牛抵抗素克隆及高效可溶性表达研究

doi:10.16036/j.issn.1000-2650.2018.06.014

摘要/Abstract

摘要： 【目的】抵抗素（resistin,RETN）在机体脂糖代谢和促炎过程中都发挥着重要作用。获得有活性、高效表达的可溶性牛抵抗素并鉴定其正确性,以期为后续其相关疾病的研究和防治奠定基础。【方法】以肉牛大网膜脂肪组织为原料提取总RNA,经RT-PCR获得【目的】基因,克隆至原核表达载体PET-21a中,构建重组表达质粒PET-Resistin。经双酶切及测序鉴定正确后,对宿主菌、IPTG浓度、诱导温度及时间进行优化,用抗His抗体鉴定融合蛋白表达正确。【结果】在宿主菌Rosetta,IPTG终浓度1.0 mmol/L,20 ℃诱导14 h时,牛抵抗素的表达量最高;在上述条件下,牛抵抗素存在于上清液中,为可溶性表达。【结论】成功克隆表达出仅含His标签的可溶性牛抵抗素,为进一步研究牛抵抗素参与相关疾病的调控途径和防治奠定了基础。

关键词: 牛抵抗素, 原核表达, 条件优化, 基因克隆

Abstract: 【Objective】 Resistin plays an important role in the process of proinflammatory and metabolism of lipid and sugar. Our study aims to obtain soluble expression of bovine resistin with high activity and high expression and identify it for further study and prevention of related diseases. 【Method】 Total RNA was extracted from bovine omental adipose tissue, the cDNA sequence obtained by PCR was cloned into the prokaryotic expression vector PET-21a to recombinant cloning vector PET-RETN. After identifying with double-enzyme digestion and sequence analysis, we optimizated host bacteria, IPTG concentration, induction temperature and time and then analyzed by Western blotting. 【Result】 When the host bacteria was Rosetta, the final concentration of IPTG was 1.0 mmol/L, the induction temperature was 20 ℃, the induction time was 14 h and the expression of bovine resistin was the highest. Under the above-mentioned condition, the bovine resistin was soluble expression which presented in the supernatant. 【Conclusion】 Through the PET-21a vector,the bovine resistin only with His-tag was successfully cloned and expressed, which laid a foundation for further study of bovine resistin regulation and control of related diseases.

Key words: bovine resistin, prokaryotic expression, condition optimization, gene cloning

中图分类号:

S852.4

阳明贤, 左之才, 李碧, 王宇. 牛抵抗素克隆及高效可溶性表达研究[J]. 四川农业大学学报, 2018, 36(06): 802-807.

YANG Mingxian, ZUO Zhicai, LI Bi, WANG Yu. Bovine Resistin Cloning and Its High Level of Soluble Expression[J]. Journal of Sichuan Agricultural University, 2018, 36(06): 802-807.

