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旋毛虫丝氨酸蛋白酶基因的克隆与分析
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李莲瑞1，付宝权2，卢强3，韩文瑜3，刘明远3*
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（1.新疆生产建设兵团 塔里木畜牧科技重点实验室，新疆 阿拉尔843300；2.中国农业科学院
兰州兽医研究所,甘肃 兰州730046;3.吉林大学 人兽共患病研究所，吉林 长春130062）
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摘要：
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以旋毛虫感染猪血清为抗体探针，对旋毛虫3日龄成虫cDNA文库进行免疫筛选，将获得的阳性克隆进行测序，用分子生物学软件对所得cDNA序列进行了分析。序列分析结果显示，阳性克隆Zh68 cDNA全长为1372bp，含有1个1287bp的完整的开放阅读框架，编码由429个氨基酸组成的多肽，理论分子质量为47.5ku，等电点为8.45。SMART分析表明，1～18位氨基酸为信号肽序列，37～277位氨基酸为典型的丝氨酸蛋白酶胰蛋白酶类结构域，其中His88、Asp142、Ser233为催化中心的3个主要氨基酸残基，78～80位氨基酸为N-糖基化位点（NCS），另外还有6个保守的Cys形成二硫键。BLASTn同源性分析表明，与其他生物丝氨酸蛋白酶的基因序列无明显的同源性，为1个新的cDNA分子。BLASTp蛋白质同源性分析表明，与丝氨酸蛋白酶的同源性为30%左右。Southern-blot杂交表明，此基因在旋毛虫基因组中属于多基因家族，具有基因多态性。cDNA的PCR结果表明，此基因在旋毛虫肌幼虫、新生幼虫及成虫期均有表达。 |
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关键词：
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旋毛虫；丝氨酸蛋白酶基因；克隆 |
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中图分类号：S 852.731:Q 785 文献标识码：
A 文章编号：1673-4696(2008)03-0200-06 |
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Cloning and analysis of serine protease gene of Trichinella spiralis |
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LI Lian-rui1,FU Bao-quan2,LU Qiang3,HAN
Wen-yu3,LIU Ming-yuan3
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（1.Key Laboratory of Tarim Animal Science and Technology,Xinjiang Production and Construction Army Group,
Alar 843300,China;2.Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,
Lanzhou 730046,China;3.Institute of Zoonoses,Jilin University,Changchun 130062,China） |
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Abstract: The cDNA library of three-day-old adult Trichinella spiralis was immunoscreened using the anti-sera from swine experimentally infected with T.spiralis.The positive clones were sequenced and analyzed with bioinformatics softwares.Sequence analysis showed that clone Zh68 contained a cDNA of 1372bp in length with an open reading frame(ORF) of 1287bp.The ORF encoded a polypeptide of 429 amino acid residues with the predicted molecular mass of 47.5ku and pI of 8.45.SMART analysis showed that 1st to 18th amino acid residues of the peptide represent signal peptide sequences,and amino acids 37 to 277 represent a conserved domain of serine protease.His88,Asp142 and Ser233 were three amino acid residues of catalysis active sites,and amino acid 78 to 80 was a N-glycosylation site.There were six conserved Cys residues related to the formation of disulfide bridge.BLASTn homology analysis indicated that it was a novel cDNA without obvious identity with those of serine protease genes of other animals and BLASTp homology analysis showed 30% identity to serine protease.Southern-blot hybridization of genomic DNA of T.spiralis showed that this gene was a member of a gene family with polymorphism.cDNA PCR amplification revealed that this gene was expressed at all developmental stages examined. |
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Key words: Trichinella spiralis;serine protease gene;cloning |
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