奶山羊硬脂酰辅酶A去饱和酶单克隆抗体的制备与鉴定

发布时间：2018-05-25 18:07

  本文选题：奶山羊SCD基因 + 原核表达　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：硬脂酰辅酶A去饱和酶(SCD)既是乳中调控不饱和脂肪酸合成的限速酶,同时又是反刍动物肉及乳中共轭亚油酸内源合成的关键酶,能催化饱和脂肪酸发生去饱和反应,对羊奶脂质组成及代谢具有重要的调控作用,山羊特异性SCD抗体对该基因的脂肪酸调控功能研究等十分必要。本研究旨在用原核表达纯化后的SCD重组蛋白免疫小鼠,制备针对奶山羊(Capra hircus)SCD的单克隆抗体并进行鉴定,明确该抗体的特性为研究SCD的功能提供了有效的工具。通过生物信息学分析选取SCD基因目标序列,采用PCR方法扩增SCD基因,连接至pET32a(+)原核表达载体上,重组质粒pET32a(+)-SCD在大肠杆菌(Escherichia coli)Rosetta(DE3)中诱导表达;使用组氨酸标签蛋白纯化试剂盒纯化融合蛋白,纯化的SCD融合蛋白经常规免疫法免疫4~6周龄的雌性Balb/c小鼠,酶联免疫吸附测定(ELISA)检测得到的免疫血清效价;将脾细胞及SP2/0用促融剂融合制备杂交瘤克隆,ELISA鉴定阳性克隆孔,并进行3次亚克隆;选用12周龄个体较大的小鼠注射鉴定结果为阳性的杂交瘤克隆诱生腹水,纯化腹水中单抗,再对纯化后得到的SCD单抗进行一系列生物学特性鉴定。本研究的主要结果如下:1.PCR扩增获得了SCD N端210bp序列,成功构建了pET32a(+)-SCD重组载体。重组质粒转化至Rosetta(DE3),IPTG诱导后,成功生产得到了His-SCD目标融合蛋白,大小为30 kD。2.重组蛋白的最佳表达条件为:温度25℃,IPTG 1 mmol/L,诱导表达时间12 h。在此条件下,由Rosetta(DE3)上清中获得了可溶的SCD重组蛋白,纯度高,浓度为2.5mg/mL,满足免疫需求。3.用获得的SCD重组蛋白当作抗原注射小鼠,用PEG诱导发生成功免疫应答的脾细胞与SP2/0进行细胞融合,利用ELISA方法筛选阳性杂交瘤克隆,经过至少3次亚克隆得到了一株阳性杂交瘤细胞系。4.小鼠接种阳性克隆细胞诱生腹水,并纯化腹水中单抗,获得了纯度高,浓度为2mg/mL的抗体。将纯化后的SCD单抗进行效价、亚类、特异性等生物学特性鉴定,结果表明制备的单抗效价达106,能针对特定抗原表位特异性检测乳腺上皮细胞中表达的天然SCD蛋白。综上所述,本试验成功生产并纯化得到SCD重组蛋白,同时获得了高效价及高特异性的山羊SCD单抗,为进一步研究SCD在羊奶短中链脂肪酸代谢过程中的调控功能机制提供了重要的试验工具。
[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，

本文编号：1934167