猪肌生成抑制素C末端80氨基酸编码基因的合成及其原核表达  

C-terminal 80 Amino Acid Chemical Synthesis and Prokaryotic Expression of Swine Myostatin Gene

作　　者：吕文发[1] 张英霞[1] 欧阳红生[1] 梁冠生[1] 李树伟[1] 张永亮[1] 

机构地区：[1]解放军军需大学军事兽医系,吉林长春130062

出　　处：《兽医大学学报》2003年第3期265-267,共3页

基　　金：国家自然科学基金资助项目 ( 3 0 0 70 5 63 ) ;军队医药卫生基金资助项目 ( 98Q0 79)

摘　　要：将猪肌生成抑制素编码序列下游 883～ 1 1 2 6 bp按大肠杆菌嗜好密码子进行优化 ,将优化后序列双链分成 1 2个单链片段 (F1 、F2 、F3、R6 、R5、R4 、F4 、F5、F6 、R3、R2 、R1 ) ,采用化学合成方法合成 ,每对相邻互补片段之间有 2 0 bp序列交叉重叠。F1 长 38bp,R1 长 36 bp,其他片段均 4 0 bp长 ,F1 和 R1 片段两端分别加上限制性内切酶 Nco 和 Xho 的识别位点序列。经 PCR扩增出 2 5 4 bp片段 ,该片段与 p MD1 8- T载体连接 ,转化 JM1 0 9感受态细胞 ,所得阳性克隆进行测序分析 ,得到的克隆序列与设计的序列完全一致 ,表明成功地获得了猪肌生成抑制素下游编码序列克隆载体。从阳性细菌中提取质粒 ,经 Nco 和 Xho 酶切 ,回收 2 5 4 bp目的片段 ,定向克隆到 p ET2 8a中 ,转化 DH5α受体菌 ,提取质粒 ,再转化到 BL2 1 (DE3)中 ,成功筛选出阳性克隆。阳性菌经 IPTG诱导 ,通过 SDS- PAGE。The coding sequence of swine myostatin optimized according to common codons of E.coli was divided 12 single segments,F 1,F 2,F 3,R 6,R 5,R 4,F 4,F 5,F 6,R 3,R 2 and R 1.These segments were synthesised by chemical means.Each pair of adjancent segments overlaps with 20 bp.The end of F 1 and R 1 are designed with recognition site sequence of restriction enzyme,NcoⅠ and XhoⅠ.A construction Mstnd pMD 18T was generated by inserting the sequence of 254 bp obtained by a PCR into pMD 18T vector and selecting the sense clones.The result showed that the cloned sequence coincides with the designed sequence by sequencing.This construction was digested with NcoⅠ and XhoⅠ and ligated the pET 28a vector digested with the same enzymes using T4 DNA ligase.And a construction was generated by transforming the plasmid MSTN pET 28a to the competent cell of BL21(DE3).The sense clone was induced with IPTG.The expression of swine myostatin gene was determined on SDS PAGE.

关 键 词：猪 肌生成抑制素 C末端 氨基酸编码基因 合成 原核表达 

分 类 号：Q786[生物学—分子生物学] S828[农业科学—畜牧学]