[1]张青苗,王雯雯,张会勇,等.分枝杆菌热休克蛋白65重组质粒的构建与原核表达[J].新乡医学院学报,2016,33(12):1028-1030.[doi:10.7683/xxyxyxb.2016.12.003]
　ZHANG Qing-miao,WANG Wen-wen,ZHANG Hui-yong,et al.Construction and prokaryotic expression of recombinant plasmid of heat shock protein 65 from Mycobacterium[J].Journal of Xinxiang Medical University,2016,33(12):1028-1030.[doi:10.7683/xxyxyxb.2016.12.003]
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分枝杆菌热休克蛋白65重组质粒的构建与原核表达
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
33
期数:
2016年12
页码:
1028-1030
栏目:
基础研究
出版日期:
2016-12-05

文章信息/Info

Title:
Construction and prokaryotic expression of recombinant plasmid of heat shock protein 65 from Mycobacterium
作者:
张青苗1; 王雯雯1; 张会勇2; 张光谋2
（1.新乡医学院，河南　新乡　453003；2.新乡医学院生命科学技术学院，河南　新乡　453003）
Author(s):
ZHANG Qing-miao1; WANG Wen-wen1; ZHANG Hui-yong2; ZHANG Guang-mou2
（1.Xinxiang Medical University,Xinxiang 453003,Henan Province,China;2.College of Life Science and Technology,Xinxiang Medical University,Xinxiang 453003,Henan Province,China）
关键词:
热休克蛋白; 重组质粒; 融合蛋白; 原核表达; 分枝杆菌
Keywords:
heat shock protein; recombinant plasmid; fusion protein; prokaryotic expression; Mycobacterium
分类号:
Q813.6
DOI:
10.7683/xxyxyxb.2016.12.003
文献标志码:
A
摘要:
目的　研究分枝杆菌热休克蛋白65（HSP65）重组质粒的构建与原核表达。方法　HSP65的开放阅读框经聚合酶链反应（PCR）扩增后，经Nco Ⅰ与Not Ⅰ内切酶依次酶切，连接至酶切后的原核表达载体PET-28a中，然后将重组质粒转入大肠杆菌BL-21中，PCR筛选阳性克隆，并经DNA测序进一步验证。阳性克隆进一步扩大培养，并进行乳糖诱导，十二烷基磺酸钠聚丙烯酰胺凝胶电泳对菌体蛋白进行检测。结果　成功扩增出1 623 bp的目的片段HSP65；测序结果显示，该片段与目的序列完全相符，重组子PET-28a/HSP65构建成功；乳糖诱导后的HSP65在大肠杆菌中实现高表达。结论　分枝杆菌HSP65蛋白的成功表达，为研究其在抗肿瘤疫苗中的应用奠定了基础。
Abstract:
Objective　To study the construction and prokaryotic expression of recombinant plasmid of heat shock protein(HSP)65 from Mycobacterium.Methods　The open reading frame of HSP65 was amplified by polymerase chain reaction(PCR),cleaved by restriction enzyme Not Ⅰ and Nco Ⅰ,and then ligated with the prokaryotic expression vector PET-28a which cleaved with the same restriction enzyme.The recombinant plasmid was transformed into E.coli BL-21,and the positive clones were selected by PCR,and further confirmed by DNA sequencing.The positive clones was further expanded and induced by lactose.The bacterial protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis.Results　The target fragment HSP65(1 623 bp)was successfully amplified.The results of sequencing showed that the fragment was consistent with the target sequence,and the recombinant plasmid PET-28a/HSP65 was successfully constructed.The high expression of HSP65 in E.coli was successfully induced by lactose.Conclusion　The successful expression of HSP65 protein of Mycobacterium can lay a foundation for studying its application in anti tumor vaccine.

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更新日期/Last Update: 2016-12-05