论文部分内容阅读
神经生长因子(NGF)是最早发现的神经趋化因子。它对神经细胞具有营养与保护作用,对损伤神经具有调节与修复功能。为大量获得重组人NGF,本研究采用构建杂合克隆因子(hybrid clonemid)进行DNA操作,使NGF基因与pET-16 b载体重组。根据人NGF基因序列合成一对PCR引物,并在引物的5’端加上与载体互补的一小段序列(21bp)。用此引物进行PCR扩增,获得NGF基因扩增产物。将载体pET-16b用Nde I酶切使其成为线性载体(为避免克隆过程中载体自身转化的本底,此步酶切要完全)。为使NGF基因位于正确的阅读框架上并与因子Xa特异酶切位点相接,在得到酶切完全的线性载体后,应用T4DNA聚合酶的3’→5’外切酶活性,将3’端AC去除
Nerve growth factor (NGF) is the earliest discovered neurochemotactic factor. It has nourishment and protection of nerve cells, damage the nerves with regulation and repair functions. In order to obtain a large amount of recombinant human NGF, a hybrid clonemid was constructed for DNA manipulation to recombine the NGF gene with the pET-16b vector. A pair of PCR primers was synthesized based on the human NGF gene sequence, and a small sequence complementary to the vector (21 bp) was added to the 5 ’end of the primer. Using this primer for PCR amplification, the NGF gene amplification product was obtained. The vector pET-16b was digested with Nde I to make it a linear vector (this step should be completely digested to avoid the background of the vector self-transformation during cloning). In order to locate the NGF gene in the correct reading frame and connect with the factor Xa-specific restriction enzyme site, after obtaining the linear vector with the complete digestion, the 3 ’→ 5’ exonuclease activity of T4 DNA polymerase was used to convert the 3 ’ Side AC removal