摘要
根据GenBank中鸡卵泡抑制素α亚基序列设计引物,对该序列按大肠杆菌嗜好密码子进行优化,在设计的引物两端分别加上限制性内切酶SacⅠ和XhoⅡ的识别位点序列。利用RT PCR技术从溆浦鹅卵泡的颗粒细胞总RNA中扩增出抑制素α亚基成熟区序列,经PCR扩增出354 bp片段,该片段与pMD18 T载体连接,转化JM109感受态细胞,所得阳性克隆进行双酶切鉴定和PCR鉴定,并进行了测序分析,得到的克隆序列与设计的序列基本一致。表明成功地获得了抑制素α亚基编码序列的克隆载体。从阳性细菌中提取质粒,经SacⅠ和XhoⅠ酶切,回收354 bp目的片段,定向克隆到pET28a中,转化DH5α受体菌,提取质粒,再转化到BL21(DE3)中,成功筛选出阳性克隆。阳性菌经IPTG诱导,通过SDS PAGE,检测出溆浦鹅卵泡抑制素α亚基编码区的表达。
The Inhibin α-subunit coding sequence of Xupu goose optimized according to common codons of E.coli.The ends of F_1 and R_1 were designed with recognition sites restriction enzyme,SacⅠ and XhoⅡ.Maturation zone of Inhibin α-subunit was amplificated from granular cell of Xupu goose ovarian follicle by RT-PCR.The recombinant plasmid pMD-18T-IB was constructed by inserting the fragment of 354 bp obtained by PCR into pMD-18T vector.The sense clone from JM109 cell was evaluated by double enzyme digestion and PCR.DNA sequence analysis showed that the cloned sequence was the very fragment we designed. Those showed we obtained clone vector of Xupu goose inhibin α subunit.This construction was digested with SacⅠ and XhoⅠ and ligated the pET-28a vector digested with the same enzymes using T4 DNA ligase.One construction was generated by transforming the plasmid IB-pET-28a to the competent cell of BL21(DE3).The sense clone was induced by IPTG.The expression of Swine myostatin was observed on SDS-PAGE.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2005年第6期536-539,共4页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
吉林省自然科学基金项目(20020654)