高原鼠兔(Ochotona curzoniae)leptin的原核表达、纯化及真核表达载体构建

NWIPB OpenIR

	高原鼠兔(Ochotona curzoniae)leptin的原核表达、纯化及真核表达载体构建
其他题名	Prokaryotic Expression, Purification of Plateau Pika(Ochotona curzoniae) Leptin and Establishment of Its Eucaryotic Expression Vector
	邓治莲
学位类型	硕士
导师	赵新全
	2009-06-01
学位授予单位	中国科学院西北高原生物研究所
学位授予地点	西北高原生物研究所
关键词	高原鼠兔 Leptin 原核表达载体真核表达载体表达纯化
摘要	高原鼠兔（Ochotona curzoniae）是生活在青藏高原3000m以上区域的小型非冬眠植食性哺乳动物，是青藏高原上的关键种和特有种，具有极强的低温低氧耐受能力。Leptin由ob基因编码，是主要由白色脂肪组织分泌的167个氨基酸组成的蛋白，其可与分布于中枢和外周的多个组织器官中的leptin受体结合，发挥多种重要的生物学功能，其中最主要的功能是调节能量代谢。高原鼠兔leptin被看做冷压力敏感蛋白，与高原鼠兔的低温耐受性密切相关。高原鼠兔leptin原核表达以高原鼠兔leptin cDNA为模版PCR扩增高原鼠兔ob基因编码区序列，利用DNA基因重组技术将目的片段克隆到原核表达载体pET30a(+)中，构建高原鼠兔leptin蛋白原核表达载体pET30a(+)/ppleptin，然后将其转化到BL21大肠杆菌中，诱导外源性目的蛋白表达并纯化。高原鼠兔的真核表达以高原鼠兔leptin cDNA为模版PCR扩增高原鼠兔ob基因编码区序列，利用DNA基因重组技术将其克隆到真核表达载体pPICZα中，构建高原鼠兔leptin蛋白真核表达载体pPICZα/ppleptin，并利用电转化法将其整合到毕赤酵母的基因组中，筛选酵母重组子。本研究中，高原鼠兔leptin的原核表达纯化以及真核表达载体的构建，为进一步研究高原鼠兔leptin功能奠定了基础。主要研究结论如下： 1. 高原鼠兔重组质粒pET30a(+)/ppleptin经质粒双酶切、菌液PCR和测序表明原核载体构建正确；SDS-PAGE凝胶电泳图显示，高原鼠兔leptin原核表达成功且纯化获得较高纯度目的蛋白。 2. 琼脂糖凝胶电泳结果、测序和电转化入毕赤酵母后菌液PCR均显示高原鼠兔leptin真核表达载体pPICZα/ppleptin构建成功。
其他摘要	Plateau pika (Ochotona curzoniae) is an endemic and keystone species living at 3000-5000 m above sea level in the Qinghai-Tibet plateau. In evolution, plateau pika has become a hypoxia and low temperature tolerant mammal with markedly high resting metabolic rate (RMR), nonshivering thermogenesis (NST), and a high ratio of oxygen utilization to cope with its cold and hypoxic plateau environment. Leptin, an adipocyte-derived hormone, plays an important role in body energy homeostasis acting on its receptors in hypothalamus and other several peripheral tissues, exerts diverse biological effects. The full-length pika leptin cDNA was 3015 with 504 bp open-reading frame encoding the precursor peptide of 167 amino acids including 21 residues of signal peptide. Plateau pika leptin is closely related to cold tolerance. It’s necessary to expression recombination plateau pika leptin for studying its molecular mechanism. The gene sequence encoding pika leptin was constructed into the prokaryotic expression vector pET30a(+) by DNA recombination techniques. When the construction was confirmed correctly by enzymatic digestion and sequence analysis, the recombinant plasmid of pika leptin was transformed into E. coli BL21 for expression under the induction of IPTG. The recombinant protein was purified and identified by SDS-PAGE. The gene sequence encoding pika leptin was constructed into the eucaryotic expression vector pPICZα by DNA recombination techniques. Linearize the vector pPICZα/ppleptin with Sac I to transform pichia pastoris strain X-33 by eletroporation. Positive transformants were selected on the YPDS plates with Zeocin. This study used molecular biology methods to make pika leptin gene express and purify in the prokaryotic and constructe eucaryotic expression vector, which provided base materials of the deeper research in this filed. This study maybe summarized as follows: 1. The prokaryotic expression vector of pleteau pika leptin was correctly constructed and the higher purity leptin was obtained successfully. 2. The construction of pPICZα/ppleptin was confirmed correctly by enzymatic digestion and sequence analysis; it’s successfully constructed of transformed X-33 by PCR analysis.
页数	50
语种	中文
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/3240
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	邓治莲. 高原鼠兔(Ochotona curzoniae)leptin的原核表达、纯化及真核表达载体构建[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2009.

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