DNA标记杂交技术筛选抗HBs-IFN-α融合蛋白表达载体

为在无抗药性改变 ,转化细胞无生物指示系统改变的克隆载体中扩大筛选范围 ,提高筛选阳性率 ,本文应用地高辛标记DNA检测系统筛选人源单抗片段与干扰素融合蛋白表达质粒 (Anti HBs IFN α ) ,取得了理想的效果。纯化制备载体的插入段DNA ,用地高辛标记系统进行化学标记 ,制成标记探针。挑取单克隆菌扩增 ,以溶菌酶加煮沸法批量制备载体DNA ,点样于硝酸纤维膜上并经 80℃烤 2h ,分别用预杂交液和标记探针进行 42℃ 2h、 42℃ 6h的膜预杂交和杂交反应 ,洗膜后用酶标抗地高辛抗体显色。未带插入片段的抗体表达载体为阴性对照 ,IFN α质粒DNA作阳性对照 ,在 40株转化细胞中筛选出 7个目的克隆。为进一步核实该法的筛选效率和可靠性 ,分别用单酶切和双酶切法对筛选出的阳性载体进行鉴定 ,琼脂糖电泳结果显示 ,两者的符合率为 10 0 %。该系统筛选结果可靠 ,尤其在克隆载体转化细胞后无抗药改变 ,无其他筛选特征的载体构建中有一定的优势

The Screening of Anti-HBs-IFN-a Fusion Protein Expression Vector by Dig-labeled DNA Hybridization in Situ

In this paper,the screening for subclonal IFNα sequence with the Diglabeled probes was demonstrated The IFNα DNA prepared from the IFNα plasmids by PCR method was subcloned into an antiHBs Fab expression vector to express antibodyIFNα fusion proteins Single clone selected,prepared for plasmid DNA,fixed on nitrocellulose membrane,and the PCR product of IFNα was Diglabeled with Boerhinger kit as probes In situ hybridization was performed as the protocol required The experimental results showed that 7 strains of clones with inserts were obtained from 40 strains of the transformed cells,and the hybridization results were confirmed by restriction fragment electrophoresis and the PAGE analysis of the expression products of positive clones