發(fā)布時(shí)間：2018-05-25 18:07

  本文選題：奶山羊SCD基因 + 原核表達(dá)�。� 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：硬脂酰輔酶A去飽和酶(SCD)既是乳中調(diào)控不飽和脂肪酸合成的限速酶,同時(shí)又是反芻動物肉及乳中共軛亞油酸內(nèi)源合成的關(guān)鍵酶,能催化飽和脂肪酸發(fā)生去飽和反應(yīng),對羊奶脂質(zhì)組成及代謝具有重要的調(diào)控作用,山羊特異性SCD抗體對該基因的脂肪酸調(diào)控功能研究等十分必要。本研究旨在用原核表達(dá)純化后的SCD重組蛋白免疫小鼠,制備針對奶山羊(Capra hircus)SCD的單克隆抗體并進(jìn)行鑒定,明確該抗體的特性為研究SCD的功能提供了有效的工具。通過生物信息學(xué)分析選取SCD基因目標(biāo)序列,采用PCR方法擴(kuò)增SCD基因,連接至pET32a(+)原核表達(dá)載體上,重組質(zhì)粒pET32a(+)-SCD在大腸桿菌(Escherichia coli)Rosetta(DE3)中誘導(dǎo)表達(dá);使用組氨酸標(biāo)簽蛋白純化試劑盒純化融合蛋白,純化的SCD融合蛋白經(jīng)常規(guī)免疫法免疫4~6周齡的雌性Balb/c小鼠,酶聯(lián)免疫吸附測定(ELISA)檢測得到的免疫血清效價(jià);將脾細(xì)胞及SP2/0用促融劑融合制備雜交瘤克隆,ELISA鑒定陽性克隆孔,并進(jìn)行3次亞克隆;選用12周齡個(gè)體較大的小鼠注射鑒定結(jié)果為陽性的雜交瘤克隆誘生腹水,純化腹水中單抗,再對純化后得到的SCD單抗進(jìn)行一系列生物學(xué)特性鑒定。本研究的主要結(jié)果如下:1.PCR擴(kuò)增獲得了SCD N端210bp序列,成功構(gòu)建了pET32a(+)-SCD重組載體。重組質(zhì)粒轉(zhuǎn)化至Rosetta(DE3),IPTG誘導(dǎo)后,成功生產(chǎn)得到了His-SCD目標(biāo)融合蛋白,大小為30 kD。2.重組蛋白的最佳表達(dá)條件為:溫度25℃,IPTG 1 mmol/L,誘導(dǎo)表達(dá)時(shí)間12 h。在此條件下,由Rosetta(DE3)上清中獲得了可溶的SCD重組蛋白,純度高,濃度為2.5mg/mL,滿足免疫需求。3.用獲得的SCD重組蛋白當(dāng)作抗原注射小鼠,用PEG誘導(dǎo)發(fā)生成功免疫應(yīng)答的脾細(xì)胞與SP2/0進(jìn)行細(xì)胞融合,利用ELISA方法篩選陽性雜交瘤克隆,經(jīng)過至少3次亞克隆得到了一株陽性雜交瘤細(xì)胞系。4.小鼠接種陽性克隆細(xì)胞誘生腹水,并純化腹水中單抗,獲得了純度高,濃度為2mg/mL的抗體。將純化后的SCD單抗進(jìn)行效價(jià)、亞類、特異性等生物學(xué)特性鑒定,結(jié)果表明制備的單抗效價(jià)達(dá)106,能針對特定抗原表位特異性檢測乳腺上皮細(xì)胞中表達(dá)的天然SCD蛋白。綜上所述,本試驗(yàn)成功生產(chǎn)并純化得到SCD重組蛋白,同時(shí)獲得了高效價(jià)及高特異性的山羊SCD單抗,為進(jìn)一步研究SCD在羊奶短中鏈脂肪酸代謝過程中的調(diào)控功能機(jī)制提供了重要的試驗(yàn)工具。
[Abstract]:Stearyl coenzyme A desaturase (SCD) is not only a rate-limiting enzyme for regulating the synthesis of unsaturated fatty acids in milk, but also a key enzyme for endogenous synthesis of conjugated linoleic acid in ruminant meat and milk, which can catalyze the desaturation of saturated fatty acids. It is important to regulate lipid composition and metabolism of goat milk. It is necessary to study the fatty acid regulation function of goat specific SCD antibody. The purpose of this study was to immunize mice with the purified SCD recombinant protein and to prepare and identify the monoclonal antibody against capra hircus)SCD in dairy goats. The characterization of the antibody provides an effective tool for the study of the function of SCD. The target sequence of SCD gene was selected by bioinformatics analysis. The SCD gene was amplified by PCR and ligated to the prokaryotic expression vector pET32a (pET32a). The recombinant plasmid pET32a (pET-SCD) was induced to express in E. coli Escherichia coli Rosettade3. The fusion protein was purified by using histidine label protein purification kit. The purified SCD fusion protein was immunized with conventional immunoassay to female Balb/c mice aged 4 to 6 weeks. The spleen cells and SP2/0 were fused with thawing agent to prepare hybridoma clone Elisa to identify the positive clones, and to carry out three subclones, and to purify the monoclonal antibody in ascites by injecting the hybridoma clones identified as positive in 12-week-old mice by injecting the positive hybridoma clones into ascites. The purified SCD McAb was identified by a series of biological characteristics. The main results of this study were as follows: 1. The N-terminal 210bp sequence of SCD was amplified by PCR, and the recombinant vector pET32a- SCD was successfully constructed. After the recombinant plasmid was transformed into Rosetta-DE3 and induced by IPTG, the target fusion protein of His-SCD was successfully produced, with the size of 30 kD.2. The optimal expression conditions of the recombinant protein were as follows: temperature 25 鈩，

本文編號：1934167