Cloning and Expression of Leptin Gene and Immunoassay of Leptin in Serum/Plasma
| Autor: | Wu, May-Hua, 吳美華 |
|---|---|
| Rok vydání: | 1998 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 86 Leptin, a 16-kDa peptide hormone secreted by adipocytes, plays a key role in regulation of energy intake, thermogenesis, and body weight. It has been identified to be the products of the leptine gene. Mice with nonsense mutation in the obese gene, which products inactive truncated leptin, have been found to be obese and diabetic and to result in reducting locomotion activity, metabolism and body temperature. Human leptin shares approximately 84% sequence identity with the mouse leptin. But little is known about the physiologic actions of leptin in humans. Therefore, we develop a non-isotopic assay system for monitoring the concentration of leptin in human/rat serum (plasma). In my thesis, we have isolated human leptin cDNA from the cDNA library of fat cell by PCR. The cDNA fragment was cloned into pGEM3Zf(+) and verified by DNA sequencing. Then, the cDNA was regenerated by PCR using primers containing cutting sites and cloned into pQE-31 expression vector. E. coli SG13009 was transfored with the recombinant vector. Recombinant leptin was produced upon IPTG induction. A large amount of recombinant leptin, which formed inclusion body, was ioslated and dissolved in 6M guanidin. Highly purified recombinant-leptin was obtined after passing NTA-Ni column and was used as antigen to immunize rabbit. The anti-serum were then purified by NTA-Ni-recobinant leptin column and protein A column. The titers of antibodies obtained have been determined. An immunoprecipitation mehtod was used to determine the concentration of leptin in human serum/plasma. It appeared that R2 was 0.94 from the standard curve. On the other hand, dot immunoassay method was applied to determine the concentration of leptin im mouse or rat serum/plasma and R2 = 0.98 from the standard curve was achieved. Thereby, the correlation of leptin concentration with some metabolic disease as well as interaction of leptin with other hormone can be studied. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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