仿刺参补体AjC3部分基因的原核表达及多克隆抗体的制备

发布时间：2018-06-07 05:18

  本文选题：仿刺参 + AjC3　； 参考：《大连海洋大学》2016年硕士论文

【摘要】：补体C3(AjC3)在仿刺参的固有免疫系统中扮演着重要角色,是补体系统中含量最高的组成成分。补体AjC3产生的一系列裂解物C3a,C3b等能调节仿刺参免疫系统。补体AjC3还能够通过形成溶膜复合物清除靶细胞。目前还没有检测仿刺参补体AjC3比较灵敏的工具。本实验的目的是克隆AjC3部分基因片段,原核表达并纯化重组蛋白,制备补体AjC3的多克隆抗体,为进一步研究仿刺参补体AjC3的免疫机制奠定基础。利用PCR技术扩增AjC3基因片段,将该片段与表达载体pGS-21a连接,构建原核表达重组质粒。将鉴定正确的重组表达质粒转化到Transetta(DE3)中,在不同温度,时间,IPTG浓度下诱导表达。超声破碎后,SDS-PAGE分析重组蛋白的表达形式。表达的重组蛋白经镍柱纯化后,作为抗原免疫小鼠制备AjC3多克隆抗体。间接ELISA检测抗体的效价,Western blot检测抗体的特异性及AjC3蛋白在各组织中的分布。PCR扩增得到长度分别为1185bp(1d),873bp(2d),555bp(3d)的目的片段;基因测序证明成功的构建了pGS-21a-1d,pGS-21a-2d,pGS-21a-3d重组质粒;其中pGS-21a-1d未表达蛋白质,pGS-21a-2d,pGS-21a-3d最佳诱导条件是30℃,0.2 mmol/L IPTG,诱导6h;SDS-PAGE分析表明pGS-21a-2d,pGS-21a-3d均以包涵体的形式表达。镍柱纯化后重组蛋白纯度达到90%,pGS-21a-2d表达的重组蛋白分子量约为67KDa,pGS-21a-3d表达的重组蛋白分子量约为56KDa;间接ELISA检测抗体效价分别为1:400,1:25600;Western blot结果显示多克隆抗体具有良好的特异性,AjC3蛋白在体腔液及体壁中均有分布。构建了重组表达载体pGS-21a-1d,pGS-21a-2d,pGS-21a-3d;纯化获得了重组蛋白;成功制备了具有高效价和特异性的补体AjC3多克隆抗体;研究了补体AjC3在仿刺参不同组织中的分布。为补体AjC3的进一步研究提供了检测工具。
[Abstract]:Complement C _ 3 (AjC _ 3) plays an important role in the innate immune system of Acanthopsis japonicus and is the most abundant component in the complement system. Complement AjC3 produced a series of cleavage products, such as C _ 3a C _ 3b, which could regulate the immune system of Acanthopsis japonicus. Complement AjC3 can also scavenge target cells by forming a membrane complex. At present, there is no sensitive tool for the detection of AjC3 imitating the complement of Acanthopsis japonicus. The purpose of this experiment was to clone part of AjC3 gene fragment, express and purify recombinant protein in prokaryotic, prepare polyclonal antibody of complement AjC3, and lay a foundation for further study on immune mechanism of complement AjC3. The AjC3 gene fragment was amplified by PCR and ligated with the expression vector pGS-21a to construct the prokaryotic expression plasmid. The identified recombinant plasmids were transformed into Transettafil _ 3 and induced by IPTG at different temperature and time. The expression of recombinant protein was analyzed by SDS-PAGE after ultrasonic fragmentation. The recombinant protein was purified by nickel column and immunized with AjC3 polyclonal antibody as antigen. The titer of the antibody was detected by indirect ELISA. The specificity of the antibody and the distribution of AjC3 protein in various tissues were detected by Western blot. The length of the fragment was 1185bp1, 873 bp1, 555 bp3 d, respectively. The recombinant plasmid pGS-21a-1dGS-21a-2dGS-21a-3d was successfully constructed by gene sequencing. The results showed that the recombinant plasmid pGS-21a-2dGS-21a-2dGS-21a-2dGS-21a-3d was constructed successfully. The optimal induction condition of pGS-21a-1d unexpressed protein, pGS-21a-2dGS-21a-3d, was 0.2 mmol/L IPTGat 30 鈩，

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