猪肥胖基因在大肠杆菌中的高效融合表达  

HIGH EXPRESSION OF PORCINE OBESE GENE IN E. COLI

作　　者：昔奋攻[1] 李宁[2] 戴茹娟[3] 刘兆良[2] 吴常信[3] 

机构地区：[1]新疆农科院微生物所,乌鲁木齐830000 [2]中国农业大学农业生物技术国家重点实验室,北京100094 [3]中国农业大学动物科技学院遗传组,北京100094

出　　处：《动物学报》2001年第5期547-552,共6页ACTA ZOOLOGICA SINICA

基　　金：国家自然科学基金资助项目 (No .39870 5 88)~~

摘　　要：采用PCR方法扩增猪肥胖基因编码的成熟蛋白cDNA序列 ,并在 5′端加上BamHⅠ位点 ,3′端加上EcoRⅠ位点 ,将 5′端密码子CCC转变为大肠杆菌常见密码子CCG ,扩增得到 4 5 9bp的片段 ,克隆于融合表达载体pGEX 2TBamHⅠ和EcoRⅠ位点 ,酶切、测序正确 ,经 0 1mmol/LIPTG诱导表达出一条约 4 2kD的融合蛋白 ,其中 2 6kD为 pGEX 2T中带有的谷胱苷肽转移酶 ,16kD是猪肥胖基因表达产物瘦蛋白。利用非融合表达产品制备抗血清 ,检测融合表达的重组蛋白 ,Western blot为阳性。A 459 bp fragment of porcine obese cDNA, which encodes ob mature protein without signal peptide, was amplified by PCR technique. The DNA fragment has been modified as follow: an EcoR Ⅰsite was included in the 5′ end and the codon CCC of it was changed to CCG, which was more frequently used in E.coli, and a BamH Ⅰsite was designed in the 3′ end. Then the fragment was cloned into expression vector pGEX 2T and the correct construction was confirmed by sequencing. 0 1 mmol/L IPTG can induce high level expression of GST OB fused protein under the control of P tac promote, which consisted of 26 kD GST and 16 kD leptin. The yield of recombinant protein was over 49 1% of total cellular and expressed as inclusion bodies. The recombinant protein was purified with the different pH. Western blotting testing of the porcine expression of pGEMX POB in E.coli BL21 was positive.

关 键 词：猪 肥胖基因 大肠杆菌 高效表达 融合表达 

分 类 号：Q959.842[生物学—动物学]