| 鲤肠道sglt1基因的表达与抗体制备 |
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| 引用本文： | 聂国兴,王贝,闫潇,侯彩霞,张建新,张新胜,郑俊林,王俊丽.鲤肠道sglt1基因的表达与抗体制备[J].水产学报,2012,36(3):329-335. |
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| 作者姓名： | 聂国兴 王贝 闫潇 侯彩霞 张建新 张新胜 郑俊林 王俊丽 |
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| 作者单位： | 河南师范大学生命科学学院,河南新乡,453007 |
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| 基金项目： | 国家自然科学基金项目(30972252);河南省高校科技创新杰出人才项目(2010HASTIT020);河南省重点科技攻关项目(112102210106,112102310321) |
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| 摘 要： | 钠/葡萄糖共转运载体1(sodium/glucose cotransporter 1,Sglt1)是协助葡萄糖吸收的主要蛋白。实验首先采用RT-PCR获取sglt1基因全长,克隆至PGME-T载体进行序列及免疫原性分析,选择长度为92个氨基酸(544～637)的多肽作为目的片段(sglt1-P)),扩增sglt1-P,引入双酶切位点EcoRⅠ和HindⅢ后,连接至pET-32a(+)上,构建表达载体pET-32a(+)-sglt1-P,转化至E.coli Rosetta中,获得重组基因工程菌,通过IPTG诱导表达,获得目标多肽,并以此为抗原制备Sglt1特异性抗体。SDS-PAGE电泳分析表明,目标多肽分子量约为30 ku。采用耳缘静脉结合皮下注射,免疫新西兰长耳兔,免疫总时长为38 d。制备了鲤Sglt1抗体,ELISA测得效价为1∶105;免疫组化结果表明,抗体具有较高亲和力和特异性,可以应用于鲤Sglt1的表达定位研究。该抗体的获得为鲤肠道Sglt1表达及转运活性的系统研究奠定了基础,同时,获取的Sglt1抗体亦可用于其它鱼类Sglt1转运蛋白表达定位和定量研究。
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| 关 键 词： | 鲤 钠/葡萄糖共转运载体1 原核表达 ELISA 抗体效价 |
| 收稿时间： | 2011/11/7 0:00:00 |
| 修稿时间： | 2011/12/9 0:00:00 |
Expression of sglt1 gene in Cyprinus carpio and preparation of its polyclonal antibody |
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| NIE Guo-xing,WANG Bei,YAN Xiao,HOU Cai-xi,ZHANG Jian-xin,ZHANG Xin-sheng,ZHENG Jun-lin and WANG Jun-li.Expression of sglt1 gene in Cyprinus carpio and preparation of its polyclonal antibody[J].Journal of Fisheries of China,2012,36(3):329-335. |
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| Authors: | NIE Guo-xing WANG Bei YAN Xiao HOU Cai-xi ZHANG Jian-xin ZHANG Xin-sheng ZHENG Jun-lin and WANG Jun-li |
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| Institution: | (College of Life Sciences,Henan Normal University,Xinxiang 453007,China) |
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| Abstract: | The high-affinity Na+/glucose contransporter Sglt1 is one of the important members of the sodium:solute symporter family(SSF),belonging to the homologous family 5(SLC5).The Sglt1 plays an important role in accumulating glucoses from intestinal or kidney epithelial cells against an adverse concentration gradient and maintaining the adjustment of metabolism.So far,few studies on Na+/glucose cotransporter have been reported in the freshwater fishes.This research would develop special antibodies of Sglt1,which could supply the foundation for the research of glucose metabolism in Cyprinus carpio intestines on molecular level.To study the molecular mechanism of glucose metabolism in freshwater fishes,the full-length cDNA of sglt1 in C.carpio with an ORF of 1 977 bp was cloned and the antigenicity of Sglt1 was first predicted.92(544-637)amino acids with strong antigenicity and immunogenicity were selected as target section of sglt1 in C.carpio and were cloned into plasmid pET-32a(+)vector which had restriction sites for EcoRⅠ and HindⅢ.The rcombinant plasmid named pET-32a(+)-sglt1-P was transformed into E.coli Rosetta.The Sglt1-P fusion protein of approximately 30 ku was highly expressed in E.coli Rosetta after being induced with IPTG.The purified fusion protein was used as antigen to immunize New Zealand Rabbits with ear margin veins by subcutaneous injection with power.The results of ELISA showed that the titer of the antiserum was about 1∶ 105.The Sglt1-P polyclonal antibody was used to determine the expression of Sglt1 protein in C.carpio intestines through immunohistochemical method.The result revealed that the antibody performed high affinity and specificity and could be applied to study the expression and locating of Sglt1 in the C.carpio.The Sglt1-P polyclonal antibody also could serve as an important research tool to study Sglt1 expression and transshipment activity of C.carpio.Meanwhile,the Sglt1-P polyclonal antibody could also be used to exploratively study the expression location and quantity of the Sglt1 transporters in other fishes. |
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| Keywords: | Cyprinus carpio sodium/glucose cotransporter 1(Sglt1) prokaryotic expression ELISA antibody titer |
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