人降钙素在昆虫细胞中的表达

Expression of Recombinant Human Calcitonin in Cultural Insect Cells

目的 :利用昆虫细胞杆状病毒系统表达重组人降钙素 (recombinanthumanCalcitonin,rhCT) ,探索一条真核生物表达生物活性rhCT的新途径。方法 :将人降钙素基因重组到pFastBac杆状病毒穿梭载体(baculovirusshuttlevector ,Bacmid)中 ,构建重组人降钙素杆状病毒表达载体即rhCTBacmid ,并将其转染昆虫细胞Sf9 ,表达目的蛋白。并对表达产物进行分子量及免疫反应性鉴定 ,在初步纯化后对人降钙素表达产物进行大鼠体内低血钙活性检测。结果 :证实目的基因在昆虫细胞中获得了表达,表达产物经大鼠体内低血钙活性测定具有生物活性。结论

Objective:To study the expression of recombinant human calcitonin(rhCT)in cultural insect cells.Methods:The encoding fragment of human calcitonin gene was amplified from plasmid pGex-3x-hCT by means of PCR and inserted into baculovirus transfer vector pFastBacl,formed a recombinant plasmid pFastBaˉcl-hCT.And then,pFastBacl-hCT was transposited with baculovirus shuttle vector(bacmid)in the Max Effiˉciency DH10Bac Competent cells,constructed the expression vector,rhCT Bacmid.The rhCT Bacmid was idenˉtified by PCR and used to transfect the Sf9insect cells to produced the interesting peptide.Results:The exˉpression product was identified with Tricine SDS PAGE and estimated with radioimmunological assay as hCT.The rhCT product showed hypocalcmic activity,in vitro,in rate.Conclusion:The hCT gene was successˉfully expressed in insect cells by the baculovirus eukaryotic expression vector system.The expression product hCT is bioactive.