乌苏里蝮蛇丝氨酸蛋白酶基因克隆和序列分析  

Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland

作　　者：孙德军[1] 刘珊珊[1] 杨春伟[1] 赵轶卓[1] 常淑芳[1] 颜炜群[1] 

机构地区：[1]吉林大学再生医学科学研究所生化与生物技术药物教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2005年第3期325-329,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题 (39870 375 )

摘　　要：目的:构建乌苏里蝮蛇(Gloydius ussuriensis)毒腺c DNA文库,克隆、分析丝氨酸蛋白酶基因,为进一步基因研究和操作奠定基础。方法:采用链转化法和长距离PCR技术,构建乌苏里蝮蛇毒腺c DNA文库。用TRIzol试剂盒从乌苏里蝮蛇毒腺提取总RNA,再用Oligo(d T) -生物素-链菌素-磁珠法从总RNA中分离m RNA ,反转录合成c DNA第一链,长距离PCR合成第二链c DNA。回收c DNA,用限制性内切酶消化产生粘性末端,层析去除小于5 0 0 bp片段,纯化的c DNA插入载体p Bluescript- sk,电转化E.coli DH 5α,得到乌苏里蝮蛇毒腺c DNA文库,测定丝氨酸蛋白酶上游氨基酸序列,并推导其基因序列,合成引物。从该c DNA文库克隆丝氨酸蛋白酶基因,并进行序列测定和分析。结果:该文库库容为2 .3×10 6 ,该c DNA文库为筛选新的目的基因和进一步基因操作提供了有效平台。序列测定和分析显示丝氨酸蛋白酶基因开框读码序列全长70 2个核苷酸,编码2 34个氨基酸,活性中心His4 1 、Asp86 、Ser1 80 和6对二硫键进化上高度保守。结论:该文库库容大于通用的10 5标准,符合规定可作为进一步基因操作平台,克隆的丝氨酸蛋白酶基因与Gene Bank中其他蛇毒丝氨酸蛋白酶氨基酸序列同源性在85 %以上。Objective To construct a cDNA library by using mRNA from Gloydius ussuriensis(G. ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescript-sk. The recombinant cDNA was transformed into E.coli DH5α. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. 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 2.3×10 6. Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. The query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase Ⅰ of D. acutus and serine protease catroxase Ⅱ of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His 41 ,Asp 86 , Ser 180 ; and six disulfide bridges Cys 7 -Cys 139 , Cys 26 -Cys 42 , Cys 74 -Cys 232 , Cys 118 -Cys 186 , Cys 150 -Cys 165 , Cys 176 -Cys 201 . Conclusion The capacity of cDNA library of venom gland is above 2.3×10 6, overtop the level of 10 5 capicity, The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The clo

关 键 词：蝮蛇属 毒腺 CDNA文库 安克洛酶 序列分析 蛋白质 

分 类 号：Q785[生物学—分子生物学]