部分蜘蛛牵丝Ⅰ型蛋白基因的合成及其聚合体在大肠杆菌中的表达  

Synthesis of Spider Dragline Silk Genes and Their Expression in Escherichia coli

作　　者：昔奋攻[1] 朱洪云 李宁[1] 吴常信[3] 

机构地区：[1]中国农业大学农业生物技术国家重点实验室,北京100094 [2]西藏农业大学动物科技学院,灵芝860000 [3]中国农业大学动物科技学院,北京100094

出　　处：《农业生物技术学报》2005年第5期624-628,共5页Journal of Agricultural Biotechnology

基　　金：高技术研究与发展计划(863)项目(No.2002AA206111)资助

摘　　要：已知蜘蛛牵丝Ⅰ型蛋白N端序列不完整,有大量多聚丙氨酸基序组成的重复区。根据已报道的金纺者(Nephila clavipes)相应基因序列,合成若干寡核苷酸链,通过PCR获得编码涵盖三组多聚丙氨酸基序的基因片断。并以此为单体,经过酶切和连接获得重复区分别为2、4、8拷贝的聚合体。序列及RN A二级结构比对显示:所获8聚体与已报道基因编码的氨基酸同源性高达84%,但8聚体基因m RN A具有较低能级。将上述3种聚合体基因分别克隆大肠杆菌(Escherichia coli)表达载体pET-30a(+),经IPTG诱导、利福平阻断菌体基因表达及在培养基中添加35S-M et,通过放射自显影可见由T7启动子引发的特异目的基因表达带。Although complete sequence have not been reported, it has been known that polyalanine repeat domains are evident in the N terminal of spidroin Ⅰ that comprise the dragline silk of Nephila clavipes. 5 oligonucleotides were synthesized on the basis of published sequence data from cDNA clones for spidroin Ⅰ and used to amplification of the monomer sequence in corresponding to three polyalanine blocks. Multimer containing 2, 4 and 8-unit of monomer were obtained respectively using a “head-to-tail” constmction strategy. Sequence analysis showed that the 8-unit multimer had 84% similarity to that of the MaSp Ⅰ, but the former one had a lower mRNA secondary structure energy. Then, these three kinds of multimer genes were cloned respectively into pET-30a （＋）, and induced with IPTG. Protein expression was characterized by the method of labeling and electrophoresis, since the addition of rifampin and 35S-Met together with IPTG blocks the expression of the Escherichia coli genome and only synthesis of the plasmid genes was detected. The result showed that special bands did exist in the cell lysate transformed with interest clone.

关 键 词：牵丝Ⅰ型蛋白基因 基因合成 基因表达 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]