【作者】 谭树华； 吴梧桐； 刘景晶； 濮颖辉；

【Author】 Tan Shuhua,Wu Wutong,Liu Jingjing,Pu Yinghui (Department of Biological Pharmacy,China Pharmaceutical University,Nanjing 210009)

【机构】 中国药科大学生物制药教研室；

【摘要】 设计合成了两对(四条)寡核苷酸链,将之退火、拼连成一种新型E.coli信号肽简并基因,同时利用密码子的简并生在该基因3’端基因内引入一个NheI限制酶切位点。此外,用PCR技术在水蛭素Ⅲ基因5’端引入NheI限制酶切位点,利用该酶切位点将信号肽基因与水蛭素Ⅲ基因进行连接且保持原有阅读框架不变,构建了重组质粒pUCSH,测序结果表明,融合基因核苷酸序列与设计要求完全一致,将该融合基因插入表达载体pTA中构建成水蛭素Ⅲ分泌表达质粒pTASH,该质粒在大肠杆菌中表达后,表达产物有效分泌至细胞外培养基中,抗凝血酶生物活性达350ATU/ml培养液以上。更多还原

【Abstract】 For the secretory manufacture of HV3,a DNA fragment coding for a novel E.coli signal peptide was assembled from four chemically synthesized oligonucleotides.A single NheI restriction site was designed at the 3’ end of the signal sequence by using the degeneracy of codons.HV3 gene was joined to the signal sequence and the hybrid gene was placed under the control of the tac promotor in a recombinant plasmid pTASH.Flask-shaking fermentation results indicate that HV3 was expressed and secreted into the culture medium,and the yield of biologically active HV3 was>350 ATU/ml culture.更多还原