摘要
目的：探讨白细胞介素18（IL-18）在毕赤酵母中的表达条件及其对BRL-3A细胞的增殖作用和作用机制.方法：构建表达载体p PIC9K-IL-18,电转化至毕赤酵母GS115,PCR、Western blotting和SDS-PAGE方法鉴定表达产物的正确性,并对发酵条件进行优化,再经双水相萃取偶联DEAE离子交换层析分离纯化表达产物.然后,MTT法检测重组大鼠IL-18（rr IL-18）对BRL-3A细胞的增殖作用,RT-PCR和Western blotting方法检测IL-18信号通路相关基因的表达变化.结果：在3 L发酵罐规模下600的菌体密度、p H=6.0、诱导温度23℃、体积分数分别为20%DO和0.25%甲醇浓度条件下表达质量浓度最高约为280 mg/L,经分离纯化后纯度可达95%.此外,质量浓度为15-20 ng/m L rr IL-18孵育48 h,能明显促进BRL-3A细胞增殖（P〈0.5）,且IL18R、My D88、NF-κB及其下游靶蛋白cyclin B1和cyclin B2的表达量均显著高于对照组（P〈0.5）.同时,p38途径的ATF2及其下游靶蛋白cyclin A2和Bcl-2的表达量也随之升高（P〈0.5）.结论：成功构建大鼠IL-18表达载体,优化在毕赤酵母中的发酵条件和纯化工艺,并首次证实IL-18能通过NF-κB和p38/ATF2途径激活细胞增殖相关靶蛋白cyclin B1、cyclin B2、cyclin A2和Bcl-2,进而促进BRL-3A大鼠肝细胞增殖。
Aim：To explore the expression conditions of rat interleukin-18 （rIL-18）in Pichia pastoris and its effects and mechanism on proliferation of BRL-3A cells.Methods：pPIC9K-IL-18 expression vec-tor was constructed and then transformed to GS115 by electroporation.Expression level of the recombi-nant fusion protein was determined by PCR,SDS-PAGE and Western blotting.The induction conditions were optimized and the expression product was isolated and purified by the method of aqueous two-phase extraction coupled with DEAE ion exchange chromatography.The effect of the recombinant rat IL-18＆amp;nbsp;（rrIL-18）on rat liver cells was detected by MTT assay.The expression changes of IL-18-associated genes in the signaling pathway were determined by RT-PCR and Western blot.Results：Under the condi-tions of cell density at 600,pH =6.0,23？C,20% dissolved oxygen （DO）and 0.25% methanol in a 3L bioreactor,and after it was induced for 5d,the highest expression level of rrIL-18 reached 280 mg/L. The purity of rrIL-18 was 95% after purification.It was found that rrIL-18 promoted proliferation of BRL-3A cells at the concentration of 15 -20 ng/mL （P 〈0.05）.Furthermore,at 48 h after treatment,IL-18 receptor increased at both mRNA and protein levels.The expression levels of transcription factor NF-κB, MyD88,and the downstream targets cyclin B1 and cyclin B2 were found remarkably increased.The tran-scription factor ATF2 of the p38 pathway and its targets cyclin A2 and Bcl-2 were also remarkably in-creased.Conclusion：Rat IL-18 expression vector was successfully constructed and the expression condi-tions and purification method were optimized in Pichia pastoris system.It was demonstrated for the first time that IL-18 can promote proliferation of BRL-3A cells via NF-κB and p38 /ATF2 pathways by targe-ting cyclin B1,cyclin B2,cyclin A2 and Bcl-2.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2015年第6期458-466,共9页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家973计划前期研究专项基金项目(2012CB722304)
河南省重大科技攻关项目(111100910600)
