碩士 / 國立臺灣大學 / 生化科學研究所 / 90 / Alpha-crystallins are classified as a group of small heat proteins. Joseph Horwitz has demonstrated that they played a role as molecular chaperone to prevent thermal aggregation of other crystallins and enzymes in animal lens. The main purpose of the thesis is to study the characterization of carpfish α-crystallins . To isolate and determine the primary sequences of α-crystallins, cDNA mixture was synthesized from ploy(A) mRNA prepared freshly from carpfishs and cDNA encoding α-crystallin was then amplified by polymerase chain reaction (PCR) based the two primers designed according to the relatively conserved N-terminal sequences of known crystallin from other vertebrates and oligo-T anchor . PCR-amplified products corresponding to α-crystallin were obtained and subcloned into pGEM vector; and then transformed into E. Coli stain JM109. Plasmids purified from the positive clones were prepared for DNA sequencing.
Three DNA sequences were obtained including one each of α-A ，α-B crystallins and one special DNA sequence α-Bd. There is a notable similarity between αB andαBd crystallin. The difference betweenαB andαBd is αBd lock 41 amino acid in the middle of sequence. Make a comparison with carpfish α-crystallin sequence and terrestrial vertebrateα-crystallin sequence , we find carpfish α-crystallin sequence length is shorter than other vertebrate’s. Next, We assay the chaperone activity of α-crystallin expression by E. Coli . Finally, we found three α-crystallin protein all process chaperone activity and αBd chaperone activity is lower than others .In the thermal stability expermint , we find thermal stability of αA and αBd are very good . After 90℃ heating , they didn’t aggregation. However, thermal stability of αB is not good. After 60℃ heating , αB formed aggregation and insoluilization. The characterization analysis of these carofish α-crystallin proteins points to a greater sequence diversity of fish than other vertebrate species.
| Identifer | oai:union.ndltd.org:TW/090NTU00103005 |
| Date | January 2002 |
| Creators | Shih-Hao Tseng, 曾士豪 |
| Contributors | Shih-Hsiung Wu, Fu-Ming Pan, 吳世雄, 潘扶明 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 0 |
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