致倦库蚊后期胰蛋白酶cDNA克隆与序列分析

CLONING AND SEQUENCING OF THREE LATE TRYPSIN cDNA INDUCED FROM THE MIDGUT OF CULEX PIPIENS QUINQUEFASCIATUS

应用逆转录聚合酶链反应 (RT PCR)技术 ,从吸血 2 4h的致倦库蚊广州株蚊虫总RNA中反转录合成后期胰蛋白酶cDNA第 1链。采用自动DNA分析仪进行序列分析 ,并与其他胰蛋白酶进行同源性比较。结果表明 :致倦库蚊后期胰蛋白酶cDNA部分序列分别长 2 16bp、 2 2 9bp和 2 70bp ,命名为Ctry1、Ctry2和Ctry3。Ctry1和Ctry2均可编码 5 0个氨基酸 (1～ 15 0bp) ,氨基酸组成完全相同。Ctry1、Ctry2与致倦库蚊美国株后期胰蛋白酶前体物氨基酸同源性达 86 %。催化中心位点高度保守 (Ser 5 ) ,具胰蛋白酶特异位点(Gly 2 4,Gly 32 )。而Ctry3可编码 76个氨基酸 (1～ 2 2 8bp)。Ctry3编码的氨基酸与果蝇胰蛋白酶前体物的同源性为 6 8%。催化中心位点高度保守 (Ser 5 ) ,具胰酶特异位点 (Gly 2 4,Gly 32 )。以上表明已克隆出致倦库蚊后期胰蛋白酶cDNA。

The cDNA sequence of late trypsin induced by blood meal was amplified by RT PCR from the Culex pipiens quinquefasciatus 24h after blood meal. The cDNA was inserted into pGEM T easy vector and transferred to E.coli JM109. The positive clone sequence was analyzed by automatic DNA sequencing method. The deduced amino acid sequence from the 216bp, 229bp cDNA of late trypsin showed 86% homologous with the late trypsin of Culex pipien pallens, and the deduced amino acid sequence from the 270bp cDNA showed 72% homologous with the late trypsin from Drosophial melanogaster. All the three late trypsins are homologous to that of other trypsins in those residues around the catalytic pocket and the trypsin specificity pocket.