猪生长激素促分泌素受体基因真核表达载体的构建及鉴定

中国畜牧兽医

猪生长激素促分泌素受体基因真核表达载体的构建及鉴定

石琳，蔺帅，周东庆，孙艳君，王志强

（扬州大学兽医学院，江苏扬州 225009）

出版日期:2014-01-20 发布日期:2014-02-27
通讯作者: 王志强，男，河南人，副教授，研究方向：兽医药理与毒理学。E-mail: zqwang@yzu.edu.cn
作者简介:石琳（1988—），女，江苏人，硕士生，研究方向：兽医药理与毒理。
基金资助:
江苏高校优势学科建设工程资助项目(PAPD)；扬州大学科技创新培育基金(2011CXJ065、2012CXJ080)。

Construction and Identification of Eukaryotic Expression Vector of Porcine Growth Hormone Secretagogue Receptor Gene

SHI Lin, LIN Shuai, ZHOU Dong-qing, SUN Yan-jun, WANG Zhi-qiang

(College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China)

Online:2014-01-20 Published:2014-02-27

摘要/Abstract

摘要： 本试验旨在构建猪生长激素促分泌素受体（pGHS-R）真核表达系统，并瞬时转染人源胚胎肾细胞（HEK293T）观察其表达情况。以猪基因组为模板，通过剪接重叠延伸聚合酶链式反应（SOE-PCR）克隆出pGHS-R的编码区序列，插入真核表达载体pcDNA3.1(+)中，构建重组真核表达质粒pcDNA3.1(+)/pGHS-R，酶切鉴定并测序，加myc标签，瞬时转染HEK293T细胞，用Western blotting鉴定该重组质粒是否能在真核细胞中表达相应的目的蛋白。结果显示，本试验成功扩增出pGHS-R编码序列，酶切和测序结果表明pcDNA3.1(+)-myc/pGHS-R构建正确，Western blotting方法证实转染的该质粒能在HEK293T细胞中正确表达目的蛋白。结果表明，本试验成功构建了pGHS-R真核表达载体，并正确表达蛋白，为进一步研究GHS-R的功能奠定了基础。

关键词: 生长激素促分泌素受体; 真核表达; 剪接重叠延伸聚合酶链式反应; 人源胚胎肾细胞

Abstract: This test was intended to construct eukaryotic expression vector of porcine growth hormone secretagogue receptor (pGHS-R), and observe its expression in transiently transfected HEK293T cells. The coding DNA sequence of GSH-R gene was amplified by SOE-PCR technology from porcine genomic DNA and cloned into pcDNA3.1(+) vector, then inserted myc-tag. The positive clone was confirmed by enzyme digestion and sequencing. The expression vector was transiently transfected into HEK293T cells. The expression of pGHS-R protein was identified by Western blotting. The results showed that the CDS of pGHS-R was successfully cloned; the construction of pcDNA3.1(+)-myc/pGHS-R was correct and its expression of pGHS-R protein was confirmed by Western blotting. The results showed that the eukaryotic expression vector pcDNA3.1(+)-myc/pGHS-R was successfully constructed and pGHS-R protein was successfully expressed. The study laid a foundation for further study on GHS-R gene function in vitro.

Key words: growth hormone secretagogue receptor; eukaryotic expression; splicing overlapping extension PCR; human emborynic kidney

石琳，蔺帅，周东庆，孙艳君，王志强. 猪生长激素促分泌素受体基因真核表达载体的构建及鉴定[J]. 中国畜牧兽医.

SHI Lin, LIN Shuai, ZHOU Dong-qing, SUN Yan-jun, WANG Zhi-qiang. Construction and Identification of Eukaryotic Expression Vector of Porcine Growth Hormone Secretagogue Receptor Gene[J]. China Animal Husbandry & Veterinary Medicine.

参考文献

1 王维刚，王志刚，邱文才，等. 小鼠生长激素促分泌素受体真核表达载体的构建及表达鉴定［J］. 中国普通外科杂志，2009，18(5)：473～476.
2 邱文才，王维刚，王志刚，等. 生长激素促分泌素受体及其内源性配体ghrelin与胃肠运动［J］. 中国普通外科杂志，2007，16(10)：1007～1009.
3 詹凯，许月英，赵瑞宏. 家鸭GHSR基因部分编码序列的克隆及变异检测［J］. 中国畜牧兽医，2008，35(6)：34～36.
4 Cassoni P, Papotti M, Catapano F, et al. Specific binding sites for synthetic growth hormone secretagogues in non-tumoral and neoplastic human thyroid tissue［J］. J Endocrinol, 2000, 165(1): 139.
5 Dass N B, Munonyara M, Bassil A K, et al. Growth hormone secretagogue receptors in rat and human gastrointestinal tract and the effects of ghrelin［J］. Neu roscience, 2003, 120(1): 443～453.
6 Dixit V D, Taub D D. Ghrelin and immunity: A young player in an old field［J］. Exp Gerontol, 2005, 40(11): 900.
7 Ghigo E, Broglio F, Arvat E, et al. Ghrelin: More than a natural GH secretagogue and/or an orexigenic factor［J］. Clinical Endocrinology, 2005, 62(1): 1～17.
8 Guan X M, Yu H, Palyha O C, et al. Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues［J］. Brain Res Mol Brain Res, 1997, 48(1): 23～29.
9 Howard A D, Feighner S D, Cully D F, et al. A receptor in pituitary and hypothalamus that functions in growth hormone release［J］. Science, 1996, 273(5277): 974～977.
10 Jeffery P L, Murray R E, Yeh A H, et al. Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines［J］. Endocr Relat Cancer, 2005, 12(4): 839～850.
11 Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth hormone releasing acylated peptide from stomach［J］. Nature, 1999, 402(6762): 656～660.
12 Leung P K, Chow K B, Lau P N, et al. The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor［J］. Cell Signal, 2007, 19(5): 1011.
13 Malagon M M, Lu Que R M, Ruiz-Guerrero E, et al. Intracellular signaling mechanisms mediating ghrelin stimulated growth hormone release in somatotropes［J］. Endocrinology, 2003, 144: 5372～5380.
14 Nakazato M, Murakami N, Date Y, et al. A role for ghrelin in the central regulation of feeding［J］. Nature, 2001, 409(6817): 194.
15 Rak A, Szczepankiewicz D, Gregoraszczuk E L. Expression of ghrelin receptor, GHSR-1a, and its functional role in the porcine ovarian follicles［J］. Growth Hormone & IGF Research, 2009, 19(1): 68～76.
16 Sun Y, Butte N F, Garcia J M, et al. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance［J］. Endocrinology, 2008, 149(2): 843～850.
17 Unniappan S, Lin X, Cervini L, et al. Goldfish ghrelin: Molecular characterization of the complementary deoxyribonucleic acid, partial gene structure and evidence for its stimulatory role in food intake［J］. Endocrinology, 2002, 143(10): 4143～4146.
18 Zigman J M, Nakano Y, Coppari R, et al. Mice lacking ghrelin receptors resist the development of diet-induced obesity［J］. J Clin Invest, 2005, 115(12): 3564～3572.

