| 一种新的精子膜蛋白sp18基因的克隆及其在E.coli 中的表达 |
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| 引用本文： | 柏家林,苟克勉,安晓荣,李瑞国,侯健,陈永福.一种新的精子膜蛋白sp18基因的克隆及其在E.coli 中的表达[J].农业生物技术学报,2003,11(3):268-272. |
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| 作者姓名： | 柏家林 苟克勉 安晓荣 李瑞国 侯健 陈永福 |
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| 作者单位： | 1. 中国农业大学农业生物技术国家重点实验室,北京,100094
2. 中国农业大学农业生物技术国家重点实验室,北京,100094;中国农业大学生物学院,北京,100094 |
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| 基金项目： | 北京市自然科学基金(512007),国家"863"计划(2001AA213021)和国家基金(30270647,30270956)资助项目. |
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| 摘 要： | 摘要：利用sp18 mAb筛选小鼠睾丸λgt11-cDNA表达文库,命名为sp18（GenBank登录号：AF129872）的阳性克隆由 1 468 bp组成,开放阅读框（open reading frame, ORF）长约429 bp,编码142个氨基酸。起始密码ATG位于第694 bp,终止密码位于第1 122 bp,polyA位于1 245~1 250 bp。核苷酸同源性分析表明,sp18 cDNA的10~651 bp与人视神经Hr44的532~ 1 173 bp、sp18 cDNA的10~816 bp、864~1 444 bp与牛视神经Hr44的1 408~2 208 bp、2 250~2 309 bp的cDNA序列有很高的同源性, 高达98%。推测sp18基因可能是生殖系统存在的一种新基因。sp18编码蛋白的理论分子量约为15.7 kD,有8个N-糖基化位点和12个O-糖基化位点,亲水性分析表明sp18多肽是一种精子跨膜蛋白,跨膜区位于第17~33氨基酸处。将sp18 cDNA亚克隆到原核表达载体pET-30a(+),在E. coli BL21（DE3）中得到诱导表达,其表观分子量约为16.5 kD,表达量约占菌体总蛋白的32%。Western blot分析表明sp18 mAb能识别重组的sp18膜蛋白,表明重组蛋白具有免疫原性,为进一步研究sp18基因的功能打下了基础。
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| 关 键 词： | 关键词：精子 sp18基因 分子克隆 表达 |
| 修稿时间： | 2002年4月24日 |
Cloning of a New Sperm Membrane Protein sp18 Gene and Its Expression in E. Coli |
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| Abstract: | Abstract: cDNA encoding for a sperm membrane antigen, designated sperm membrane protein sp18, was cloned and sequenced from murine testis λgt11-cDNA expression library using sp18 mAb. Computr-generated translation analysis of 1 468 bp cDNA yielded an open reading frame(ORF) of 429 bp which deduced 142 amino acids with the first ATG(Met) start codon at nucleotide 694 bp, the stop codon TAA at nucleotide 1 122 bp and a small polyadenylation tail at nucleotide from 1 245 to 1 250 bp. The translated protein, which consists of 142 amino acids, has a calculated molecular mass of 15.7 kD, 8 potential N-linked glycosylation sites and 12 O-linked glycosylation sites. The hydropathy plot generated from deduced amino acids sequence indicated it to be a sperm span-membrane peptide. Comparing the nucleotides of sp18 cDNA with other nucleotides deposited in GenBank , the 10 to 651 bp of sp18 cDNA and 532 to 1 173 bp of human Hr44, the 10 to 816 bp and 864 to 1 444 bp of sp18 cDNA and 1 408 to 2 208 bp and 2 250 to 2 309 bp of bovine Hr44 had 98% similarity respectively. This indicated that sp18 cDNA was a new gene(GenBank accession number:AF129872) specifically localized in reproductive system.The sp18 cDNA was subcloned into pET-30a(+) vector and expressed in BL-21(DE3) of E.coli with molecular mass of 16.5 kD. Western blot indicated that sp18 mAb could recognize sp18 protein and protein possesse immunogenicity. All above laid a foundation of further investigating the function of sp18 gene. |
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