本文關(guān)鍵詞： SARS Spike蛋白 受體結(jié)合域 表達(dá) 多肽 抗體　出處：《廣州醫(yī)學(xué)院》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的 利用大腸桿菌BL21原核表達(dá)系統(tǒng)對(duì)spike蛋白的受體結(jié)合域(RBD)片段進(jìn)行融合表達(dá),為進(jìn)一步研究spike蛋白免疫原性及抗原特性提供了物質(zhì)基礎(chǔ),同時(shí)建立HIS融合表達(dá)實(shí)驗(yàn)室系統(tǒng);運(yùn)用Fmoc技術(shù)合成純化抗原多肽,免疫小鼠獲得多克隆抗體,為進(jìn)一步研究抗體特性并應(yīng)用于SARS疾病的預(yù)防、預(yù)后診斷及治療提供了物質(zhì)基礎(chǔ)。 方法 1、構(gòu)建PET32(a)-RBD重組質(zhì)粒進(jìn)行原核表達(dá)依賴(lài)高效T4連接酶連接PET32(a)和RBD,轉(zhuǎn)化感受態(tài)BL21大腸桿菌。提取質(zhì)粒酶切電泳初步確定獲得PET32(a)-RBD重組質(zhì)粒,送TaKaRa公司測(cè)序。以IPTG誘導(dǎo)BL21大腸桿菌對(duì)融合蛋白進(jìn)行表達(dá),運(yùn)用聚丙烯酰胺凝膠電泳及Western-blotting來(lái)檢測(cè)表達(dá)產(chǎn)物,保存菌種。 2.SARS-CoV的S1蛋白抗原表位多肽合成及其抗體的制備 以DNAstar軟件對(duì)S1蛋白R(shí)BD片段的原始序列進(jìn)行軟件分析,選擇三段親水性強(qiáng),抗原指數(shù)高的多肽,運(yùn)用Fmoc固相法合成三條肽鏈:CQ19(CFSNVYADSFVVK GDDVRQ)、TY-14(TRNIDATSTGNYNY)和LV-11 (LRPFERDISNV),分別與KLH和BSA偶聯(lián)成三條具有免疫原性多肽:KLH(BSA)-CQ19 KLH(BSA)-LV11 KLH(BSA)-TY14,免疫BABL/c小鼠剪尾取血檢測(cè)抗體產(chǎn)生情況(ELISA法)。小鼠在第一次免疫注射之前,首先剪尾取血,該血清作為陰性對(duì)照加0.05% NaN3-70℃保存。 結(jié)果 1、PET32(a)-RBD重組質(zhì)粒構(gòu)建及其表達(dá): (1)重組質(zhì)粒構(gòu)建及鑒定:瓊脂糖凝膠電泳及測(cè)序表明重組質(zhì)粒PET32(a)-RBD構(gòu)建成功。 (2)IPTG誘導(dǎo)融合蛋白表達(dá):SDS-PAGE及Western-blotting實(shí)驗(yàn)表明融合蛋白在IPTG誘導(dǎo)下大量表達(dá),未經(jīng)IPTG誘導(dǎo)的細(xì)菌對(duì)融合蛋白幾乎不表達(dá)。(見(jiàn)圖1.3)。 2、合成多肽制備抗體: (1)分析S蛋白:根據(jù)DNAstar軟件分析篩選S蛋白三段多肽:CQ19(378～396AA)、TY14(425～438AA)、LV11(448～458AA)具有較強(qiáng)抗原性和親水性。 (2)多肽合成:氨基酸組分析表明合成正確序列CQ19、TY14、LV11多肽。 (3) KLH-CQ19免疫小鼠獲得抗CQ19多克隆抗體:ELISA檢測(cè)血清結(jié)果表明抗體產(chǎn)生為弱陽(yáng)性。1:10~3和1:10~4滴度OD值約等于陰性對(duì)照2倍。 (4) KLH-LV11、KLH-TY14免疫小鼠均無(wú)抗體產(chǎn)生。 結(jié)論 1、本研究構(gòu)建了PET32(a)-RBD重組質(zhì)粒,獲得融合蛋白在IPTG誘導(dǎo)下的穩(wěn)定表達(dá),建立本實(shí)驗(yàn)室PET32(a)載體融合的平臺(tái),為進(jìn)一步對(duì)SARS-CoVspike蛋白的研究奠定了基礎(chǔ),但其純化效果及蛋白在純化后所具有的免疫特性還有待進(jìn)一步研究。 2、應(yīng)用軟件分析技術(shù),合成高純度抗原多肽片斷,免疫動(dòng)物實(shí)驗(yàn)可獲得抗CQ19多克隆抗體,為抗spike蛋白抗體的后續(xù)研究及臨床應(yīng)用抗體治療及預(yù)防SARS疾病提供了可能。
[Abstract]:Purpose. E. coli BL21 prokaryotic expression system was used to express the receptor binding domain of spike protein, which provided a material basis for the further study of immunogenicity and antigenic characteristics of spike protein. At the same time, the HIS fusion expression laboratory system was established. Fmoc technique was used to synthesize and purify antigenic polypeptides and to immunize mice to obtain polyclonal antibodies, which provided a material basis for further study of antibody characteristics and application in the prevention, prognosis diagnosis and treatment of SARS diseases. Method. 1. The PET32(a)-RBD recombinant plasmid was constructed for prokaryotic expression dependent on high efficient T4 ligase ligation with PET32A) and RBD. the recombinant plasmid was obtained by restriction endonuclease digestion, and the recombinant plasmid was obtained by restriction endonuclease electrophoresis, and the recombinant plasmid was transformed into BL21 Escherichia coli. The fusion protein was expressed in BL21 Escherichia coli induced by IPTG. Polyacrylamide gel electrophoresis and Western-blotting were used to detect the expressed product and preserve the strain. 2. Synthesis of SARS-CoV S _ 1 protein epitope peptide and preparation of its antibody. The original sequence of S1 protein RBD fragment was analyzed by DNAstar software. Three segments of polypeptides with strong hydrophilicity and high antigen index were selected. Three peptide chains: CQ19, CFSNVYADSFVVK GDDVRQK TY-14 TRNIDATSTGNYYNYY) and LV-11 LRPFERDISNVY were synthesized by Fmoc solid state method. Three immunogenicity polypeptides, KLHBSA-CQ19 KLH(BSA)-LV11 KLHSABSA-TY14, were coupled with KLH and BSA respectively. First, the blood was taken from the tail, and the serum was stored as a negative control at 0.05% NaN3-70 鈩，

本文編號(hào)：1528503