Cloning and Characterization of a Batroxobin-like cDNA from Trimeresurus mucrosquamatus

• Main Authors: Liang, Mei-Chih, 梁美智
• Other Authors: Chao, Chung-Fays; Guo, Yaw-Wen
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/00616696247889587043

碩士 === 國防醫學院 === 解剖學研究所 === 82 === Trimeresurus mucrosquamatus, one of the commonest land venomous snake in Taiwan, belongs to the Subfamily Crotalinae. The venom contains many valuable components including the thrombolytic enzymes which have therapeutic potential in stroke and human heart disease, and neurotransmitters used as tools in neurobiological research. However,it is hard to get quantitatively protein material for investigate.In this study, we constract a λ gt11 cDNA library from Trimeresurus mucrosquamatus venom glands and analyzing the cloned cDNAs.The optimal time forRNA expression in the venom gland were analyzed by histological observation using hematoxylin and eosin, and wheat germ agglutinin stained paraffin sections. Morphological evidence indicate that maximal RNA expression was 24 hours after venom milking were presumably in correlated with maximum RNA synthesis. Poly (A) RNAs purified at this period were used to construct a cDNA library. The phage titer of the cDNA library was 1.0 x 108 pfu/ml, and the recombinant efficiency was higher than 80%. Polyclonal antiserum against the whole venom were used to screen the cDNA library and identified two positive clones (TMV1 and TMV2). DNA sequence of TMV1b shared 90% homology with batroxobin, a thrombin-like snake venom protein and TMV2 exhibited 92% similarity with trigramin, a platelet aggregation inhibitor. Northern blot analysis revealed a tissue specific expression of the TMV1. The 1.4 Kb TMV1 mRNA was identified only in venom gland among 6 organs of the snake investigated. These results indicated that a high quality of constructed.