大鼠IP-10-PE38 KDEL重组免疫毒素真核表达载体的构建及表达  

Construction of eukaryotic expression vector of recombinant immunotoxin rat IP-10-PE38KDEL and its expression in NIH 3T3 cells

作　　者：孙岚[1] 祁志荣[1] 张琴[1] 林媛[1] 李明远[1] 张林[2] 蒋忠华[1] 李虹[1] 

机构地区：[1]四川大学基础医学与法医学院微生物学教研室,成都610041 [2]四川大学华西医院生物治疗国家重点实验室,成都610041

出　　处：《免疫学杂志》2007年第5期482-485,共4页Immunological Journal

基　　金：国家863高科技计划(2002AA214101);国家自然科学基金(30470613)资助

摘　　要：目的构建大鼠干扰素诱导蛋白10(rIP-10)基因和铜绿假单胞菌外毒素衍生物(PE38KDEL)基因的真核融合表达载体,并研究其在NIH3T3细胞中的表达情况。方法通过PCR获得本实验需要的rIP-10基因片段,通过酶切原核表达载体PRKL459K-IL-4-PE38KDEL获得铜绿假单胞菌外毒素衍生物PE38KDEL基因,再通过适当的酶切及连接反应,构建rIP-10与PE38KDEL融合基因的真核表达载体pcDNA3.1(+)-rIP-10-PE38KDEL。重组质粒经限制性性内切酶,PCR及DNA序列测定证实构建成功后,用PolyFect脂质体转染NIH3T3细胞,然后用免疫荧光技术检测rIP-10-PE38KDEL融合蛋白在NIH3T3细胞的表达。结果酶切分析、PCR及DNA序列测定证实,rIP-10-PE38KDEL融合基因被成功克隆入真核表达质粒载体pcDNA3.1(+),免疫荧光技术检测证实重组基因可在NIH3T3细胞表达。结论rIP-10-PE38KDEL融合基因可在NIH3T3细胞中表达,为进一步研究其对自身免疫病的Th1细胞靶向毒性及临床应用奠定基础。Objective To construct a new recombinant immunotoxin expression vector by fusing rat WN-γ-inducible protein gene （rIPlO） and a truncated pseudomonas exotoxin A ramification （PE38KDEL） gene, and explore the expression of the rIP-10-PE38KDEL fusion protein in NIH 3T3 cells. Methods riP- 10 was cloned by polymerase chain reaction （PCR）. PE38KDEL gene was gained from an prokaryotic expression vector plasmid PRKIA59K-IL-4-PE38KDEL by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pcDNA3.1（ ＋ ）. After the eukaryotic recombinant vector pcDNA3.1（ ＋ ）-rIP-10-PE38KDEL was identified by PCR, restriction endonuclease digestion, and sequence analysis, then the vector was transfected into NIH 3T3 cells by liposome protocol. Immunofiuorescence method was used to confmn the expression of the fusion gene in the NIH 3T3 cells. Results PCR, restriction endonuclease digestion, and sequence analysis revealed the rIP-10-PE38PE38KDEL fusion gene was cloned into the eukaryotic expression plasmid vector pcDNA3.1 （ ＋ ） successfully. The pcDNA3.1（ ＋ ）-rIP-10-PE38PE38KDEL fusion gene could express in the NIH 3T3 cell. Conclusion The result provide the basis for research of the targeted cytotoxic activity to Thl cell and may have some potential value in clinical application.

关 键 词：rIP-10 PE38KDEL 重组免疫毒素 NIH 313细胞 真核表达 

分 类 号：R392.11[医药卫生—免疫学]