长爪沙鼠CST3序列同源性分析及蛋白体外表达
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摘要
目的分析长爪沙鼠半胱氨酸蛋白酶抑制剂C (Cystatin C,CST3)cDNA序列同源性,并建立CST3蛋白原核表达体系,为长爪沙鼠CST3抗体制备和后续基因功能研究奠定基础。方法对长爪沙鼠Cst3 cDNA序列进行克隆、同源性分析及密码子优化;将优化后序列酶切连接到pET28a载体,完成重组CST3蛋白表达载体构建;将该载体转化到感受态细胞中,通过异丙基硫代半乳糖苷(Isopropylβ-D-Thiogalactoside,IPTG)诱导实现CST3蛋白的原核表达,并用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)验证。结果长爪沙鼠Cst3基因与人和小鼠Cst3基因的序列同源性较低。密码子优化后的长爪沙鼠Cst3表达序列插入到pET28a质粒中,获得了CST3蛋白表达载体。经1 mmol/L IPTG 37℃诱导12 h,可获得大量CST3蛋白。结论成功地构建了长爪沙鼠CST3蛋白的体外表达体系。
Objective To analyze the sequence homology of CST3 and establish a prokaryotic expression system of CST3 protein in vitro for producing anti-Mongolian gerbil Cystatin C(CST3) antibodies and perform function studies in future. Method After cloning and homology analysis, the condon optimization of Cst3 cDNA sequences of Mongolian gerbils were conducted. The recombinant CST3 protein expression vector was constructed by inserting the optimized sequences into pET28a vectors, which were then transformed into competent cells. The prokaryotic expression of CST3 protein was induced by Isopropyl β-D-Thiogalactoside(IPTG) and verified by SDS-PAGE gel electrophoresis and Western blotting. Result Both nucleotide and amino acid sequences of CST3 in Mongolian gerbils displayed low identity with human or mice CST3. After codon optimization, the Cst3 expression sequences of Mongolian gerbils were inserted into the pET28a plasmids, and a large amount of CST3 protein was obtained after induced expression with 1 mmol/L IPTG at 37 ℃ for 12 h as an optimized condition. Conclusion We successfully established a prokaryotic expression system of CST3 protein in vitro.
Objective To analyze the sequence homology of CST3 and establish a prokaryotic expression system of CST3 protein in vitro for producing anti-Mongolian gerbil Cystatin C(CST3) antibodies and perform function studies in future. Method After cloning and homology analysis, the condon optimization of Cst3 cDNA sequences of Mongolian gerbils were conducted. The recombinant CST3 protein expression vector was constructed by inserting the optimized sequences into pET28a vectors, which were then transformed into competent cells. The prokaryotic expression of CST3 protein was induced by Isopropyl β-D-Thiogalactoside(IPTG) and verified by SDS-PAGE gel electrophoresis and Western blotting. Result Both nucleotide and amino acid sequences of CST3 in Mongolian gerbils displayed low identity with human or mice CST3. After codon optimization, the Cst3 expression sequences of Mongolian gerbils were inserted into the pET28a plasmids, and a large amount of CST3 protein was obtained after induced expression with 1 mmol/L IPTG at 37 ℃ for 12 h as an optimized condition. Conclusion We successfully established a prokaryotic expression system of CST3 protein in vitro.
引文
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[20] Hua Y,Zhao H,Lu X,et al.Meta-Analysis of the Cystatin C(CST3) Gene G73A Polymorphism and Susceptibility to Alzheimer's Disease[J].Int J Neurosci,2012,122(8):431-438.
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[2] Zorio D A R,Monsma S,Sanes D H,et al.De novo sequencing and initial annotation of the Mongolian gerbil (Meriones unguiculatus) genome[J].Genomics,2018,S0888-7543(17)30161-1.
[3] Fujisawa N,Maeda Y,Yamamoto Y,et al.Newly established low seizure susceptible and seizure-prone inbred strains of Mongolian gerbil[J].Exp Anim,2003,52(2):169-172.
[4] Li X,Lu J,Wang Y,et al.Establishment and Characterization of a Newly Established Diabetic Gerbil Line[J].PLOS ONE,2016,11(7):e159420.
[5] Du X,Wang D,Li Y,et al.Newly breeding an inbred strain of ischemia-prone Mongolian gerbils and its reproduction and genetic characteristics[J].Exp Anim,2018,67(1):83-90.
[6] Li Z,Huo X,Zhang S,et al.Selection of Genes Associated with Variations in the Circle of Willis in Gerbils Using Suppression Subtractive Hybridization[J].PLOS ONE,2015,10(5):e127355.
[7] Szpaderska A M,Frankfater A.An intracellular form of cathepsin B contributes to invasiveness in cancer[J].Cancer Res,2001,61(8):3493-3500.
[8] Premzl A,Zava?nik-Bergant V,Turk V,et al.Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro[J].Experimental Cell Research,2003,283(2):206-214.
[9] Xu Y,Schnorrer P,Proiett A,et al.Regulatory Factor 8 Expression Inflammation through Regulation of IFN Blood Concentration in Response to IL-10 Controls Cystatin C Synthesis and blood concentration in response to inflammation through regulation of IFN regulatory factor 8 expression[J].The Journal of Immunology,2011,186(6):3666-3673.
[10] 朱旭,郑利平,杨兰,等.胱抑素C及其基因多态性与广西地区壮族人群冠心病的相关性[J].检验医学与临床,2018,15(3):293-296.
[11] 何琳莉,蹇顺海,文彬.cathepsin B与cystatin C在青年及老年人胃癌发生、发展中的意义[J].临床与实验病理学杂志,2016,32(4):380-383.
[12] 王梦茹,张源明.血清胱抑素C与高血压关系研究进展[J].海南医学,2018,29(4):513-516.
[13] Mathews P M,Levy E.Cystatin C in aging and in Alzheimer’s disease[J].Ageing Research Reviews,2016,32:38-50.
[14] Leto G,Crescimanno M,Flandina C.On the role of cystatin C in cancer progression[J].Life Sciences,2018,202:152-160.
[15] Yang S,Song L,Zhao L,et al.Predictive value of cystatin C in people with suspected or established coronary artery disease:A meta-analysis[J].Atherosclerosis,2017,263:60-67.
[16] 张学德,侯彦丽,牛泽群,等.检测肺癌患者血清Cathepsin X及Cystatin C的临床意义[J].中国肺癌杂志,2013,(8):411-416.
[17] Hooton H,Dubern B,Henegar C A,et al.Ssociation between CST3 rs2424577 Polymorphism and Corpulence Related Phenotypes during Lifetime in Populations of European Ancestry[J].Obes Facts.2011,4(2):131-44.
[18] Magnusson M,Molvin J,Engstr?m G,et al.Cystatin C and Risk of Diabetes and the Metabolic Syndrome-Biomarker and Genotype Association Analyses[J].PLoS One,2016,11(5):e0155735.
[19] Li J,Wei J,Xu P,et al.Impact of diabetes-related gene polymorphisms on the clinical characteristics of type 2 diabetes Chinese Han population[J].Oncotarget,2016,7(51):85464-85471.
[20] Hua Y,Zhao H,Lu X,et al.Meta-Analysis of the Cystatin C(CST3) Gene G73A Polymorphism and Susceptibility to Alzheimer's Disease[J].Int J Neurosci,2012,122(8):431-438.
[21] 陈颖,沈敏,刘宝林.包涵体蛋白纯化方法的探讨 [J].放射免疫学杂志,2011,24(1):62-64.