本文選題：RegⅢγ + 重組表達　； 參考：《湖南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：Reg3 Y是含有149個氨基酸殘基的Reg (regenerating gene再生基因)家族成員,屬于C型凝集素的第Ⅳ家族,主要在哺乳動物的腸道和皮膚的上皮組織表達,具有抗菌、抗炎和促進細胞增殖等功能,在機體對病原體的免疫過程中發(fā)揮了重要的作用。鑒于這一蛋白的多種功能,Reg3 γ在新生斷奶仔豬疾病防治方面具有非常廣闊的前景。本研究通過構(gòu)建pET-EI-Reg3 γ質(zhì)粒,在大腸桿菌BLR菌系里表達重組Reg3γ蛋白,用硫酸銨鹽沉淀法來純化重組Reg3γ蛋白,并測定其抗菌活性。取健康的0到5周的新生仔豬腸道不同部位,提取蛋白,通過western blotting來確定Reg3γ基因在仔豬不同發(fā)育階段腸道不同部位的表達情況。本課題主要研究內(nèi)容包括以下幾個方面：1.Reg3γ基因在新生仔豬腸道不同部位不同時期的表達水平為了比較Reg3γ基因在新生仔豬腸道不同部位不同時期的表達水平,取0到5周健康的新生仔豬的腸道不同部位(十二指腸,空腸,回腸,結(jié)腸)組織,加入RIPA強裂解液和蛋白酶抑制劑,用電動勻漿器和細胞超聲粉碎儀裂解組織,提取蛋白液。用western blot檢測Reg3γ蛋白在不同腸道部位和不同時期的表達量,結(jié)果表明：在仔豬的回腸空腸內(nèi)Reg3γ蛋白表達量的變化趨勢接近,隨著年齡周期的增加蛋白的表達量逐漸升高,并且在3到5周的仔豬體內(nèi)蛋白的表達量相對剛出生仔豬的表達量差異顯著。而相對仔豬腸道的其他部位,十二指腸的蛋白表達量變化較小,在結(jié)腸內(nèi),3-4周的蛋白表達量相對0周仔豬差異性極顯著,而在第5周相對前兩周有所下降。另外,同一時期內(nèi),十二指腸的表達量最高,其中在0到2周空腸的表達量僅次于十二指腸,而到了3周以后回腸的表達量開始升高超過空腸,而在0到5周的整個時期Reg3 γ蛋白在結(jié)腸內(nèi)的表達量都相對最低。2.利用ELP-intein系統(tǒng)表達和純化重組Reg3γ蛋白為了通過簡單經(jīng)濟的方法得到純化的重組Reg3γ蛋白,本試驗利用限制性內(nèi)切酶HindⅢ和Bsr G1將ELP-intein載體切割下來,再用T4連接酶將其與合成的RegⅢγ基因片段連接起來構(gòu)建成pET-EI-RegⅢγ質(zhì)粒。將合成的質(zhì)粒轉(zhuǎn)化到大腸桿菌BLR菌系內(nèi)進行原核表達,利用ELP-intein表達系統(tǒng)的鹽沉析特性和自我剪切特性,用硫酸銨鹽沉淀法得到純化的重組RegⅢγ蛋白。將純化的上清液進行SDS-PAGE電泳,考馬斯亮藍染色后在16kD附近有一蛋白條帶與RegⅢγ蛋白的分子量接近,再將上清液進行免疫印跡試驗,同Ⅰ樣在16kD附近得到蛋白條帶。3.重組Reg3 γ蛋白體外的抗菌活性測定為了確定重組RegⅢγ蛋白的抗菌譜,利用紙片法測定ELP-intein系統(tǒng)表達純化的重組RegⅢγ蛋白和抗生素(氨芐青霉素和卡那霉素)分別對金黃色葡萄球菌和大腸桿菌的抑菌作用。試驗結(jié)果表明：重組蛋白對革蘭氏陰性的大腸桿菌沒有抑菌效果,對革蘭氏陽性的金黃色葡萄球菌有明顯抑菌效果。
[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 緯 has a very broad prospect in disease prevention and treatment of newborn weaning piglets. The recombinant Reg3 緯 protein was expressed in Escherichia coli BLR strain by constructing pET-EI-Reg3 緯 plasmid. The recombinant Reg3 緯 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 緯 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 緯 gene in different parts of intestine of newborn piglets at different stages in order to compare the expression level of Reg3 緯 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 緯 protein in the ileum jejunum of piglets was detected by western blot. The results showed that the expression level of Reg3 緯 protein in ileum jejunum of piglets was similar to that in the ileum of piglets, and the expression of Reg3 緯 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 緯 protein in colon was the lowest in the whole period of 0 to 5 weeks. Expression and purification of Recombinant Reg3 緯 protein by ELP-intein system in order to obtain the purified Recombinant Reg3 緯 protein by simple and economical method, the recombinant Reg3 緯 protein was digested by restriction endonuclease Hind 鈪，

本文編號：2001162