家蚕丝素重链C末端序列分析、克隆及表达纯化  

Sequence Analysis,Cloning,Expression and Purification of Fibroin Heavy Chain C-terminal

作　　者：刘青松[1,2] 李瑜[1,2] 李珍珍[1,3] 刘莉娜 王叶菁[1,2] 何华伟[1,2,3] 

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400716 [2]西南大学生物技术学院,重庆400716 [3]中国科学院生物物理研究所生物大分子国家重点实验室,北京100101

出　　处：《蚕学通讯》2015年第2期1-6,共6页Newsletter of Sericultural Science

基　　金：西南大学"国家级大学生创新创业训练计划"项目(201410635046);生物大分子国家重点实验室开放基金(2015kf08);中央高校基本科研业务费(SWU112111;XDJK2013A019;SWU112086;XDJK2013C049)

摘　　要：蚕丝主要由丝素和丝胶蛋白组成,其中丝素包括丝素重链,丝素轻链和P25蛋白。丝素重链蛋白分子量大,体外表达困难,难以分离纯化,很难对其结构进行研究。本文通过对丝素重链基因的分析,以家蚕5龄3d后部丝腺cDNA为模板,克隆获得了丝素重链C末端碱基序列,并将其构建到pET-50b(+)表达载体上,转入到大肠杆菌中进行表达,通过镍柱亲和层析纯化获得了丝素重链C末端的融合蛋白,为进一步研究蚕丝蛋白的折叠和组装奠定了研究基础。Silk is mainly composed of fibroin and sericin proteins. Fibroin includes fibroin heavy chain, light chain and P25 protein. It is difficult to study the structure of fibroin heavy chain because it is hard to express and purify such a high molecular weight protein in vitro.. In t：his study, we analyzed the sequence of fibroin heavy chain gene and cloned its C-terminal nucleotide fragment by using the eDNA from the posterior silk gland of fifth instar 3d silk- worm as a template. Then the fragment was subcloned into pET-50b（＋） expression vector. Subsequently, the vector was transferred into E. coli and induced by IPTG to express the C- terminal fragment of fibroin heavy chain. The recombinant NusA-fused protein was purified by nickel affinity chromatography column. Our research will shed light on the folding and as： sembling of silk protein.

关 键 词：家蚕 丝素重链 C末端 克隆 表达纯化 

分 类 号：Q78[生物学—分子生物学]