大鼠IP-10-PE38KDEL重组免疫毒素原核表达载体的构建及表达

Construction and prokaryotic expression of a recombinant immunotoxin IP-10-PE38KDEL

目的构建大鼠γ干扰素诱导蛋白10(IP-10)基因和绿脓杆菌外毒素衍生物PE38KDEL基因的原核融合表达载体,并对其表达进行鉴定。方法通过PCR获得本实验所需要的IP-10基因片段,通过酶切含有PE38KDEL的质粒PRKL459K获得绿脓杆菌外毒素衍生物PE38KDEL基因,再通过适当的酶切及连接反应,构建IP-10与PE38KDEL融合基因的原核表达载体pET-32a(+)-IP-10-PE38KDEL,重组质粒经限制性内切酶、PCR及DNA序列测定证实构建成功后,转化感受态大肠杆菌BL21,经IPTG诱导表达,表达产物经SDS-PAGE及Western-blotting分析证实其相对分子质量和特异性。结果酶切分析、PCR及DNA序列测定证实,IP-10-PE38KDEL融合基因被成功克隆入原核表达载体pET-32a(+),并在大肠杆菌中稳定表达,表达产物的相对分子质量与预期值相符,且所表达蛋白可被抗PE的特异性抗体识别。结论成功构建了原核表达载体pET-32a(+)-IP-10-PE38KDEL,其融合基因在大肠杆菌中高效表达,为进一步研究其功能奠定了基础。

Objective To construct a new recombinant immunotoxin expression vector by fusing rattus IFN-γ-inducible protein gene and a truncated pseudomonas exotoxin A ramification （PE38KDEL） gene, and examine the expression of IP-10-PE38KDEL fusion protein in E coli BL21. nethords IP-10 gene was cloned by polymerase chain reaction （PCR） and the PE38KDEL gene was cleaved from a vector PRKL459K containing PE38KDEL by restriction endonucleases, then inserted to the prokaryotic expression vector pET-32a（ ＋ ）. The prokaryotic recombinant vector pET-32a（ ＋ ）-IP-10-PE38KDEL was identified by PCR, restriction endonuclease digestion, and sequence analysis, and then transformed into E coli BL21 and induced by IFTG, The expressed product was obtained and the molecular weight and specificity of the protein were detected by SDS-PAGE and Western blotting, Results PCR, restriction endonuclease digestion, and sequence analysis revealed that the IP-10-PE38PE38KDEL fusion gene was cloned into the prokaryotic expression plasmid vector pET-32a（ ＋ ） successfully. The IP-10- PE38PE38KDEL fusion protein was expressed in E coli BI221, which could react with the specific antibody. The molecular weight of the expression product was identical to the expected value. Conclusion A prokaryotic expression vector pET-32a（ ＋ ）-IP-10-PE38KDEL is constructed successfully and the fusion protein is expressed stably in E coli BL21, which will provide the basis for the further study on function of the protein.