定点突变的猪源性胰高血糖素样肽-2融合蛋白的原核表达与鉴定

doi:10.16431/j.cnki.1671-7236.2019.11.001

中国畜牧兽医 ›› 2019, Vol. 46 ›› Issue (11): 3137-3143.doi: 10.16431/j.cnki.1671-7236.2019.11.001

定点突变的猪源性胰高血糖素样肽-2融合蛋白的原核表达与鉴定

韩斐¹, 赵睿骁¹, 王刚², 江明锋¹

1. 西南民族大学青藏高原研究院, 成都 610041;
2. 四川大学国家生物医学材料工程研究中心, 成都 610064

收稿日期:2019-05-05 出版日期:2019-11-20 发布日期:2019-11-25
通讯作者: 江明锋 E-mail:mingfengjiang@vip.sina.com
作者简介:韩斐(1993-),男,河南安阳人,硕士,研究方向:GLP-2的高效利用与家禽生产,E-mail:grantfei@outlook.com
基金资助:
西南民族大学研究生创新型项目（CX2018SZ47）；四川省国际科技创新合作/港澳台科技创新合作项目"牦牛复胃关键发育阶段分子机制研究及胃溶菌酶开发"（2019YFH0035）

Prokaryotic Expression and Identification of Porcine Derived Glucagon Like Peptide-2 Fusion Protein with Site Directed Mutagenesis

HAN Fei¹, ZHAO Ruixiao¹, WANG Gang², JIANG Mingfeng¹

1. Institute of Qinghai-Tibet Plateau, Southwest Minzu University, Chengdu 610041, China;
2. National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China

Received:2019-05-05 Online:2019-11-20 Published:2019-11-25

摘要/Abstract

摘要： 为提高胰高血糖素样肽-2（GLP-2）生产效率以适应畜牧生产应用的需求，本试验利用基因工程技术表达出定点诱变的p[Gly2]GLP-2。用甘氨酸取代GLP-2氨基末端第2位的丙氨酸后，再根据大肠杆菌的偏好性对p[Gly2]GLP-2序列进行优化并在目标肽序列N-端添加肠激酶识别位点，合成基因序列，通过Kpn Ⅰ和Xho Ⅰ双酶切位点连接到pET-40b（+）构建原核重组表达载体p[Gly2]GLP-2-pET-40b（+），重组质粒转化大肠杆菌BL21（DE3）感受态细胞，诱导表达重组蛋白。优化后最适表达条件为：菌液D_{600 nm}值为0.6时，用0.2 mmol/L IPTG在37℃诱导培养5 h；最终得到p[Gly2]GLP-2重组蛋白表达量较高的基因工程菌株。通过镍离子亲和层析柱纯化及分梯度洗脱获得纯度较高的p[Gly2]GLP-2融合蛋白，本结果为后续p[Gly2]GLP-2功能性的研究及其在畜牧生产的应用奠定了基础。

关键词: 胰高血糖素样肽-2(GLP-2); 原核表达; 诱导条件优化; 亲和层析; 梯度洗脱

Abstract: In order to improve the production efficiency of GLP-2 to meet the application needs of animal husbandry,the porcine derived[Gly2]GLP-2(p[Gly2]GLP-2) was induced by site directed mutagenesis using genetic engineering technology.After replacing alanine at the second amino end of GLP-2 with glycine residue,the codon sequence of p[Gly2]GLP-2 was optimized according to the codon preference of Escherichia coli,and enterokinase recognition sites were added to the N end of the target peptide sequence to synthesize the gene sequence.Prokaryotic recombinant expression vector p[Gly2]GLP-2-pET-40b(+) was constructed by connecting Kpn Ⅰ and Xho Ⅰ digestion sites to pET-40b(+).The recombinant plasmid was transformed in E.coli BL21(DE3) competent cell to induced the expression of recombinant protein.The optimum expression parameters were that 0.2 mmol/L IPTG was used to induce the expression of p[Gly2]GLP-2-pET-40b(+) recombinant plasmid for 5 h when the bacterial solution D_{600 nm} value was 0.6,and the recombinant strain with high expression of p[Gly2]GLP-2 was finally obtained.High purity p[Gly2]GLP-2 fusion protein was obtained by purification with nickel ion affinity chromatography column and gradient elution,which laid a foundation for further functional research and application in animal husbandry.

Key words: GLP-2; prokaryotic expression; induction conditions optimization; affinity chromatography; gradient elute

中图分类号:

韩斐, 赵睿骁, 王刚, 江明锋. 定点突变的猪源性胰高血糖素样肽-2融合蛋白的原核表达与鉴定[J]. 中国畜牧兽医, 2019, 46(11): 3137-3143.

