RegⅢγ基因在仔猪肠道的表达特点及RegⅢγ的重组表达与抗菌活性分析

发布时间：2018-06-09 23:16

  本文选题：RegⅢγ + 重组表达　； 参考：《湖南农业大学》2016年硕士论文

【摘要】：Reg3 Y是含有149个氨基酸残基的Reg (regenerating gene再生基因)家族成员,属于C型凝集素的第Ⅳ家族,主要在哺乳动物的肠道和皮肤的上皮组织表达,具有抗菌、抗炎和促进细胞增殖等功能,在机体对病原体的免疫过程中发挥了重要的作用。鉴于这一蛋白的多种功能,Reg3 γ在新生断奶仔猪疾病防治方面具有非常广阔的前景。本研究通过构建pET-EI-Reg3 γ质粒,在大肠杆菌BLR菌系里表达重组Reg3γ蛋白,用硫酸铵盐沉淀法来纯化重组Reg3γ蛋白,并测定其抗菌活性。取健康的0到5周的新生仔猪肠道不同部位,提取蛋白,通过western blotting来确定Reg3γ基因在仔猪不同发育阶段肠道不同部位的表达情况。本课题主要研究内容包括以下几个方面：1.Reg3γ基因在新生仔猪肠道不同部位不同时期的表达水平为了比较Reg3γ基因在新生仔猪肠道不同部位不同时期的表达水平,取0到5周健康的新生仔猪的肠道不同部位(十二指肠,空肠,回肠,结肠)组织,加入RIPA强裂解液和蛋白酶抑制剂,用电动匀浆器和细胞超声粉碎仪裂解组织,提取蛋白液。用western blot检测Reg3γ蛋白在不同肠道部位和不同时期的表达量,结果表明：在仔猪的回肠空肠内Reg3γ蛋白表达量的变化趋势接近,随着年龄周期的增加蛋白的表达量逐渐升高,并且在3到5周的仔猪体内蛋白的表达量相对刚出生仔猪的表达量差异显著。而相对仔猪肠道的其他部位,十二指肠的蛋白表达量变化较小,在结肠内,3-4周的蛋白表达量相对0周仔猪差异性极显著,而在第5周相对前两周有所下降。另外,同一时期内,十二指肠的表达量最高,其中在0到2周空肠的表达量仅次于十二指肠,而到了3周以后回肠的表达量开始升高超过空肠,而在0到5周的整个时期Reg3 γ蛋白在结肠内的表达量都相对最低。2.利用ELP-intein系统表达和纯化重组Reg3γ蛋白为了通过简单经济的方法得到纯化的重组Reg3γ蛋白,本试验利用限制性内切酶HindⅢ和Bsr G1将ELP-intein载体切割下来,再用T4连接酶将其与合成的RegⅢγ基因片段连接起来构建成pET-EI-RegⅢγ质粒。将合成的质粒转化到大肠杆菌BLR菌系内进行原核表达,利用ELP-intein表达系统的盐沉析特性和自我剪切特性,用硫酸铵盐沉淀法得到纯化的重组RegⅢγ蛋白。将纯化的上清液进行SDS-PAGE电泳,考马斯亮蓝染色后在16kD附近有一蛋白条带与RegⅢγ蛋白的分子量接近,再将上清液进行免疫印迹试验,同Ⅰ样在16kD附近得到蛋白条带。3.重组Reg3 γ蛋白体外的抗菌活性测定为了确定重组RegⅢγ蛋白的抗菌谱,利用纸片法测定ELP-intein系统表达纯化的重组RegⅢγ蛋白和抗生素(氨苄青霉素和卡那霉素)分别对金黄色葡萄球菌和大肠杆菌的抑菌作用。试验结果表明：重组蛋白对革兰氏阴性的大肠杆菌没有抑菌效果,对革兰氏阳性的金黄色葡萄球菌有明显抑菌效果。
[Abstract]:Reg3Y is a member of the Reg3Y family of regenerating gene regeneration genes containing 149 amino acid residues, belonging to the fourth family of C-type lectin. Reg3Y is expressed mainly in the intestinal tract and the skin of mammals. The functions of anti-inflammation and promoting cell proliferation play an important role in the immune process of organism against pathogens. In view of the multifunction of this protein, Reg3 纬 has a very broad prospect in disease prevention and treatment of newborn weaning piglets. The recombinant Reg3 纬 protein was expressed in Escherichia coli BLR strain by constructing pET-EI-Reg3 纬 plasmid. The recombinant Reg3 纬 protein was purified by ammonium sulfate precipitation and its antibacterial activity was determined. The protein was extracted from different parts of intestine of healthy newborn piglets from 0 to 5 weeks, and the expression of Reg3 纬 gene in different parts of intestine of piglets at different stages of development was determined by western blotting. The main contents of this study include the following aspects: 1. The expression level of Reg3 纬 gene in different parts of intestine of newborn piglets at different stages in order to compare the expression level of Reg3 纬 gene in different parts of intestine of newborn piglets at different stages. The tissues of different parts of intestine (duodenum, jejunum, ileum, colon) of healthy newborn piglets from 0 to 5 weeks were added with RIPA strong lytic fluid and protease inhibitor. The expression of Reg3 纬 protein in the ileum jejunum of piglets was detected by western blot. The results showed that the expression level of Reg3 纬 protein in ileum jejunum of piglets was similar to that in the ileum of piglets, and the expression of Reg3 纬 protein increased gradually with the increase of age cycle. The expression of protein in piglets at 3 to 5 weeks was significantly different from that in newborn piglets. Compared with other parts of piglets intestinal tract, the protein expression in duodenum changed slightly, and the protein expression at 3-4 weeks in colon was significantly different from that in 0-week piglets, but decreased in the fifth week compared with the first two weeks. In addition, the highest level of duodenal expression was observed in the same period, in which the expression of jejunum was second only to that of duodenum at 0 to 2 weeks, and after 3 weeks, the expression of ileum began to increase more than that of jejunum. However, the expression of Reg3 纬 protein in colon was the lowest in the whole period of 0 to 5 weeks. Expression and purification of Recombinant Reg3 纬 protein by ELP-intein system in order to obtain the purified Recombinant Reg3 纬 protein by simple and economical method, the recombinant Reg3 纬 protein was digested by restriction endonuclease Hind 鈪，

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