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白兆星,么宏强,罗雨晨,张雅文,张光杰,斯日古楞.2022.双峰驼NUCB2/Nesfatin-1抗体的制备及其表达分布.动物学杂志,57(3):401-411.
双峰驼NUCB2/Nesfatin-1抗体的制备及其表达分布
Antibody Preparation and Expression Distribution of NUCB2/Nesfatin-1 in Bactrian Camel
投稿时间:2021-11-02  修订日期:2022-04-15
DOI:10.13859/j.cjz.202203009
中文关键词:  Nesfatin-1  多克隆抗体  双峰驼  组织分布
英文关键词:Nesfatin-1  Polyclonal antibodies  Bactrian camel  Tissue distribution
基金项目:国家自然科学基金项目(No. 31860693,32160821)
作者单位E-mail
白兆星 内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室 呼和浩特 010000 948970897@qq.com 
么宏强 内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室 呼和浩特 010000 yaohq66@126.com 
罗雨晨 内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室 呼和浩特 010000 1393030385@qq.com 
张雅文 内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室 呼和浩特 010000 1161095060@qq.com 
张光杰 内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室 呼和浩特 010000 940184598@qq.com 
斯日古楞 内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室 呼和浩特 010000 srgl_imau@163.com 
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中文摘要:
      双峰驼(Camelus bactrianus)经过长期的自然选择,具备了许多特殊的生物学特性,比如极强的耐渴、耐饥饿以及适应恶劣气候等能力。Nesfatin-1是一种由82个氨基酸组成的肽,通过其前体物质NUCB2在Lys 83 ~ Arg 84位点的前激素转化酶(PCs)的蛋白质水解而来,其可以调节机体的能量代谢效率,对食欲有抑制作用。研究双峰驼体内NUCB2/Nesfatin-1的分布与表达,以探究双峰驼体内是否具有特有的能量代谢方式,是否与其不会发生代谢性疾病有一定的联系。使用化学合成的方法合成双峰驼NUCB2/Nesfatin-1蛋白特定表位的半抗原多肽,使用马来酰亚胺法将半抗原与血蓝蛋白(KLH)偶联,通过免疫动物制备针对NUCB2/Nesfatin-1蛋白单一抗原表位的多克隆抗体,应用Western Blot方法检测NUCB2/Nesfatin-1蛋白在双峰驼下丘脑(弓状核、孤束核、腹内侧核)、前峰脂肪、后峰脂肪、胃(胃底腺周围组织)、十二指肠、空肠、回肠、盲肠、结肠、直肠、胰腺、肝以及腹部脂肪组织中的表达情况,使用荧光定量PCR技术检测NUCB2/Nesfatin-1 mRNA在双峰驼上述组织中的表达情况。结果采用GraphPad Prism 5.0软件的t检验分析。合成的双峰驼NUCB2/Nesfatin-1蛋白特定表位的多肽杂峰很少,经过计算其纯度大于95%。多肽的质荷比[M + 4H]4+和[M + 3H]3+符合预期。经过间接ELISA法测定制备的针对NUCB2/Nesfatin-1蛋白的多克隆抗体的效价为5.12 ′ 105,成功制备多克隆抗体。使用制备的NUCB2/Nesfatin-1多克隆抗体检测双峰驼体内的NUCB2/Nesfatin-1蛋白的分布,其中,在双峰驼脂肪组织和胰腺组织中表达较为显著。荧光定量PCR检测双峰驼体内的NUCB2/Nesfatin-1 mRNA的分布,在所检测的组织中均有基因表达,其中,在腹部脂肪和肝组织的相对表达量较高。本研究通过分析抗原表位及合成多肽的方式,成功制备了针对双峰驼NUCB2/Nesfatin-1蛋白的特异性抗体,且该抗体的效价高、特异性强。通过成功制备的特异性抗体在蛋白层次上检测NUCB2/Nesfatin-1在双峰驼体内的分布情况,再使用荧光定量PCR方法在基因层次检测NUCB2/Nesfatin-1在双峰驼体内的分布情况。通过对结果的分析后发现,NUCB2/Nesfatin-1可能在双峰驼的耐渴、耐饥饿的机制调节中起到了抑制食欲的作用,使得双峰驼可以忍受长时间的饥饿,在双峰驼的外周脂肪组织中高表达,推测NUCB2/Nesfatin-1蛋白在双峰驼体内可能通过抑制脂肪细胞的分化,促进脂肪细胞中脂滴的水解为机体提供能量,其具体在脂肪细胞中的功能有待我们的进一步研究。
英文摘要:
      [Objectives] After long-term natural selection, the Bactrian Camel (Camelus bactrianus) has many special biological characteristics, such as strong thirst tolerance, hunger tolerance and the ability to adapt to bad weather. Nesfatin-1 is a peptide composed of 82 amino acids. It is derived from the protein hydrolysis of its precursor NUCB2 in prohormone convertases (PCS) at Lys 83 - Arg 84 site. It can regulate the efficiency of energy metabolism and inhibit appetite. To study the distribution and expression of NUCB2/Nesfatin-1 in Bactrian camels, in order to explore whether Bactrian camels have a unique way of energy metabolism and whether it is related to the absence of