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

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PLA2

Synthetic 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.