HAN Fei, ZHAO Ruixiao, WANG Gang, JIANG Mingfeng. Prokaryotic Expression and Identification of Porcine Derived Glucagon Like Peptide-2 Fusion Protein with Site Directed Mutagenesis[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3137-3143.

参考文献

[1] CONNOR E E,EVOCK-CLOVER C M,WALKER M P,et al.Comparative gut physiology symposium:Comparative physiology of glucagon-like peptide-2:Implications and applications for production and health of ruminants[J].Journal of Animal Science,2015,93(2):492-501.
[2] RING L L,NERUP N,JEPPESEN P B,et al.Glucagon like peptide-2 and neoplasia:A systematic review[J].Expert Review of Gastroenterology & Hepatology,2017,12(3):257-264.
[3] BRUBAKER P L.Glucagon-like peptide-2 and the regulation of intestinal growth and function[J].Comprehensive Physiology,2018,8(3):1185-1210.
[4] 韩斐,江明锋,王刚.胰高血糖素样肽-2及其在畜牧生产中的应用[J].中国畜牧兽医,2019,46(6):1612-1618. HAN F,JIANG M F,WANG G.Glucagon-like peptide-2 and its application in animal production[J].China Animal Husbandry & Veterinary Medicine,2019,46(6):1612-1618.(in Chinese)
[5] 陈渝,赵云.胰高血糖素样多肽-2的重组表达及鉴定[J].西南国防医药,2009,19(9):875-878. CHEN Y,ZHAO Y.Expression and identification of recombinant glucagons like peptide-2 prokaryotic[J].Medical Journal of National Defending Forces in Southwest China,2009,19(9):875-878.(in Chinese)
[6] HANSEN L,HARE K J,HARTMANN B,et al.Metabolism of glucagon-like peptide-2 in pigs:Role of dipeptidyl peptidase Ⅳ[J].Regulatory Peptides,2007,138(2):126-132.
[7] 马先君,杨梅佳,仝爱平.人源抗菌肽AP-57的原核表达与纯化[J].科学技术与工程,2016,16(23):248-252. MA X J,YANG M J,TONG A P.Expression and purification of antimicrobial peptides AP-57[J].Science Technology and Engineering,2016,16(23):248-252.(in Chinese)
[8] 任霞,吴忠义,黄丛林,等.拟南芥FT基因原核表达载体的构建、表达和蛋白纯化[J].生物技术通报,2010,3:99-103. REN X,WU Z Y,HUANG C L,et a1.Expression and purification of Arabidopsis FT gene in prokaryotic cells[J].Biotechnology Bulletin,2010,3:99-103.(in Chinese)
[9] 潘滨,吴建祥,李桂新,等.烟草曲茎病毒复制相关蛋白基因原核表达条件优化[J].浙江大学报(农业与生命科学版),2007,33(1):24-28. PAN B,WU J X,LI G X,et al.Optimization of prokaryotic expression of Tobacco curly shoot virus replication-associated protein gene[J].Journal of Zhejiang University(Agriculture & Life Sciences) 2007,33(1):24-28.(in Chinese)
[10] 张赭,李健,王芸,等.中国对虾细胞色素P450基因CYP4原核表达条件优化[J].海洋科学,2011,35(9):49-55. ZHANG Z,LI J,WANG Y,et al.Optimization of prokaryotic expression of the CYP4 gene of Chinese shrimp (Fenneropenaeus chinensis)[J].Marmine Sicence,201l,35(9):49-55.(in Chinese)
[11] 岳盈盈,李鹏,李志会,等.风疹病毒包膜糖蛋白E1的原核表达及条件优化[J].山东医药,2010,50(3):18-19. YUE Y Y,LI P,LI Z H,et a1.Prokaryotic expression and condition optimizing of rubella virus glycoprotein E1[J].Shandong Medical Journal,2010,50(3):18-19.(in Chinese)
[12] DRUCKER D J,SHI Q,CRIVICI A,et al.Regulation of the biological activity of glucagon like peptide 2 by dipeptidyl peptidase Ⅵ[J].Nature Biotechnology,1997,15:673-677.
[13] OKAWADA M,MARIA H M,TEITELBAUM D H.Use of a DPP-Ⅳ inhibitor leads to accelerated intestinal adaptation in a mouse model of short bowel syndrome[J].Journal of Surgical Research,2010,165(2):212.
[14] PETHIYAGODA C L,WELCH D R,FLEMING T P.Dipeptidyl peptidase Ⅳ (DPPIV) inhibits cellular invasion of melanoma cells[J].Clinical & Experimental Metastasis,2000,18(5):391-400.
[15] MIETLICKI-BAASE E G,MCGRATH L E,KOCH-LASKOWSKI K,et al.Hindbrain DPP-Ⅳ inhibition improves glycemic control and promotes negative energy balance[J].Physiology & Behavior,2017,173:9-14.
[16] PODUST V N,BALAN S,SIM B C,et al.Extension of in vivo half-life of biologically active molecules by XTEN protein polymers[J].Journal of Controlled Release,2016,240:52-66.
[17] SCHELLENBERGER V,WANG C W,GEETHING N C,et al.A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner[J].Nature Biotechnology,2009,27(12):1186-1190.
[18] ALTERS S E,BRYANT M L,BENJAMIN S,et al.GLP2-2G-XTEN:A Pharmaceutical Protein with improved serum half-life and efficacy in a rat Crohn's disease model[J].PLoS One,2012,7(11):e50630.
[19] BENDELE A,SEELY J,RICHEY C,et al.Short communication:Renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins[J].Toxicological Sciences,1998,42(2):152-157.
[20] RICHTER A W,AKERBLOM E.Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins[J].International Archives of Allergy and Immunology,1983,70(2):124-131.
[21] SRODA K,RYDLEWSKI J,LANGNER M,et al.Repeated injections of PEG-PE liposomes generate anti-PEG antibodies[J].Cellular & Molecular Biology Letters,2005,10(1):37-47.
[22] BURNESS C B,MCCORMACK P L.Teduglutide:A review of its use in the treatment of patients with short bowel syndrome[J].Drugs,2013,73(9):935-947.

