%0 Journal Article %T 牛胰腺<i>DNase</i>Ⅰ基因的合成、表达及功能验证<br>Synthesis, Expression and Function Identification of Bovine Pancreatic Gene <i>DNase</i>Ⅰ %A 覃鸿妮 %A 谢钰珍 %A 薛高旭 %A 梅啸 %A 沈为琴 %A 蔡一林< %A br> %A QIN Hong-ni %A XIE Yu-zhen %A XUE Gao-xu %A MEI Xiao %A SHEN Wei-qin %A CAI Yi-lin %J 西南大学学报(自然科学版) %D 2018 %R 10.13718/j.cnki.xdzk.2018.06.004 %X 从NCBI下载牛胰腺DNaseⅠ蛋白序列，利用密码子优化软件(Codon Optimization)进行序列优化，设计并合成24条首尾重叠的引物合成786 bp的<i>DNase</i>Ⅰ基因，构建重组载体pET30a-DNaseⅠ，转入BL21(DE3)，ArcticExpress(DE3)，BL21(DE3)pLysS 3种感受态中诱导表达后，纯化目的蛋白并验证其活性.结果表明：BL21(DE3)，ArcticExpress(DE3)两种感受态菌落生长异常，且未纯化到目的蛋白，BL21(DE3)pLysS感受态菌落生长正常，但未经诱导和葡萄糖诱导表达的菌液中也未纯化到目的蛋白，IPTG诱导扩大培养的菌液中纯化到0.15 mg/mL的目的蛋白.经验证，酶原液能彻底消化λ-DNA和不同大小的质粒DNA，酶稀释液消化产物电泳检测显示条带拖尾弥散，表明酶稀释液只能消化部分λ-DNA.<br>Deoxyribonulease I (DNaseⅠ) sequence from bovine pancreas, 786 bp in size, acquired from NCBI and optimized by Codon Optimization, was synthesized, using SOE PCR(gene splicing by overlap extension PCR)with 24 overlapped primers. The recombinant expression vector pET30a-DNaseⅠ was successfully constituted, and was transformed into the competent cells of <i>E. coli</i>, BL21 (DE3), ArcticExpress(DE3) and BL21(DE3) pLysS. Then, DNaseⅠwas purified and its activity was detected. The results showed that the colonies in BL21(DE3) and ArcticExpress(DE3) did not grow properly and no target protein was gained. The colony in BL21(DE3) pLysS induced by glucose or without induction grew normally, but no purified target protein was obtained. In amplification culture of BL21(DE3) pLysS induced by IPTG, 0. 15 mg/mL DNaseⅠwas purified. DNaseⅠactivity detection indicated that the raw liquid of the enzyme could completely dissolveλ-DNA and plasmid DNA of various sizes, while its diluted liquid partially dissolvedλ-DNA only %K < %K i> %K DNase< %K /i> %K Ⅰ %K 基因合成 %K 基因表达 %K 基因功能验证< %K br> %K < %K i> %K DNase< %K /i> %K Ⅰ %K gene synthesis %K gene expression %K gene function identification %U http://xbgjxt.swu.edu.cn/jsuns/html/jsuns/2018/6/201806004.htm