MSTN纳米抗体的筛选和功能验证

发布时间：2018-05-18 01:14

本文选题：肌肉生长抑制素 + 纳米抗体　；参考：《石河子大学》2015年硕士论文

【摘要】：本研究以抑制绵羊肌肉生长抑制素(MSTN)的功能为出发点,通过制备MSTN纳米抗体,探究一条有效促进动物肌肉生长的新途径。利用噬菌体展示技术表达绵羊MSTN纳米抗体基因文库,经过四轮亲和淘选(Panning)获得MSTN高特异性的纳米抗体片段,利用大肠杆菌表达MSTN纳米抗体并分离纯化,在细胞水平和小白鼠动物试验中验证MSTN纳米抗体对肌肉生长作用的影响。主要研究方法与结果如下:1、绵羊MSTN的诱导表达,确定ELISA包被抗原用量。利用IPTG在大肠杆菌中诱导表达MSTN成熟肽蛋白并纯化,通过ELISA和Western blot方法检测抗原反应原性,优化亲和淘选包被抗原蛋白的浓度。2、绵羊MSTN纳米抗体的淘选和鉴定。扩繁M13K07噬菌体侵染原始文库,构建噬菌体展示抗体文库,噬菌体文库容经滴度测定达到1.8×1012pfu/m L。利用重组MSTN蛋白抗原对噬菌体抗体库进行富集、筛选,经过4轮亲和淘选,获得了一株亲和力高、特异性强的阳性克隆,测序后经BLAST比对分析证实该克隆序列为骆驼单域重链抗体序列。3、MSTN纳米抗体在大肠杆菌中诱导表达与鉴定。利用PCR克隆出目的抗体基因序列,构建两种原核表达载体,转化大肠杆菌后经IPTG诱导表达,表达产物经HIS镍柱纯化重组抗体,经ELISA和Western blot验证其与MSTN抗原结合的亲和活性和特异性。4、MSTN纳米抗体对小白鼠生长效果研究。通过MTT细胞计数法验证其对成肌细胞C2C12无毒性作用。实验分组对小白鼠肌肉定量注射纳米抗体,比较各试验组小白鼠体重增长变化,对试验小白鼠肌肉组织切片和免疫荧光观察其肌肉形态学变化和MSTN蛋白表达量变化情况。结果表明纳米抗体可以明显促进小白鼠的体重增长,注射纳米抗体的小白鼠肌纤维横截面积高于未注射纳米抗体组和对照组,表明MSTN纳米抗体对肌肉生长具有明显促进作用。综上所述,本研究通过四轮亲和淘选成功筛选到驼源绵羊MSTN纳米抗体,并利用大肠杆菌成功表达重组MSTN纳米抗体,其对肌肉细胞无毒性,能够有效促进小白鼠肌肉生长。该项研究为研发抗体促生长生物制剂提供了候选材料。
[Abstract]:In order to inhibit the function of sheep muscle growth inhibitor (MSTN), this study explored a new way to promote the growth of animal muscle by preparing MSTN nano-antibody. The phage display technique was used to express sheep MSTN antibody gene library. After four rounds of affinity panning, a high specific MSTN antibody fragment was obtained, and the MSTN nano-antibody was expressed and purified by E. coli. The effects of MSTN nanoparticles on muscle growth were tested at cell level and in mice. The main methods and results were as follows: 1: 1, induced expression of sheep MSTN and determined the amount of ELISA coated antigen. IPTG was used to induce the expression and purification of MSTN mature peptide protein in Escherichia coli. The antigen reactivity was detected by ELISA and Western blot methods. The concentration of antigen protein of affinity panning coating was optimized. The panning and identification of sheep MSTN nano-antibody were carried out. The expanded M13K07 phage infected the original library and constructed the phage display antibody library. The phage display antibody library reached 1.8 脳 1012pfu/m L. The phage antibody library was enriched and screened by recombinant MSTN protein antigen. After four rounds of affinity panning, a positive clone with high affinity and strong specificity was obtained. The cloned sequence was confirmed by BLAST alignment analysis as camel single-domain heavy chain antibody sequence. 3MSTN nanoparticles were induced to express and identify in Escherichia coli. Two prokaryotic expression vectors were constructed by using PCR to clone the target antibody gene sequence. After transformed into E. coli, the recombinant antibody was induced by IPTG. The recombinant antibody was purified by HIS nickel column. The effects of ELISA and Western blot on the growth of mice were studied. The MTT cell count method was used to verify its nontoxic effect on myoblast C2C12. Nano-antibody was injected into the muscle of mice. The changes of body weight in each group were compared. The morphologic changes of muscle and the expression of MSTN protein were observed on muscle tissue sections and immunofluorescence of experimental rats. The results showed that nano-antibody could significantly promote the weight gain of mice, and the cross-sectional area of muscle fiber of mice injected with nano-antibody was higher than that of control group and non-injection group, indicating that MSTN nano-antibody could obviously promote muscle growth. In conclusion, MSTN nanoparticles of camel sheep were successfully screened by four-round affinity panning, and recombinant MSTN nano-antibody was successfully expressed by E. coli, which was non-toxic to muscle cells and could effectively promote muscle growth of mice. The study provides candidate materials for the development of antibody-stimulating biologics.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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