奶牛骨钙素基因的克隆与原核表达

Cloning and Prokaryotic Expression of Bovine Osteocalcin Gene

以奶牛骨组织提取的RNA为模板,利用RT-PCR技术扩增出奶牛骨钙素全长cDNA,然后将扩增产物重组到PMD-18T载体中,测定了全基因的核苷酸序列。序列分析表明,奶牛骨钙素全长cDNA为303bp,编码100个氨基酸,与GenBank中的X53699的序列完全相同。通过加端PCR技术连接单链DNA片段人工定点同义突变,将奶牛骨钙素成熟蛋白基因中的大肠杆菌稀有密码子同义突变为大肠杆菌常用密码子并亚克隆至PET-32a表达载体,转化到宿主菌BL21(DE3)中,经IPTG诱导后,成功表达出奶牛骨钙素融合蛋白。

Bovine osteocalcin eDNA gene was amplified from bone of bovine by RT-PCR .PCR product was cloned into PMD-18T plasmid, Sequencing analysis showed that bovine osteocalcin eDNA gene was 303 bp and encoded 100 amino acids, completely matched with X53699 in GenBank. through connecting three artificial single strands DNA which contain fixed-point mutated sites by add-on PCR,we changed some rare codons to the codons that Escherichia coli frequently used in mature bovine osteocalcin gene,then recombined the connected production into PET-32a plasmid and transformed into the competent expressive cells of E.coli BL21 （DE3）.Recombinant E.coli BL21 was induced by IPTG and osteocalcin fusion protein was successfully expressed.