广西师范大学学报(自然科学版) ›› 2016, Vol. 34 ›› Issue (1): 150-155.doi: 10.16088/j.issn.1001-6600.2016.01.024

• • 上一篇    下一篇

人白介素24融合载体的构建、突变纠正和表达优化

范秋丽, 宋德贵   

  1. 广西师范大学生命科学学院,广西桂林541006
  • 收稿日期:2015-09-29 发布日期:2018-09-14
  • 通讯作者: 宋德贵(1954—),男,广西博白人,广西师范大学教授。E-mail:sondegui@163.com
  • 基金资助:
    “桂科重”研究项目 (14121003-3-1)

Prokaryotic Expression Optimization, Point Mutation Correctionand Cloning of Human Interleukin-24

FAN Qiuli, SONG Degui   

  1. College of Life Science,Guangxi Normal University,Guilin Guangxi 541006, China
  • Received:2015-09-29 Published:2018-09-14

摘要: 以武汉大学病毒学国家重点实验室保存的白介素24(IL-24)为模板,构建原核表达载体pET28a-IL-24,转化大肠杆菌DH5α,挑取单克隆,测序结果显示:在IL-24第18位A→G,21位C→T,412位G→A,18位和21位的突变没有使该位上的氨基酸发生改变,而412位的突变导致了该位点上的氨基酸发生改变——由丙氨酸A变为苏氨酸T。通过设计突变引物,二次PCR将其纠正,转化大肠杆菌BL21。通过改变诱导剂IPTG的浓度、诱导时间和诱导温度摸索最佳诱导条件,SDS-PAGE、Western blot检测显示有融合蛋白6his-IL-24表达。 结果表明:通过二次PCR成功构建了IL-24原核表达载体,表达产物主要以包涵体的形式存在,最佳诱导温度为28 ℃,最佳诱导剂浓度为1 mmol/L,最佳诱导时间是8 h。

关键词: 大肠杆菌, 人白介素24, 原核表达, 二次PCR, 融合蛋白

Abstract: Taking the interleukin (IL-24) in the lab as the template, recombinant prokaryotic vector of expressing pET28a-IL-24 was constructed, the vector was transferred into E.coli DH5α and the monoclones was picked out. The mutant(G→A in IL-24 cDNA)resulted in the change of IL-24 protein(A412T), The mutants(A→G, C→T, in IL-24 cDNA)did not change the amino acids on the bit. By designing mutation primers, correction of mutation was performed by a two-step PCR reaction, the correct recombinant vector was transformed into E.coli. BL21. The optimal condition was confirmed by changing the concentration of IPTG and induction time and temperature, the fusion protein 6his-IL-24 was detected by SDS-PAGE and Western blotting. As a result, by two-step PCR, the prokaryotic expression vector was constructed, the expression product was existed in the form of inclusion body, the best induction temperature was 28 ℃, the optimal IPTG concentration was 1 mmol/L and the best induction time was 8 h.

Key words: Escherichia coli, interleukin-24(IL-24), prokaryotic expression, two-step PCR, fusion protein

中图分类号: 

  • Q51
[1] JIANG H,LIN J J,SU Z Z,et al.Substraction hybridization identifies a novel melanoma-differentiation gene,mda7, modulated during human melanoma differentiation, growth and progression[J].Oncogene,1995,11(12):2477-2486.
[2] XIAO B,LI W,YANG J,et al.RGD-IL-24,a novel tumor-targeted fusion cytokine: expression,purification and functional evaluation[J].Mol Biotechool,2009,41(2):138-144.
[3] 曹亚玲,黄茂涛,黄一,等.靶向肿瘤病毒介导IL-24抑制人胃癌细胞增殖[J].西南国防医药,2014,24(1):9-12.
[4] 祁秋干,周清华,李印,等.人骨髓间充质干细胞表达外源IL-24基因对肺癌细胞A549增殖的影响[J].中国现代医学杂志,2014,24(9):11-16.
[5] SIEGER K A,MHASHIKLAR A M, STEWART A,et al.The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells[J].Mol Ther,2004,9(3):355-367.
[6] SAUANE M,GOPALKRISHNAN R V,SARKAR D,et al.MDA-7/IL-24: novel cancer growth suppressing and apoptosis inducing cytokine[J].Cytokine Growth Factor Rev,2003,14(1):35-51.
[7] 李正袆,缪竞诚,盛伟华,等.Ad-ING4-IL-24对骨肉瘤生长抑制效应的实验研究[J].实用医学杂志,2014,30(3):349-353.
[8] 马薇,李莉,王朝霞.IL-24基因联合顺铂对人宫颈癌Siha细胞淋巴转移抑制作用的研究[J].现代妇产科进展,2015,24(5):343-346.
[9] 于明磊,依莫安,张家骊,等.人白介素24(IL-24)的表达与纯化及其初步活性评价[J].药物生物技术,2013,20(4):314-317.
[10] 杨珺,张家敏,黄卫平,等. N-糖基化IL-24的表达及体外诱导肿瘤细胞凋亡的研究[J].中国药理学通报,2009,25(7): 915-919.
[1] 赵志常, 胡福初, 胡桂兵, 王惠聪, 杨转英, 苏纯兰, 李加强. 荔枝类黄酮糖基转移酶(UFGT)基因的克隆及其原核表达研究[J]. 广西师范大学学报(自然科学版), 2011, 29(4): 104-110.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!
版权所有 © 广西师范大学学报(自然科学版)编辑部
地址:广西桂林市三里店育才路15号 邮编:541004
电话:0773-5857325 E-mail: gxsdzkb@mailbox.gxnu.edu.cn
本系统由北京玛格泰克科技发展有限公司设计开发