参考文献 24

[1]	STEPPAN C M, BAILEY S T, BHAT S, et al.The hormone re-sistin links obesity to diabetes[J]. Nature, 2001, 409(6818): 307-312.
[2]	PRAVENEC M, MLEJNEK P,ZA-DEK V, et al.Autocrine effects of transgenic resistin reduce palmitate and glucose oxidation in brown adipose tissue[J]. Physiological Genomics, 2016, 48(6): 420-427.
[3]	MOON B, KWAN J J, DUDDY N, et al.Resistin inhibits glucose uptake in L6 cells independently of changes in insulin signaling and GLUT4 translocation[J]. American Journal of Physiology Endocrinology & Metabolism, 2003, 285(1): 106-115.
[4]	王婉奕, 朱森, 杨吉春, 等. 抵抗素与肥胖和2型糖尿病[J]. 生理科学进展, 2009, 40(2): 157-160.
[5]	SILSWAL N, SINGH A K, ARUNA B, et al.Human resistin stimulates the pro-inflammatory cytokines TNF-alpha and IL-12 in macrophages by NF-kappaB-dependent pathway[J]. Biochemical & Biophysical Research Communications, 2005, 334(4): 1092-1101.
[6]	高振秋, 张巧, 徐彤, 等. 嗜中性粒细胞是人抵抗素表达的主要细胞[J]. 中国生物化学与分子生物学报, 2011, 27(1): 48-54.
[7]	BERTOLANI C, SANCHOBRU P, FAILLI P, et al.Resistin as an intrahepatic cytokine : overexpression during chronic injury and induction of proinflammatory actions in hepatic stellate cells[J]. American Journal of Pathology, 2006, 169(6): 2042-2053.
[8]	DI S N, DI N F, SANGUINETTI M, et al.Resistin regulates human choriocarcinoma cell invasive behaviour and endothelial cell angiogenic processes[J]. Journal of Endocrinology, 2006, 189(3): 691-699.
[9]	DEGAWA-YAMAUCHI M, BOVENKERK J E, JULIAR B E, et al.Serum resistin (FIZZ3) protein is increased in obese humans[J]. J Clin Endocrinol Metab, 2003, 88(11): 5452-5455.
[10]	BANERJEE R R, RANGWALA S M, SHAPIRO J S, et al.Regulation of fasted blood glucose by resistin[J]. Science, 2004, 303(5661): 1195-1198.
[11]	黄建龙. 牛抵抗素基因的原核表达及其活性研究[D]. 长春: 吉林大学, 2007.
[12]	范翠英, 冯利兴, 樊金玲, 等. 重组蛋白表达系统的研究进展[J]. 生物技术, 2012, 22(2): 76-80.
[13]	WELKER M W, TROJAN J.Anti-angiogenesis in hepatocellular carcinoma treatment: current evidence and future perspectives[J]. World Journal of Gastroenterology Wjg, 2011, 17(26): 3075-3081.
[14]	RIBAS A V, HO P L, TANIZAKI M M, et al.High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli[J]. Biotechnol Appl Biochem, 2000, 31(2): 91-94.
[15]	SHAN L R, YANG Z, NING A N, et al.Cloning, prokaryotic expression, purification and identification of human small ubiquitin-related modifier 4 (SUMO4)[J]. Journal of Northwest A & F University, 2008, 36(9): 11-16.
[16]	KANE J F, HARTLEY D L.Formation of recombinant protein inclusion bodies in Escherichia coli[J]. Trends in Biotechnology, 1988, 6(5): 95-101.
[17]	BERLEC A, TOMPA G, SLAPAR N, et al.Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis[J]. Letters in Applied Microbiology, 2008, 46(2): 227-231.
[18]	SHARP P M, TUOHY T M, MOSURSKI K R.Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes[J]. Nucleic Acids Research, 1986, 14(13): 5125-5143.
[19]	CORRALES-GARCIA L, ORTIZ E, CASTANEDA-DELGADO J, et al.Bacterial expression and antibiotic activities of recombinant variants of human β-defensins on pathogenic bacteria and M. tuberculosis[J]. Protein Expression & Purification, 2013, 89(1): 33-43.
[20]	陈惠玲, 刘东, 马博, 等. 人Eppin蛋白在大肠杆菌中的可溶性表达[J]. 免疫学杂志, 2014(2): 114-118.
[21]	叶姣, 陈长华, 夏杰, 等. 温度对重组大肠杆菌生长及外源蛋白表达的影响[J]. 华东理工大学学报(自然科学版), 2002, 28(4): 364-367.
[22]	URBAN A, ANSMANT I, MOTORIN Y.Optimisation of expression and purification of the recombinant Yo1066(Rib2)protein from Saccharomyces cerevisiae[J]. Journal of Chromatography B, 2003, 786(1/2): 187-195.
[23]	DONOVAN R S, ROBINSON C W, GLICK B R.Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter[J]. Journal of Industrial Microbiology & Biotechnology, 1996, 16(3): 145-154.
[24]	唐威华, 张景六. SDS-PAPG法测定His-tag融合蛋白分子量产生偏差的原因[J]. 植物生理学报, 2000, 26(1): 64-68.