摘要
用KpnⅠ和HindⅢ双酶切pGEM -TB3 克隆质粒 ,得到为大小约为 2 2 5bp的ShT -B基因片段 ,分别将其插入到经双酶切的 pQE4 0和 pQE30表达载体中 ,构建了两个ShT -B的重组表达质粒pQE4 0 -B3 和 pQE30 -B2 ,分别转化到E .coliM15。经IPTG诱导后 ,重组质粒目的蛋白均得到表达。其中 ,pQE4 0 -B3 和pQE30 -B2 ,分别化到E .coliM 15 ,经IPTG诱导后 ,重组质粒目的蛋白均得到表达。其中 ,pQE4 0 -B3 表达蛋白约占菌体总蛋白的 37% ,主要为包涵体形式。pQE30 -B2 表达蛋白约占菌体总蛋白的 16 % ,主要为可溶性形式 ,约 9 2 %。为重组抗原的制备提供了必要的物质基础。
The gene fragment of Shiga toxin B subunit of about 225 bp in length obtained by double cleavage with Kpm and Hind III was inserted into the double-cleavaged cloning vector p GRM-ShB3 and prokaryotic expression vector pQE40 pQE30.Then,the small fragment of ShT-B gene and the large fragment of linearized pQE40 and pQE30 were ligated under T4 DNA ligase,thus forming the recombinant vectors,resigned as pQE40-B3 and pQE30-B2 respectively.These recombinant expression vectors were selected and identified by colony-PCR assay and endonuclease digestions,and finally expressed in E.coli M15 cells.It was found that the transgenetic strain of E.coli M13 cells with pQE40-ShTG3 could highly expressed a fusion protein ShTB-DHFR induced by IPTG.The size of the expressed fusion protein was similar to that of ShTB-DHFR as shown by SDS-PAGE analysis.The target fusion protein occupied 37.1% of the total cell proteins,mainly existing as an inclusion body form,and the pQE30-B2 expressed protein occupied 16% of the total cell proteins,mainly existing in soluble form,approximately 9.2% in the supernatant of cell cultures.These results might provide essential basis for the preparation of recombinant antigens.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2004年第8期679-683,共5页
Chinese Journal of Zoonoses
