猪肌生成抑制素基因成熟蛋白编码序列表达载体的构建及其转录水平的检测

Construction of expressing vectors of mature protein coding sequence of porcine myostatin gene and examination of the mRNA transcription level

构建猪肌生成抑制素 (MSTN)成熟蛋白编码序列的克隆与表达载体 ,并对mRNA进行转录水平的检测 ,为获得较好的猪肌生成抑制素抗原、制备抗体打下良好的基础。猪MSTN基因的cDNA去除信号肽后 ,用PCR扩增的 1.2kb目的片段与 pMD18 T载体连接 ,将克隆载体的质粒DNA与同样用BamHⅠ和SalⅠ限制性内切酶酶解的表达载体 pET2 8a(+ )质粒定向连接 ,对筛选出的阳性克隆用酶切及测序法鉴定。对猪MSTN基因成熟蛋白编码序列进行反转录 ,对mRNANorthern杂交进行转录水平的检测。结果 ,所得到的序列与所设计的序列完全一致 ,表明成功地进行了猪MSTN基因编码序列的克隆及筛选 ;目的片段定向插入 ,阅读框架正确 ;RT PCR成功地反转录得到约 1.2kb的目的DNA片段 ,Northern杂交后对mRNA印迹放射自显影 ,见到清晰的 1.

Myostatin is a negative regulator of skeletal muscle growth. In order to investigate the relationship between myostatin gene and high lean meat rate and plump hipped trait, The cloning and expressing vectors of mature protein coding sequence of porcine myostatin gene were amplified and constructed. Firstly, the cDNA fragment of porcine MSTN gene's mature protein coding sequence with no signal peptide was amplified, Whose length is 1.2 kb. Secondly,the target fragment was ligated to pMD18 T vector and then transformed into Esherichia coli JM109. The results showed that the cloned MSTN gene was conserved. Thirdly,The plasmid MSTN pMD18 T 029 005 was amplified. The 1.2 kb fragment was cloned into the expression vector pET28a(+). Positive clone was identified successfully. The level of mRNA transcription was examined by RT PCR and Northern blotting, The results showed the mature protein coding sequence of porcine myostatin gene had high transcription level. The results lay a good foundation for the application of MSTN gene to molecular biological technique in animal nutrition and breeding.