%0 Journal Article %T 犬弓首蛔虫卵黄原蛋白DUF1943结构域的克隆及原核表达<br>Cloning and Prokaryotic Expression of the DUF1943 Domain of <i>Toxocara canis</i> Vitellogenin %A 罗永莉 %A 朱宏宏 %A 江艾耘 %A 罗永芳 %A 旷策嫣 %A 周荣琼< %A br> %A LUO Yong-li %A ZHU Hong-hong %A JIANG Ai-yun %A LUO Yong-fang %A KUANG Ce-yan %A ZHOU Rong-qiong %J 西南大学学报(自然科学版) %D 2018 %R 10.13718/j.cnki.xdzk.2018.01.003 %X 为克隆犬弓首蛔虫<i>Toxocara canis</i>，<i>T</i>. <i>canis</i>卵黄原蛋白DUF1943结构域(The domain of unknown function 1943，DUF1943)(<i>Tc</i>-<i>duf</i>1943)，构建其原核表达载体pET-28a(+)/<i>duf</i>1943，并检测重组蛋白在大肠杆菌<i>Escherichia coli</i>，<i>E</i>. <i>coli</i>中的表达形式.该文根据<i>T</i>. <i>canis</i>的转录组数据，以<i>T</i>. <i>canis</i>雌、雄虫cDNA为模板扩增<i>Tc</i>-duf1943，然后与表达载体pET-28a(+)连接，转化<i>E</i>. <i>coli</i> BL21(DE3)，经IPTG诱导，对目的蛋白的原核表达形式和蛋白质纯化进行鉴定.结果显示<i>Tc</i>-duf1943序列长度为864 bp，含有一个完整的开放阅读框，编码288个氨基酸；SDS-PAGE电泳结果表明，重组蛋白以包涵体形式存在，相对分子质量大小约为3.5×10<sup>4</sup>；对表达条件进行优化后，以0.4 mmol/L IPTG，于37 ℃诱导4 h时可获得大量目的蛋白；利用Ni-NTA亲和层析柱纯化可以获得具有较高纯度的目的蛋白.该试验成功构建了pET-28a(+)/<i>duf</i>1943原核表达载体，为进一步研究<i>Tc</i>DUF1943的生物学功能奠定了基础.<br>To clone the DUF1943 (the domain of unknown function 1943) of vitellogenin in <i>Toxocara canis</i>, the prokaryotic expression vector pET-28a (+) /<i>duf</i>1943 of <i>Tc</i>-<i>duf</i>1943 was constructed and its expression form in <i>Escherichia coli</i> was detected. According to the transcriptome data about <i>T</i>. <i>canis</i>, and using the cDNA of female and male as templates, <i>Tc</i>-<i>duf</i>1943 was cloned successfully. Then the target gene was connected with the prokaryotic expression vector pET-28a(+) (pET-28a(+)/<i>duf</i>1943), <i>E</i>. <i>coli</i> BL21 (DE3) was transformed and the prokaryotic expression and purification of the protein was identified. The results showed that <i>Tc</i>-<i>duf</i>1943 was 864 bp in length, with a complete open reading frame, encoding 288 amino acids, and SDS-PAGE indicated that the recombinant protein was expressed in inclusion bodies, with a molecular weight of about 3.5×10<sup>4</sup>, The optimal condition of prokaryotic expression was induced by 0.4 mmol/L IPTG for 4 hours at 37 ℃. Purified with Ni-NTA affinity chromatography, target protein with high purity was obtained. In conclusion, this experiment successfully constructed the prokaryotic expression vector of pET-28a(+)/<i>duf</i>1943, which laid a foundation for further biological function study of <i>Tc</i>DUF1943 %K 犬弓首蛔虫 %K DUF1943 %K 克隆 %K 原核表达< %K br> %K < %K i> %K Toxocara canis< %K /i> %K DUF1943 %K clone %K prokaryotic expression %U http://xbgjxt.swu.edu.cn/jsuns/html/jsuns/2018/1/201801003.htm