西农萨能羊凝乳酶原前体cDNA的克隆与序列分析.pdfVIP

· 26· Animal Husbandry Veterinary Medicine 2007 Vo1．39 No．10 西农萨能羊凝乳酶原前体cDNA的 克隆与序列分析 王学清 ，杨宝进 ，罗 军 ，唐桂芬 ，李文刚 ，张丽娟 (1．西北农林科技大学动物科技学院，陕西杨凌 712100； 2．郑州牧业工程高等专科学校，河南郑州 450008) 摘要：参照GenBank登录的绵羊凝乳酶原前体cDNA序列设计引物，以新生5 d的西农萨能羊皱胃组织总RNA为模版，通过RT-PCR方法获得凝 乳酶原前体eDNA，克隆测序后进行序列比对。结果表明，克隆基因为B型凝乳酶，该基因cDNA具有l 292个碱基，编码381个氨基酸。包含l6 个氨基酸的信号肽序列和42个氨基酸的酶原序列。将其与已报道的山羊、绵羊和牛的凝乳酶原前体序列进行比对，发现核苷酸同源性分别为 99．4l％、98．74％和95．29％，氨基酸同源性为99．2l％、98．42％和93．70％。 关键词：西农萨能羊；凝乳酶；克隆；RT-PCR 中图分类号：$826．82 文献标识码：A 文章编号：0529-5130(2007)08．0026．04 Cloning and sequence analyses of preprochymosin cDNA of Xinong Saanen Goat WANG Xue．qing ，YANG Bao-jin ，LUO Jun ，TANG Gui．fen ，LI Wen—gang ，ZHANG L-juan (1．Northwest Agriculture and Forestry University，Yanglin 712100，China； 2．Zhengzhou College of Animal Husbandry Engineering，Zhengzhou 450008，China) Abstract：Two pairs of primers were designed based on the sequence of ovis aries prep~chymosin cDNA reported in GenBank．The pre— prochymosin cDNA was obtained from toltal RNA isolated from the abomasum of 5-day-old Xinong Saanen Goat by RT-PCR method and then cloned successfully in E coli．The sequencing results indicated that the cDNA gene coded for chymosin B and had 1 292 nt which coded 38 1 amino acid residues．The sequence of amino acids included a predicted signal peptide region(16 amino acids)and a prochymosin region(42 amino acids)．Comparison of the Xinong Saanen Goat preprochymosin sequence with those of the goat，sheep and cattle mveMed 99．41％， 98．74％，95．29％ and 99．21％，98．42％，94．22％ identity at the nucleotide and amino acid level，respectively． Key words：Xinong Saanen Goat；chymosin；cloning；RT-PCR 凝乳酶是从哺乳动物皱胃中提取出的一种天冬氨酸蛋白 使用 ，FDA认可了3种凝乳酶产品，Gist Brocades公司的克 酶，它可以特异性地裂解K-酪蛋白的