metabolic diseases. [Methods] The hapten polypeptide of specific epitope of Bactrian camel NUCB2/Nesfatin-1 protein was synthesized by chemical synthesis method. The hapten was coupled with keyhole limpet hemocyanin (KLH) by maleimide method, and polyclonal antibody against single antigenic epitope of NUCB2/Nesfatin-1 protein was prepared by immunizing animals, Western blot was used to detect the expression of NUCB2/Nesfatin-1 protein in the hypothalamus (arcuate nucleus, solitary tract nucleus, ventromedial nucleus), anterior peak fat, posterior peak fat, stomach (tissue around gastric fundus gland), duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver and abdominal adipose tissue of Bactrian camel. The expression of NUCB2/Nesfatin-1 mRNA in the above tissues o was also detected by fluorescence quantitative PCR. T-test analysis was used to analyze the results by using graphpad prism 5.0 software. [Results] The synthesized Bactrian Camel NUCB2/Nesfatin-1 protein had few polypeptide heteropeaks at specific epitopes, and its purity was calculated to be more than 95%. The mass charge ratio [M + 4H]4+ and [M + 3H]3+ of the polypeptide was in line with its expectation (Fig. 1, 2). The titer of the polyclonal antibody against NUCB2/Nesfatin-1 protein determined by indirect ELISA was 5.12 ′ 105, and the polyclonal antibody was successfully prepared (Fig. 4). The prepared polyclonal antibody of NUCB2/Nesfatin-1 was used to detect the distribution of NUCB2/Nesfatin-1 protein various tissues and organs mentioned above. It was significantly expressed in adipose tissue and pancreas of Bactrian Camel (Fig. 7, 8). The distribution of NUCB2/Nesfatin-1 mRNA in Bactrian camel was detected by fluorescence quantitative PCR. It was found that the gene was expressed in the detected tissues, especially in abdominal fat and liver tissues (Fig. 9). [Conclusion] In this study, a specific antibody against Bactrian Camel NUCB2/ Nesfatin-1 protein was successfully prepared by analyzing antigen epitopes and synthesizing peptides, and the antibody has high titer and strong specificity. The distribution of NUCB2/Nesfatin-1 in Bactrian Camels was detected at the protein level by the successfully prepared specific antibody, and then the distribution of NUCB2/Nesfatin-1 in Bactrian Camels was detected at mRNA level by fluorescence quantitative PCR. Through the analysis of the results, it is found that NUCB2/Nesfatin-1 is highly expressed in the peripheral adipose tissue, and it may play a role in inhibiting appetite in the regulation of thirst and hunger tolerance mechanism of Bactrian camel, so that Bactrian Camel can endure long-term hunger. It is speculated that NUCB2/Nesfatin-1 protein may inhibit the differentiation of adipocytes, promote the hydrolysis of lipid droplets in adipocytes and provide energy for the body in Bactrian Camel. Its specific function in adipocytes needs our further study.
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