[1]曹　顺,杨玉露,徐光华,等.重组人白血病抑制因子的原核表达及优化[J].新乡医学院学报,2019,36(3):228-233.[doi:10.7683/xxyxyxb.2019.03.006]
　CAO Shun,YANG Yu-lu,XU Guang-hua,et al.Prokaryotic expression and optimization of recombinant human leukemia inhibitory factor[J].Journal of Xinxiang Medical University,2019,36(3):228-233.[doi:10.7683/xxyxyxb.2019.03.006]
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重组人白血病抑制因子的原核表达及优化
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
36
期数:
2019年3
页码:
228-233
栏目:
基础研究
出版日期:
2019-03-05

文章信息/Info

Title:
Prokaryotic expression and optimization of recombinant human leukemia inhibitory factor
作者:
曹　顺1; 杨玉露1; 徐光华2; 陈思佳3; 王天云3
(1.新乡医学院临床医学专业2014级，河南　新乡　453003；2.新乡医学院临床医学专业2015级，河南　新乡　453003；3.新乡医学院生物化学与分子生物学教研室，河南　新乡　453003)
Author(s):
CAO Shun1; YANG Yu-lu1; XU Guang-hua2; CHEN Si-jia3; WANG Tian-yun3
(1.Grade 2014,Clinical Medicine Specialty,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;2.Grade 2015,Clinical Medicine Specialty,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;3.Department of Biochemistry and Molecular Biology,Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
关键词:
人白血病抑制因子; 原核表达; 优化
Keywords:
human leukemia inhibitor factor; prokaryotic expression; optimization
分类号:
R34
DOI:
10.7683/xxyxyxb.2019.03.006
文献标志码:
A
摘要:
目的　在大肠杆菌中表达重组人白血病抑制因子(HLIF)蛋白，并探索其高效表达的条件。方法　将HLIF重组质粒转化至大肠杆菌BL21，分别给予不同诱导时间（4、8、12、16、20、24 h）、不同浓度大肠杆菌（吸光度值分别为0.5、0.6、0.8、1.0）、不同诱导温度（20、25、30、37、42 ℃）、不同浓度诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)（0.1、0.2、0.3、0.4、0.5 mmol·L-1）、不同浓度乙酸（0.0、0.1、0.2、0.3、0.4 mmol·L-1）进行培养，蛋白电泳考马斯亮蓝染色后，采用 Image J 软件分析HLIF蛋白的相对表达量，观察不同干预条件对HLIF表达的影响。结果　不同诱导时间下HLIF蛋白相对表达量比较差异有统计学意义(F=22.088，P<0.05)，诱导16 h时的HLIF蛋白相对表达量显著高于诱导4、8、12、20、24 h(P<0.05)。不同浓度（以吸光度值表示）大肠杆菌诱导时HLIF蛋白相对表达量比较差异有统计学意义(F=17.153，P<0.05)；吸光度值为0.8时HLIF蛋白相对表达量显著高于吸光度值为0.5、0.6 时(P<0.05)，吸光度值为1.0 时与吸光度值为0.8时HLIF蛋白相对表达量比较差异无统计学意义（P>0.05）。不同温度诱导时HLIF蛋白相对表达量比较差异有统计学意义(F=18.053，P<0.05)，25 ℃时HLIF蛋白相对表达量显著高于20、30、37、42 ℃ 时(P<0.05)。不同浓度诱导剂诱导时HLIF蛋白相对表达量比较差异无统计学意义(F=1.181，P>0.05)。不同浓度乙酸诱导时HLIF蛋白相对表达量比较差异有统计学意义(F=27.181，P<0.05)，乙酸浓度为0.00 mmol·L-1时HLIF蛋白相对表达量显著高于乙酸浓度为0.1、0.2、0.3、0.4 mmol·L-1时（P<0.05）。结论　成功在大肠杆菌表达了HLIF，并探索出了适合最佳表达的诱导时间、大肠杆菌浓度、诱导温度、诱导剂浓度、乙酸浓度等条件。
Abstract:
Objective　To recombine and express human leukemia inhibitory factor (HLIF) recombinantly in E.coli and explore the conditions for its high expression.Methods　The HLIF plasmid was transformed into E.coli BL21.The transformed BL21 were cultured at different induction time (4,8,12,16,20,24 h),concentration of E.coli （absorption value was:0.5,0.6,0.8,1.0),induction temperature (20,25,30,37,42 ℃),concentration of isopropyl-β-D-thiogalactoside (IPTG) (0.1,0.2,0.3,0.4,0.5 mmol·L-1) and concentration of acetic acid(0.0,0.1,0.2,0.3,0.4 mmol·L-1).After protein electrophoresis with Coomassie brilliant blue staining,the relative expression of HLIF protein was analyzed by Image J software,and the effect of different intervention conditions on the expression of HLIF was observed.Results　There was significant difference in the relative expression of HLIF protein at different induction time (F=22.088,P<0.05).The relative expression of HLIF protein at 16 hours of induction was significantly higher than that at 4,8,12,20 and 24 hours of induction (P<0.05).There was significant difference in the relative expression of HLIF protein with different concentration of E.coli (F=17.153,P<0.05).The relative expression of HLIF protein at absorbancy value of 0.8 was significantly higher than that at absorbancy value of 0.5 and 0.6 (P<0.05).There was no significant difference in the relative expression of HLIF protein between absorbancy value of 1.0 and 0.8 (P>0.05).There was significant difference in the relative expression of HLIF protein at different induction temperatures (F=18.053,P<0.05).The relative expression of HLIF protein at temperature of 25 ℃ was significantly higher than that at 20,30,37 and 42 ℃ (P<0.05).There was no significant difference in the relative expression of HLIF protein between different induction concentrations of inducer (F=1.181,P>0.05).There was significant difference in the relative expression of HLIF protein at different concentrations of acetic acid (F=27.181,P<0.05).The relative expression of HLIF protein at acetic acid concentration of 0.00 mmol·L-1 was significantly higher than that at acetic acid concentrations of 0.1,0.2,0.3,0.4 mmol·L-1 (P<0.05).Conclusion　HLIF was successfully expressed in E.coli,and the optimum conditions were determined,such as the induction time,concentration of E.coli,induction temperature,concentration of inducer and acetic acid.

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更新日期/Last Update: 2019-03-05