hISO基因原核表达载体的构建及其在大肠杆菌中的表达及鉴定

doi:10.13481/j.1671-587x.20160112

Abstract

Abstract:

Objective: To construct the prokaryotic expression vector pET-28a(+)-hISO and to explore the expression of hISO fusion protein in E.coli,and to provide a foundation for follow-up experiment research.Methods: The 1 770 bp fragment of hISO gene was amplified from the total RNA of Caco-2 cells by RT-PCR and inserted into pET-28a(+)to construct the recombinant plasmid pET-28a(+)-hISO.The recombinant plasmids were transformed into E.coli BL21(DE3)to induce the protein expression with IPTG after PCR,digestion and DNA sequencing.The recombinant plasmid was analyzed and identified by SDS-PAGE and Western blotting method.Results: The recombinant plasmid pET-28a(+)-hISO was constructed successfully after identified by PCR,digestion and sequencing. The fusion protein was 68 860 when the soluble fusion protein was purified,and using elution buffer containing 40 mmol·L^-1 and 60 mmol·L^-1 imidazole could get high concentration and purity protein.Conclusion: The prokaryotic expression vector pET-28a(+)-hISO is constructed successfully and the recombinant protein is expressed in the E.coli BL21(DE3).

Key words: isomaltase, prokaryotic expression, α-glucosidase, Escherichia.coli

CLC Number:

Q786

SHAO Min, WANG Xinying, LIU Xing, WANG Yan, ZHOU Hefeng, CE Zhenglong. Construction of prokaryotic expression vector of hISO gene and its expression in E.coli and identification[J].Journal of Jilin University Medicine Edition, 2016, 42(01): 59-63.

References

[1] Lee BH,Eskandari R,Jones K,et al. Modulation of starch digestion for slow glucose release through "toggling" of activities of mucosal α-glucosidases[J].J Biol Chem,2012,287(38):31929-31938.

[2] Rodríguez D,Ramsay AJ,Quesada V,et al.Functional analysis of sucrase-isomaltase mutations from chronic lymphocytic leukemia patients[J].Hum Mol Genet,2013,22(11):2273-2282.

[3] 陈锐,聂海洋,陈锦英,等.糖尿病肾病机制的中西医研究进展[J].中国老年学杂志,2014,34(22):6515-6517.

[4] Kiadaliri AA,Gerdtham UG,Eliasson B,et al.Health utilities of type 2 diabetes-related complications: a cross-sectional study in Sweden[J].Int J Environ Res Public Health,2014,11(5):4939-4952.

[5] Sinharay K,Paul UK,Bhattacharyya AK,et al.Prevalence of diabetic foot ulcers in newly diagnosed diabetes mellitus patients[J].J Indian Med Assoc,2012,110(9):608-611.

[6] Skrepnek GH,Armstrong DG,Mills JL.Open bypass and endovascular procedures among diabetic foot ulcer cases in the United States from 2001 to 2010[J].J Vasc Surg,2014,60(5):1255-1264

[7] 王谦,张璐,边晓丽,等.α-葡萄糖苷酶抑制剂及构效关系的研究进展[J].中国新药杂志,2014,23(2):189-195.

[8] Striegel L,Kang B,Pilkenton SJ,et al.Effect of lack tea and black tea pomace polyphenols on α-glucosidase and α-amylase inhibition,relevant to type 2 diabetes prevention[J].Front Nutr,2015,2(3):1-6.

[9] Wubshet SG,Moresco HH,Tahtah Y,et al.High-resolution bioactivity profiling combined with HPLC-HRMS-SPE-NMR: α-Glucosidase inhibitors and acetylated ellagic acid rhamnosides from Myrcia palustris DC.(Myrtaceae)[J].Phytochemistry,2015,116:246-252.

[10] Cheng MW,Chegeni M,Kim KH,et al.Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing[J].J Clin Biochem Nutr,2014,54(1):55-60

[11] 郝峰,王皓,许会静,等.anoctamin 1在中国仓鼠卵巢细胞中的表达及其离子通道特性[J].吉林大学学报:医学版,2014,40(6):1226-1231.

[12] 田灵芝,徐美娟,饶志明.一株重组大肠杆菌/pET-28a-lpgad 的构建及其高效生产γ-氨基丁酸转化条件的优化[J].生物工程学报,2012,28(1): 65-75.

[13] Kim MJ,Park HS,Seo KH,et al.Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli[J].PLoS One,2013,8(2):e56168.

