【作者】 王健；

【导师】 陈韬；

【作者基本信息】 湖南农业大学 ， 基础兽医学， 2016， 硕士

【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。更多还原

【Abstract】 Reg3y is a member of regenerating gene which contains 149 amino acid residues and it belongs to C-type lectin IV family. Reg3y is expressed mainly in the epithelial tissue of the mammalian gut and skin, with antibacterial, anti-inflammatory and promoting cell proliferation and other functions. It plays an important role in the immune process of pathogens. In view of the multiple functions of this protein, Reg3γ has a very broad prospect in the prevention and treatment of neonatal piglets. In this study, the recombinant Reg3y protein was expressed in Escherichia coli BLR strain using prokaryotic expression vector the pEI-Reg3γ plasmid. The recombinant Reg3γ protein was purified with two steps of ammonium s μlfate precipitation and self-cleavage. The antibacterial activity was analysized by radial diffusion. To enclose the expression characterisitics of Reg3γ in intestine of piglets, 24 healthy 0 to 5 weeks of newborn piglets were euthanized and duodenum, jejunum, ileum, as well as colon were sampled for extracting the protein. The protein level of Reg3γ were determined by western blotting. The main contents of this paper include the following aspects:1. Reg3γ gene expression levels in different parts of the intestinal tract of newborn pigletsIn order to compare reg3γ gene expression levels in newborn piglets during different periods in different parts, we take 0 to 5 weeks healthy newborn piglets of different parts of the intestine (duodenum, jejunum, ileum and colon tissue), adding RIPA strong lysis buffer and protease inhibitors, lysising tissue with electric homogenizer and ultrasonic cell disrupter and getting the protein solution.Finally,we use western blotting detect the expression levels of Reg3γ, results as followings:In the jejunum, the expression of Reg3γ is close to ileum, with age increasing protein expression increased gradually, and in 3 to 5 weeks of piglets in vivo protein expression relative newborn piglets expression was significantly different. The piglet intestine relative to other parts, and duodenal proteins expression variation is very small, in the colon, the expression of 3-4 weeks of protein relative 0 week piglets weight difference was significant, and in the fifth week relative to the previous two weeks down. In addition, duodenal expression is the highest among others in the same period. In 0 to 2 weeks, the expression of jejunum was only inferior to that of duodenum. But the expression of the ileum increased more than the jejunum by 3 weeks. And in the whole period of 0 to 5 weeks, the expression of Reg3γ protein in the colon is relatively low.2. Using ELP-intein system to express and purify the recombinant Reg3γ proteinTo get the purified recombinant Reg3γ protein with a simple and economical method. The experiment using limit endonuclease Hind Ⅲ and Bsr G1 cut down the pET-EI vector and construct pET-EI-Reg3γ plasmid with T4 ligase to connect the vector and synthesized Reg3γ gene fragment. Transformed the plasmid into E.coli BLR strain in prokaryotic expression, using salt precipitation analysis characteristics and self shear properties of ELP-intein expression system,we get the purifed recombinant Reg3γ protein. The purified supernatant were analyzed by SDS-PAGE electrophoresis and there is a band close to 16 kD after Coomassie brilliant blue stain and western blotting test. At the same time, the concentration of RegⅢ in supernatant was determined by dot blot hybridization method is 0.36mg/ml.3. Determination of the antibacterial activity of recombinant Reg3γ protein in vitroIn order to determine the antimicrobial spectrum of recombinant Reg3γ protein,we determine the antimicrobial effect of purified recombinant Reg3γ protein and antibiotics (ampicillin and kanamycin). The results showed that the recombinant protein had no inhibitory effect on Gram negative Escherichia coli, and had obvious antimicrobial effect on Gram positive Staphylococcus aureus.更多还原