Subjects: Biology >> Bioengineering submitted time 2017-11-21 Cooperative journals: 《中国生物工程杂志》
Abstract:[目的] 亮氨酸拉链转录因子1(LZTFL1)是一种与纤毛信号传导相关的蛋白,关于其如何在信号传导中发挥作用以及作用机理目前尚不清楚。莱茵衣藻(Chlamydomonas reinhardtii)是研究纤毛信号传导的模式生物,而目前对莱茵衣藻LZTFL1蛋白的研究甚少,尚未有相应的检测抗体,因此制备LZTFL1多克隆抗体用于后续实验研究。[方法] 采用RT-PCR技术从C. reinhardtii CC125中提取总RNA,扩增981 bp的目的基因Lztfl1,插入到pET-28a(+)原核表达载体,成功构建pET-28a(+)-Lztfl1重组质粒,转入E.coli BL21(DE3)经IPTG诱导后成功表达6×His-LZTFL1融合蛋白,融合蛋白经亲和纯化后免疫新西兰大白兔制备多克隆抗体,最后采用间接ELISA法测得抗血清效价达到1:512 000,经Western blot对C. reinhardtii CC125检测具有较高特异性。[结果]首次实现了莱茵衣藻LZTFL1蛋白的原核表达,制备出一支兔抗莱茵衣藻LZTFL1蛋白的多克隆抗体,为后续研究LZTFL1蛋白在莱茵衣藻中的结构功能及在纤毛信号传导中的相互作用奠定了基础。
Subjects: Biology >> Bioengineering submitted time 2017-11-21 Cooperative journals: 《中国生物工程杂志》
Abstract:[目的] 亮氨酸拉链转录因子1(LZTFL1)是一种与纤毛信号传导相关的蛋白,关于其如何在信号传导中发挥作用以及作用机理目前尚不清楚。莱茵衣藻(Chlamydomonas reinhardtii)是研究纤毛信号传导的模式生物,而目前对莱茵衣藻LZTFL1蛋白的研究甚少,尚未有相应的检测抗体,因此制备LZTFL1多克隆抗体用于后续实验研究。[方法] 采用RT-PCR技术从C. reinhardtii CC125中提取总RNA,扩增981 bp的目的基因Lztfl1,插入到pET-28a(+)原核表达载体,成功构建pET-28a(+)-Lztfl1重组质粒,转入E.coli BL21(DE3)经IPTG诱导后成功表达6×His-LZTFL1融合蛋白,融合蛋白经亲和纯化后免疫新西兰大白兔制备多克隆抗体,最后采用间接ELISA法测得抗血清效价达到1:512 000,经Western blot对C. reinhardtii CC125检测具有较高特异性。[结果]首次实现了莱茵衣藻LZTFL1蛋白的原核表达,制备出一支兔抗莱茵衣藻LZTFL1蛋白的多克隆抗体,为后续研究LZTFL1蛋白在莱茵衣藻中的结构功能及在纤毛信号传导中的相互作用奠定了基础。
Subjects: Biology >> Bioengineering submitted time 2017-07-24 Cooperative journals: 《中国生物工程杂志》
Abstract: Polyclonal antibody preparation and application of C.reinhardtii LZTFL1 protein
Subjects: Biology >> Bioengineering submitted time 2017-06-01 Cooperative journals: 《中国生物工程杂志》
Abstract: Cholesterol oxidase is a key enzyme during cholesterol metabolism. It could be potential used to detect the cholesterol level in serum in clinical trials. The cholesterol oxidase from Rhodococcus ruber (ChOG) was transformed into BL21(DE3) and Rosetta(DE3) for protein expression, and was induced at different conditions. The results showed that ChOG was overexpressed at 16 ℃ and 0.1 mmol/L IPTG. The activity reached 8.033 U/mg after purification with Ni-NTA superflow. The product cholest-5-en-3-one was characterized by TLC and HPLC, respectively. The structural analysis showed that Glu406, together with Arg408 and Glu261 played an important role during the dehydrogenation of cholesterol C3-OH, proton transfer, and isomerization.