的研究工作是在本课题组蛋白质印迹技术研究成果基础上的进一步发展。近年来,许多研究者开展了以蛋白质为模板的分子印迹聚合物的研究,希望能够以此作为分离纯化蛋白质的一种重要手段。在高等生命体中,绝大多数蛋白质在细胞中的含量是非常微小的,一般只占细胞液中总蛋白量的万分之几或者更少,但是它们具有及其重要的生物学功能。分子克隆技术的建立和发展,使得人们可以用分子克隆的手段在大肠杆菌和酵母等低等生命体中表达高等生命体中的微量蛋白质,通过低等生物的大量培养繁衍,得到较大的收获量。尽管克隆蛋白质和天然蛋白质十分接近,但仍具有不同之处。高等生命体中的许多蛋白质都含有修饰基团,这些基团都是在基因表达蛋白质肽链合成后由专一的修饰体系完成的。而在低等生命体中不含有这些专一的修饰体系。这些结构上的差异带来功能上的差别,有时是很大的。因此纯化与鉴定这些天然的微量蛋白质一直是科学家们非常感兴趣的事情。在以前的研究工作中,我们在识别体系中引入了限长的辅助识别聚合物链,提高了对目标蛋白质识别的专一性,取得了很好的富集效果。在本工作中,我们引入带有双重识别基团的辅助识别聚合物链。在聚合物链上随机分布有羧基和吡啶基团作为识别基团。以在大肠杆菌中克隆表达的猪亲环素18蛋白质作为模板。我们将辅助识别聚合物链与模板蛋白质进行自组装,然后将组装体与作为第二单体的丙烯酰胺共同以聚乙烯醇接枝的大孔树脂为固相载体在水相中进行交联聚合,洗脱掉模板蛋白质后就得到了蛋白质印迹聚合物。我们应用该蛋白质印迹聚合物对天然细胞提取物中的蛋白质进行吸附,结果发现在吸附后的洗脱液中,目标蛋白质在总蛋白质中的含量提高了200倍。在洗脱液中目标蛋白质的浓度达到了1.2μg/mL。这样的浓度使我们可以对提取的天然猪亲环素18进行肽脯氨酰顺反异构酶活性（PPIase）的测定。我们发现提取的天然猪亲环素18的PPIase活性高于克隆的猪亲环素18,并且天然的猪亲环素18对外源配体免疫抑制剂环孢菌素A（CsA）的敏感性显著高于克隆的蛋白质。第二部分的研究工作是用nested PCR方法阐明编码猪内质网体中FKBP23的cDNA及克隆表达的研究。我们希望提高蛋白质印迹聚合物的广适性,并且进一步研究纯化的天然蛋白质的性质。我们选取猪FKBP23作为模板蛋白质,因为FKBP23是在内质网体中的一种糖蛋白,具有重要的生物学意义。在内质网体的选择方面,我们选择了猪肝中的内质网体。因为猪肝容易获得且廉价。因此需要克隆表达猪FKBP23为后续的工作奠定基础。由于猪FKBP23的序列并未报道,我们根据已知物种的FKBP23序列的同源性分析,设计引物,PCR及序列分析,得到猪FKBP23的一段序列。再根据获得的序列,应用nested PCR的方法找寻FKBP23的5’及3’端序列,接拼得到632 bp cDNA序列。已申请NCBI的GenBank得到的序列号为No.EU545235。根据序列分析猪FKBP23的结构域及性质。我们根据获得的序列构建重组质粒,在大肠杆菌中克隆表达及提纯猪FKBP23蛋白质。采用亲和吸附法研究发现重组猪FKBP23与内质网体中天然BiP可以特异性结合并且结合受Ca²⁺浓度的调控。为进一步研究蛋白质印迹聚合物的广适性,以猪FKBP23为模板的蛋白质印迹聚合物的制备,及研究纯化的天然猪FKBP23性质奠定了基础。更多还原

【Abstract】 This dissertation is composed of two parts: the first part investigated separation/enrichment of active natural low content protein using protein imprinted polymer; The second part of this dissertation studied de novo cloning of FKBP23 cDNA from pig ER using nested PCR and protein expression.Protein imprinted polymer studied in the first part is based on previous research production of our laboratory. In addition to small molecules, proteins can also be used as templates. There are thousands of types of protein within a cell and most are present at relatively low levels. Indeed, some proteins can exist in just a few copies, even though they perform important biological functions within a cell. Molecular-cloning techniques allow scientists to study these proteins by producing them in large quantities from cloned genes. However, cloned proteins are not identical to natural ones in vivo. Therefore, researchers are interested in purifying natural proteins from cell extracts all the time. In previous work, we reported a new method for protein imprinting with the assistant recognition polymer chains （ARPCs）, which shows good recognition specificity.In this work, we synthesized a new type of the assistant recogniton polymer chains with random distributed double recognition sites. Pyridyl and carboxyl groups were used as recognition sites. Cloned pig cyclophilin 18 （pCyP18） was used as template. The template protein was selectively assembled with ARPCs from their library. These assemblies of protein and ARPCs were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, the synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 200 times. The concentration of natural pCyP18 adsorbed in eluent in this work enables the assay of PPIase activity to be performed. The assay of peptidyl-prolyl cis-trans-isomerase （PPIase） activity showed that natural pCyP18 is more active than cloned pCyP18 and, in particular, it is much more sensitive to the suppressant cyclosporine A （CsA）. The second part of this thesis studied de novo cloning of FKBP23 cDNA from pig ER using nested PCR and protein expression. We hope to improve the eurytopicity of protein imprinted polymer and research natural protein. FKBP23 is a glycoprotein retained in endoplasmic reticulum （ER）. If cloned pig FKBP23 as template protein can be prepared, natural pig FKBP23 in the ER can be acquired by protein imprinted polymer. We are interested in purifying natural FKBP23 and researching the function of glycosyl in FKBP23. Pig liver is a handy and abundant source to acquire the ER.The sequence of pFKBP23 was not reported. According to two highly conservative regions of nucleotide sequences of several detected species, we designed primers and used PCR to amplify the middle segment of the pFKBP23 gene. From this sequence we used nested PCR to extend the nucleotide sequence towards 5’ and 3’ ends for detecting the full length cDNA encoding pig FKBP23. Our result is available from GenBank with accession No. EU545235. We constructed a plasmid to express pFKBP23. Furthermore, we proved that the recombinant pFKBP23 can specifically bind to natural BiP, and the binding is interrelated with the Ca²⁺concentration. These will be a foundation to research natural pFKBP23.更多还原