[1]	刘敏, 程淮, 郑珍珍, 张梦思, 张贺伟, 任静强. 犬副流感病毒N蛋白的真核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2025, 52(5): 2287-2294.
[2]	雷志刚, 潘宏, 张丹丹, 刘权辉, 孙哲, 邓珊, 黄奔. 马山黑山羊MAPK8基因克隆、生物信息学分析及真核表达载体构建[J]. 中国畜牧兽医, 2025, 52(2): 534-544.
[3]	黄丽清, 段安琴, 郑海英, 杨春艳, 尚江华. 水牛SFRP1基因序列分析、真核表达载体构建及组织表达分析[J]. 中国畜牧兽医, 2024, 51(5): 1807-1818.
[4]	邹菊红, 莫智化, 刘雨帆, 邹剑伟, 卢俊, 何海恩, 王璠, 黄艳娜, 蒋钦杨. 努比亚山羊Rheb基因克隆、生物信息学分析及真核表达载体构建[J]. 中国畜牧兽医, 2024, 51(5): 1819-1826.
[5]	潘诗雨, 尹娜, 王佳兴, 王强州, 陈世豪, 白皓, 常国斌. 鸡DCAF1生物信息学分析及对J亚群禽白血病病毒复制的影响[J]. 中国畜牧兽医, 2024, 51(5): 1837-1845.
[6]	王开开, 赵懿侔, 张含露, 邱实, 刘艺佳, 张文鑫, 杨丰利. 猫球形马拉色菌脂肪酶SMG1基因的真核表达及重组载体脂肪酶活性分析[J]. 中国畜牧兽医, 2024, 51(5): 2037-2046.
[7]	陈扬, 项燕华, 李宜海, 何秀苗. 鸡Caspase 3基因的克隆表达、酶活性及生物信息学分析[J]. 中国畜牧兽医, 2024, 51(10): 4200-4210.
[8]	马梦瑶, 贾鑫浩, 刘航, 钱梦薇, 郑兰兰. 猪NLRC5的真核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2024, 51(10): 4550-4558.
[9]	潘鹏丞, 雷宗全, 李显, 胡湘云, 秦谦涛, 覃兆鲜, 关志惠, 陈宝剑, 谢炳坤. 陆川猪MYL2基因生物信息学分析、真核表达载体构建及组织表达研究[J]. 中国畜牧兽医, 2023, 50(5): 1796-1806.
[10]	邢天琦, 许迪, 颜港, 王钰滨, 王梦影, 梁晶, 兰干球. 巴马小型猪ABCD4基因序列分析、真核表达载体构建及组织表达分析[J]. 中国畜牧兽医, 2023, 50(11): 4338-4348.
[11]	白少成, 周娟, 靳荣帅, 王璠, 卢婷婷, 汤先伟, 赵博昊, 吴信生, 陈阳. 新西兰白兔CYP11A1基因的克隆、生物信息学分析及其对繁殖相关基因的影响[J]. 中国畜牧兽医, 2022, 49(7): 2484-2496.
[12]	邹剑伟, 邹菊红, 申玉建, 韦一, 张叁保, 连子童, 徐建建, 宋颖, 黄艳娜, 韦英明, 蒋钦杨, 郑自华. 努比亚山羊LMCD1基因生物信息学分析、真核表达载体的构建及表达[J]. 中国畜牧兽医, 2022, 49(6): 2033-2042.
[13]	董媛, 于鸿绪, 陈国良, 鲍东博, 崔嘉芮, 刘磊, 王会岩. 牛妊娠相关蛋白19真核表达载体的构建及生物信息学分析[J]. 中国畜牧兽医, 2022, 49(4): 1233-1243.
[14]	庞峰, 龙琴琴, 梁绍波, 成虹松, 程振涛. 羊口疮病毒ORFV114蛋白生物信息学分析、真核表达及亚细胞定位[J]. 中国畜牧兽医, 2022, 49(11): 4150-4158.
[15]	曹颖颖, 李慧君, 李宝莹, 梁亮, 冷静, 唐海波. 瑶山亚种树鼩ISG15蛋白表达及其多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(1): 273-282.