IL-1023-57-PE40分泌表达的初步研究

将IL-1023-57-PE40基因与pelB信号肽融合置于pET-20b构建分泌表达质粒pET-20b-IL-1023-57-PE40,然后将pET-20b-IL-1023-57-PE40分别转化至BL21(DE3),BL21(DE3)pLysS,Rosetta(DE3),E·coliK12TB1,ER2566中。无论是在37℃或是在26℃,亦或在培养基中添加葡萄糖的情况下,IPTG诱导后,IL-1023-57-PE40蛋白只在BL21(DE3)pLysS菌中以可溶分泌形式表达,其中以37℃时培养基中不添加葡萄糖表达量为最高,占菌体蛋白总量的15%,说明蛋白的分泌表达与菌种的选择有关。表达产物经免疫印记检测可被抗PE40的特异抗体识别。通过质粒稳定性实验证明,pET-20b-IL-1023-57-PE40在BL21(DE3)中不稳定,导致蛋白的不表达,在Rosetta(DE3)BL21,E·coliK12TB1,ER2566中稳定但不表达,因此,以Rosetta(DE3)BL21为例,通过SDS-PAGE、DNAStar和ANThewin蛋白分析软件对本室构建的几种PE重组毒素进行比较分析,我们发现:并不是所有PE重组毒素融合信号肽序列后,就能分泌表达,PE重组毒素分泌表达还可能与导向部分的性质有关。

IL-1023-57-PE40 Secretion of Periplasm

IL-10_ 23-57 -PE40 was cloned into the prokaryotic expresion vector containing pelB signal peptide gene,The constucted plasmid pET20b- IL-10_ 23-57 -PE40 were transformed into E.coli BL21(DE3),BL21(DE3)pLysS,Rosetta(DE3),ER2566 and E. coli K12 TB1for proposed protein expression,respectively. Through changing temperature of induction or adding 0.1mol/L glucose in LB,the target protein IL-10_ 23-57 -PE40 was directed to periplasmic space as soluble form only in E.coli BL21(DE3)pLysS and was up to 15% of the total protein. Western blot analysis showed that the fusion protein may react specifically with anti-PE40 antibody. These results maybe explain translocation across the cell membrane of E.coli has to do with the characteristcs of host strain. By rationale for plasmid stability test in E.coli BL21(DE3),Rosetta(DE3),ER2566 and E.coli K12 TB1,pET20b- IL-10_ 23-57 -PE40 was only unstable target plasmids in BL21(DE3),that maybe explain why IL-10_ 23-57 -Pe40 wasn't directed to periplasmic space in BL21(DE3). However,why the target protein didn't export to periplasm of the other host strains in which have stable recombinant plasmidWhen using SDS-PAGE,DNA Star and ANThewin protein software for analysising the mechanism of immunotoxin of pseudomonas exotoxin A,our result indicate: the leader signal is necessary,but not sufficient for export into the periplasm. Translocation across the cell membrane maybe depend on characteristic of targeting agents.