摘要
目的采用链转化法和长距离PCR技术,构建岩栖蝮蛇(Gloydiussaxatilis)毒腺cDNA文库,克隆、分析降纤酶基因。方法用Trizol试剂盒从岩栖蝮蛇毒腺提取总RNA,再用oligo(dT)-生物素-链菌素-磁珠法从总RNA中分离mRNA,反转录合成cDNA第一链,长距离PCR合成第二链cDNA。回收cDNA用限制性内切酶消化产生黏性末端,色谱去除小于500bp片段,纯化的cDNA插入载体pBluescript,电转化E.coliDH5α,得到岩栖蝮蛇毒腺cDNA文库;测定制备的降纤酶上游氨基酸序列,并推导其基因序列,合成引物。从该cDNA文库克隆降纤酶基因并分析基因结构。结果岩栖蝮蛇毒腺cDNA文库的库容为2.0×106,降纤酶基因开框读码序列全长777个核苷酸,编码258个氨基酸,活性中心His65、Asp110、Ser204和6对二硫键进化上高度保守。结论该文库符合建库标准库容要求,为筛选新的目的基因和进一步基因操作提供了有效平台;克隆的降纤酶基因与GeneBank中其它蛇毒丝氨酸蛋白酶氨基酸序列同源性在86%以上。
Purpose To construct a cDNA library by using mRNA from Gloydius saxatilis venom gland, to clone and sequence analysis defibrase from the cDNA library. Methods Total RNA was isolated from venom gland of G. saxatilis , and mRNA was purified by using mRNA isolation kit. Full length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure. Lately cDAN was linked to vector pBluescript-sk. The recombinant cDNA was transformed into E.coli DH5 α. It detected and amplified the cDNA of defibrase gene in the venom gland of G. saxatilis using the hybridization in situ method. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined . Results The capacity of cDNA library of venom gland was above the 2.0×106 . Its open reading frame is composed of 777 nucleotides and codes a pre-zymogen of 258 amino acids. It contains 12 cysteine residues. Conclusion The capacity of cDNA library of venom gland was above the general level of cDNA library. The constructed cDNA library of G. saxatilis venom gland would be a helpful platform to detect new target genes and further gene manipulate. The sequence analysis indicates that the deduced amino acid sequence of the defibrase shares more than 86% identity with the Thrombin-like enzyme genes of other snakes in the gene bank.
出处
《中国生化药物杂志》
CAS
CSCD
2005年第3期151-155,共5页
Chinese Journal of Biochemical Pharmaceutics
关键词
岩栖蝮蛇
毒腺cDNA文库
降纤酶
序列分析
Gloydius saxatilis
venom gland cDNA library
defibrase
sequence analysis