[14] 苏雅拉图,张永胜,陈宇杰,等.重组小鼠IFN-β原核表达载体的构建及包涵体溶解条件的筛选[J].吉林大学学报:医学版,2013,39(4):841-846.

[15] 钟洁,陈乐然,刘洋,等.可介导多药耐药的cfr基因的原核表达及多克隆抗体的制备[J].中国兽医科学,2014,44(10):1060-1065.

Construction of prokaryotic expression vector of hISO gene and its expression in E.coli and identification

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 13

Metrics

Comments

Recommended 0

[1]	LI Huimin, YI Huanfa. Prokaryotic expression and purification of Toxoplasma gondii profilin protein [J]. Journal of Jilin University Medicine Edition, 2017, 43(06): 1109-1114.
[2]	LI Song,YANG Li-bin,ZHANG Shu-yan,JIN Yu,CHEN Xiu-hua,LI Xin,LIU Ling,LI Shu-lei,LI Shu-hong. Prokaryotic expression and identification of recombinant Cystatin of Cyprinus carpio var.Jian and preparation of its polyclonal antibody [J]. Journal of Jilin University Medicine Edition, 2014, 40(01): 204-209.
[3]	Suyalatu,ZHANG Yong-sheng,CHEN Yu-jie,HU Xiao-ping,WANG Li-ying,WAN Min,WU Qi-zhu. Construction of prokaryotic expression vector of recombinant mouse IFN-β and screening for solubilization conditions of inclusion body [J]. Journal of Jilin University Medicine Edition, 2013, 39(4): 841-846.
[4]	SONG Tian-yi*,LI Jing-hua,ZHANG Zhe,SHI Hong-yan，SUN Yan-bo. Construction of prokaryotic expression vector of nonstructural protein 1 of Tick-borne encephalitis virus [J]. Journal of Jilin University Medicine Edition, 2013, 39(3): 620-624.
[5]	LIU Tong,LI Yang,LI Dan-ni*,GENG Nan-xi,LI Feng. Construction of GST-tagged prokaryotic expression plasmids of human PAK6 gene and expressions of their recombinant proteins [J]. Journal of Jilin University Medicine Edition, 2013, 39(3): 437-440.
[6]	ZHANG Shi-yao,PU Yun,XU Kun,LI Jin-hua,YUAN Ye,JU Wen,LI Juan. Expression and antigen activity identification of severallinked antigen epitopes of consensce genes of Leptospira  [J]. J4, 2012, 38(3): 404-408.
[7]	XU Li-juan,WEN Jian-ping,SHI Hong-yan,SUN Yan-bo. Construction and expression of human papillomavirus type-16 E7 gene in prokaryotic expressing vector [J]. J4, 2011, 37(4): 661-664.
[8]	CHANG Yu-Dong, ZHANG Zheng-Yao, DING Yun-Lu, CHEN Qing-Wu, DIAO An-Hong. Inhibitory effect of ginkgo biloba extract on alpha-glucosidase and its hypoglycemic mechanism [J]. J4, 2011, 37(3): 427-432.
[9]	ZHANG Yao-Fang, WAN Xiao-Shan, ZHAO Hong-Xin, SHAO Ming-Long, YANG Ping, KONG Xiang-Xin, LIU Min, LI Xiao-Kun, WANG Hui-Yan. Cloning,expression and purification of fibroblast growth factor 21 [J]. J4, 2010, 36(6): 1088-1093.
[10]	ZHOU Jing,MA Ji-chun,ZHAO Xiao-xia,FANG Fang,SONG Xian-mei,LIU Zhong-hui,TAI Gui-xiang. Expression and identification of recombinant human IL-2 and MUC1 fusion protein in E.coli BL21 [J]. J4, 2008, 34(5): 763-766.
[11]	XU Bo,ZHENG Yong-chen,FEI Shao-yang,GAO Hang. Cloning and expression of rat heme oxygenase 1 in E. coli [J]. J4, 2007, 33(6): 982-985.
[12]	XU Bo, ZHENG Yong-chen,FEI Shao-yang. Cloning,prokaryotic expression and identification of human cell division cycle gene 2 [J]. J4, 2007, 33(5): 834-836.
[13]	GAO Yan-guang，FEI Jun-wei，FEI Rui，ZENG Xian-lu. Prokaryotic expression of human L-selectin and preparation of polyclonal antibody [J]. J4, 2006, 32(1): 145-5.