广西眼镜王蛇毒杂合性酸性磷脂酶A_2的基因克隆与序列分析  

Gene Cloning and Sequence Analysis of a Hybrid Acidic Phospholipase A_2 from Guangxi OphiophagusHannah (King Cobra) Venom

作　　者：林文珍[1] 舒雨雁[1] 庄茂辛[1] 林政炯[2] 吴祥甫[3] 周元聪[3] 

机构地区：[1]广西医科大学蛇毒研究所,南宁530021 [2]中国科学院生物物理研究所生物大分子国家重点实验室,北京100101 [3]中国科学院上海生命科学院生物化学与细胞生物学研究所,上海200031

出　　处：《现代临床医学生物工程学杂志》2002年第6期393-397,共5页Journal of Modern Clinical Medical Bioengineering

基　　金：国家自然科学资金资助(39970 1 74)

摘　　要：目的　研究广西眼镜王蛇毒杂合性酸性磷脂酶A2 (APLA2 )的基因克隆与序列分析 .方法　从广西眼镜王蛇毒腺中提取总RNA ,根据种属关系接近的台湾眼镜蛇毒APLA2 两端非翻译区的保守序列设计引物 ,利用RT -PCR进行体外扩增 ,获得磷脂酶A2 基因 ,克隆至pMD18-T载体 ,经测序筛选出APLA2 - 1,APLA2 - 2及一个新的酸性磷脂酶A2 (命名为APLA2 - 3 ) .根据测序结果确定了APLA2 - 3基因的全序列 ,并由此推导编码的氨基酸序列 .结果　利用Antheprot4 3c蛋白质序列分析软件包计算它的等电点为 4 885 ,相对分子质量为 16 5 43kD ,并预测了它的二级结构和部分理化性质 .同源性比较表明 ,APLA2 - 3的 1～ 3 9位氨基酸残基序列与APLA2 - 2相同 ,其同源性为 64 % ,而 40～ 10 8位氨基酸残基序列与APLA2 - 1相同 ,其同源性为 69% ,10 9位氨基酸残基序列皆不同于这两种APLA2 .结论揭示了APLA2 具有基因多样性 ,可能与物种进化和生物活性有关 ,并为进一步研究PLA2Synthetic oligonucleotides were used to amplify PLA 2 gene by RT-PCR from total RNA of Guangxi Ophiophagus Hannah (King Cobra) gland.The PCR product was cloned into the pMD18-T vector.The positive clones encoding APLA 2-1,APLA 2-2 and a novel APLA 2 which was designed as APLA 2-3 were selected.The complete sequences of APLA 2-3 were determined by sequencing and its amino acid sequences were deduced.Its isoelectated points,molecular mass,secondary structure and partial physic-chemistry character were predicated by Antheprot 4 3 c software.Its protein sequence showed 64% homology with APLA 2-2 and 69% homology with APLA 2-1.These would provided a good basis for further discussing the relation of structure and function,and the studying of evolution of PLA 2 from snake venom.

关 键 词：序列分析 眼镜王蛇毒 磷脂酶A2 基因 克隆 

分 类 号：Q78[生物学—分子生物学]