定点突变的猪源性胰高血糖素样肽-2融合蛋白的原核表达与鉴定

Prokaryotic Expression and Identification of Porcine Derived Glucagon Like Peptide-2 Fusion Protein with Site Directed Mutagenesis

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[2]	李灿, 李楚楚, 曾维, 张宁, 纪春晓, 陈韬. 猪RegⅢγ蛋白的重组表达及功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2414-2426.
[3]	宋越, 苏胜杰, 张帆, 白帆, 戴伶俐, 王娜, 王大伟, 杨茜雯, 赵世华, 张月梅. 溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2479-2486.
[4]	张芳源, 蔺雅婷, 杨大为, 陈虎, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立[J]. 中国畜牧兽医, 2023, 50(5): 2012-2022.
[5]	王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.
[6]	胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.
[7]	王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.
[8]	刘家兴, 韩雪英, 张鑫茹, 邓嘉昕, 姚伦广, 刘阳坤. 携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析[J]. 中国畜牧兽医, 2023, 50(4): 1567-1574.
[9]	贾新月, 马静, 张艳艳, 马勋, 王正荣, 薄新文. 细粒棘球绦虫EGR-07243和EGR-07244基因生物信息学分析及原核表达[J]. 中国畜牧兽医, 2023, 50(3): 847-858.
[10]	章志涛, 李祥龙, 孔令丽, 罗雨昕, 王冬英. 大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A的原核表达及不同时期表达水平分析[J]. 中国畜牧兽医, 2023, 50(3): 1107-1117.
[11]	甘露, 郑会珍, 诺明达来, 刘燕, 金敏, 何文文, 温丽翠, 李永畅, 巴音查汗·盖力克. 伊氏锥虫伊犁株扩繁及其PFR基因克隆表达和生物信息学分析[J]. 中国畜牧兽医, 2023, 50(2): 469-478.
[12]	吕双飞, 马勋, 王静, 孔翠莲, 寇丽君, 刘彩霞, 史唯地, 康立超, 任慧杰. 炭疽芽孢杆菌PA-LF1融合蛋白的原核表达及其反应原性研究[J]. 中国畜牧兽医, 2023, 50(2): 674-683.
[13]	张凯, 张春平, 戈胜强, 孙春喜, 程杰, 伏春雨, 王刚, 牛星, 彭军. 非洲猪瘟病毒p22蛋白表达、多克隆抗体制备及间接ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(1): 270-279.
[14]	朱丽霖, 王后坤, 陈雪阳, 刘晶, 梁雄燕, 方春, 杨玉莹. 鸡IFIT5对J亚群禽白血病病毒在DF-1细胞内复制的影响[J]. 中国畜牧兽医, 2023, 50(1): 310-316.
[15]	王宇琛, 田广原, 周雅坪, 郭婷, 赵红梅, 孙雅婕, 赵逢淼, 卞宇晨, 于佳梁, 郝永清. BPIV3 NP蛋白的原核表达及其对BPIV3灭活疫苗免疫增强作用的研究[J]. 中国畜牧兽医, 2022, 49(8): 3122-3130.