摘要
目的构建能特异性杀伤银屑病皮损中T淋巴细胞的重组免疫毒素hIL-2-Luffin P1。方法应用PCR扩增目的基因片段hIL2-Luffin P1,然后克隆到表达载体pET32a(+)中,并对重组质粒进行酶切鉴定及基因测序。测序正确的重组质粒转化到表达菌BL21中,进行诱导表达。诱导表达正确的蛋白,经过蛋白的纯化、复性、脱盐、肠激酶酶切、再纯化,最后用兔抗人IL-2多克隆抗体对目的蛋白进行Western blot鉴定。结果构建了pET32a(+)-hIL-2-Luffin P1表达载体。最后得到了表达正确的hIL2-Luffin P1蛋白。结论重组免疫毒素hIL-2-Luffin P1的成功构建为银屑病的治疗奠定了实验基础。
In order to develop a new recombinant immunotoxin hIL-2-Luffin P1 for specifically killing T lymphocytes,target gene fragments hIL2-Luffin P1 were amplified by PCR,and then subcloned into an expression plasmid pET32a（＋）.After identification by restriction enzyme digestion and DNA sequencing,the correct sequencing recombinant plasmid was transformed into the expression strain BL21 to express.The correctly expressed protein was purified,refolded,desalinated,enterokinase digested,and then repurified for Western blot analysis by using rabbit anti-human IL-2 polyclonal antibody.Finally not only expression vector pET32a（＋）-hIL-2-Luffin P1 was successfully constructed,but also the expression of right hIL2-Luffin P1 protein was obtained.Thus,the construction of hIL2-Luffin P1 fusion protein would be a foundation of psoriasis treatment.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2012年第2期128-132,137,共6页
Immunological Journal
基金
中国人民解放军324医院院管课题(201002)
重庆市科研基金资助项目(CSTC
2007BB5089)
