中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (24): 4421-4424.doi: 10.3969/j.issn.1673-8225.2010.24.012

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

犬黑皮质素受体3原核表达载体的构建与表达

谢建中，王光川，巴彩凤   

  1. 辽宁医学院实验动物中心，辽宁省锦州市  121001
  • 出版日期:2010-06-11 发布日期:2010-06-11
  • 通讯作者: 巴彩凤，硕士，教授，辽宁医学院实验动物中心，辽宁省锦州市 121001 bacaifeng@163.com
  • 作者简介:谢建中，男，1964年生，辽宁省锦州市人，汉族，1989年锦州医学院毕业，实验师，主要从事动物遗传育种方面的研究。 xiejianzhong500@163.com
  • 基金资助:

    辽宁省科技厅基金，2007408001-6，犬MC4R基因表达载体的构建及在真核细胞中的表达。

Construction and prokaryotic expression of canis melanocortin receptor-3

Xie Jian-zhong, Wang Guang-chuan, Ba Cai-feng   

  1. Experimental Animal Center, Liaoning Medical University, Jinzhou  121001, Liaoning Province, China
  • Online:2010-06-11 Published:2010-06-11
  • Contact: Ba Cai-feng, Master, Professor, Experimental Animal Center, Liaoning Medical University, Jinzhou 121001, Liaoning Province, China bacaifeng@163.com
  • About author:Xie Jian-zhong, Experimentalist, Experimental Animal Center, Liaoning Medical University, Jinzhou 121001, Liaoning Province, China xiejianzhong500@163.com
  • Supported by:

     the Foundation of Science and Technology Committee of Liaoning Province, No. 2007408001-6*

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被引次数

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摘要：

背景：近年黑皮质素受体家族在体内能量平衡中的作用越来越受到重视。黑皮质素受体3基因突变是引起体质量增加的重要因素之一，但是目前对黑皮质素受体3的深入研究很少，且未见在蛋白水平上检测其表达的单克隆抗体。
目的：构建犬黑皮质素受体3基因编码区的原核表达载体并在大肠杆菌中诱导表达。
方法：以Beagle犬基因组DNA为模板，设计特异性引物，经PCR技术扩增目的片段后，克隆到pMD18-T载体上，双酶切鉴定以后将目的基因亚克隆至原核表达载体pGEX-4T-1上，转化到E. coli DH5α，筛选阳性克隆，DNA测序检测插入序列的正确性。将测序正确的重组表达质粒pGEX-4T-1-cMC3R转化到E. coli BL21内，经IPTG诱导后，利用Western blot检测犬黑皮质素受体3融合蛋白的表达。
结果及结论：成功构建重组表达质粒pGEX-4T-1-cMC3R，经DNA测序证实插入序列与Genebank上公布的序列一致。此重组体经诱导后能在E. coli BL21内表达犬黑皮质素受体3融合蛋白，为进一步获取犬黑皮质素受体3的单克隆抗体奠定物质基础，也为研究犬黑皮质素受体3蛋白的结构和生理功能提供帮助。

关键词: 黑皮质素受体3, 犬, pGEX-4T-1, 诱导表达, 质粒构建

Abstract:

BACKGROUND: Effects of melanocortin receptor (MCR) on in vivo energy balance arose more and more attention. MC3R gene mutation plays an important role in weight gaining, but there are few studies concerning MC3R, and no monoclonal antibody has been reported to detect MC3R expression at protein level. 
OBJECTIVE: To construct an expression vector pGEX-4T-1-cMC3R and to induce its expression in E. coli.
METHODS: Using the canis MC3R DNA as template, specific primers were designed. After the PCR amplification, the product was cloned into pMD18-T vector, and identified by double enzyme digestion. The target gene was subcloned into pGEX-4T-1 vector. The recombinant pGEX-4T-1-cMC3R was transferred to E. coli DH5α, and then it was identified with double restriction enzymes digestion analysis and DNA sequencing. The recombinant pGEX-4T-1-cMC3R which was identical to what released in GeneBank was transformed into E. coli BL21. The expression of canis MC3R protein was analyzed by Western blot after IPTG induction.
RESULTS AND CONCLUSION: The prokaryotic plasmid pGEX-4T-1-cMC3R was constructed successfully. Canis MC3R sequence was mainly identical to what released in GeneBank. After induction, the recombinant pGEX-4T-1-cMC3R could express canis MC3R fusion protein in E. coli BL21. This experiment not only provides material basis for construction of canis MC3R monoclonal antibody, but also benefits the structures and physiologic functions studies of canis MC3R protein.

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