【作者】 徐岩；

【导师】 曹广力；

【作者基本信息】 苏州大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 人胰岛素样生长因子(hIGF-Ⅰ)是一个小分子多肽,具有多种生物学效应及临床应用价值。本研究将hIGF-Ⅰ基因(588 bp)克隆进原核表达载体pET-28a(+),在大肠杆菌中获得了与6×组氨酸的融合表达。Western blotting显示表达的hIGF-Ⅰ分子量约为26 kD,与理论值相符,表明hIGF-Ⅰ表达产物未经加工,表达产物中含有载体融合序列及基因中信号肽序列等编码的氨基酸序列。将hIGF-Ⅰ全cDNA基因克隆进pBacPAK8,获得了杆状病毒转移载体pBacPAK8-IGF-Ⅰ,在脂质体的介导下,与线性化的家蚕杆状病毒共转染家蚕培养细胞BmN,经空斑筛选,PCR检测,获得了重组病毒BmNPV-IGF-Ⅰ。SDS-PAGE检测表明,在感染重组病毒后,家蚕幼虫血淋巴中可以检测到两条分子量约为7.5 kD和22 kD的特异性条带,ELISA检测表达量达4.51μg/mL蚕血淋巴。在家蚕中表达的蛋白中,分子量约为7.5 kD的表达产物,与加工完善的hIGF-Ⅰ分子量一致(70个氨基酸),表明来源于真核生物的人hIGF-Ⅰ基因的信号肽也可能在家蚕杆状病毒表达系统中得到正确识别与加工;另外在感染后期,可见有一约22 kD的表达产物特异条带,与hIGF-Ⅰ完整基因推测的蛋白质分子量理论值相符,推测该产物可能未经成熟作用而保留了信号肽序列和C端部分序列。更多还原

【Abstract】 A 588bp gene sequence (hIGF-I) of the whole cDNA of human insulin-like growth factor (hIGF-I) was synthesized. The sequence was cloned into pET-28a(+). The hIGF-I expression was performed in E.coli. SDS-PAGE and Western blotting suggests that the hIGF-I has the molecular weight of about 26kDa. The hIGF-I was cloned into pBacPAK8 to form pBacPAK8-hIGF-I plasmid. Recombinant virus Bm-Bac-hIGF-I was generated by cotransfection into BmN cells with the transfer plasmid pBacPAK8-hIGF-I and the modified Bombyx mori nuclear polyhedrosis virus Bm-BacPAK6 genomic DNA. SDS-PAGE shows that recombinant protein expressed in pupae are about 7.5 kDa and 22 kDa in molecular weight, and expression level of 4.51μg per milliliter hemolymph was detected by ELISA. These results show that the insect expression system has the ability to modify the protein from senior animal.. The protein expressed in Bombyx mori of 7.5 KDa is the same as the molecular weight of the mature hIGF-I. In the end of infection period, there is a band of 22 KDa in the SDS-PAGE which is equal to the theory. The speculation is the expression can not be modified thoroughly.更多还原