本文選題：肌肉生長抑制素 + 納米抗體　； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：本研究以抑制綿羊肌肉生長抑制素(MSTN)的功能為出發(fā)點(diǎn),通過制備MSTN納米抗體,探究一條有效促進(jìn)動物肌肉生長的新途徑。利用噬菌體展示技術(shù)表達(dá)綿羊MSTN納米抗體基因文庫,經(jīng)過四輪親和淘選(Panning)獲得MSTN高特異性的納米抗體片段,利用大腸桿菌表達(dá)MSTN納米抗體并分離純化,在細(xì)胞水平和小白鼠動物試驗(yàn)中驗(yàn)證MSTN納米抗體對肌肉生長作用的影響。主要研究方法與結(jié)果如下:1、綿羊MSTN的誘導(dǎo)表達(dá),確定ELISA包被抗原用量。利用IPTG在大腸桿菌中誘導(dǎo)表達(dá)MSTN成熟肽蛋白并純化,通過ELISA和Western blot方法檢測抗原反應(yīng)原性,優(yōu)化親和淘選包被抗原蛋白的濃度。2、綿羊MSTN納米抗體的淘選和鑒定。擴(kuò)繁M13K07噬菌體侵染原始文庫,構(gòu)建噬菌體展示抗體文庫,噬菌體文庫容經(jīng)滴度測定達(dá)到1.8×1012pfu/m L。利用重組MSTN蛋白抗原對噬菌體抗體庫進(jìn)行富集、篩選,經(jīng)過4輪親和淘選,獲得了一株親和力高、特異性強(qiáng)的陽性克隆,測序后經(jīng)BLAST比對分析證實(shí)該克隆序列為駱駝單域重鏈抗體序列。3、MSTN納米抗體在大腸桿菌中誘導(dǎo)表達(dá)與鑒定。利用PCR克隆出目的抗體基因序列,構(gòu)建兩種原核表達(dá)載體,轉(zhuǎn)化大腸桿菌后經(jīng)IPTG誘導(dǎo)表達(dá),表達(dá)產(chǎn)物經(jīng)HIS鎳柱純化重組抗體,經(jīng)ELISA和Western blot驗(yàn)證其與MSTN抗原結(jié)合的親和活性和特異性。4、MSTN納米抗體對小白鼠生長效果研究。通過MTT細(xì)胞計(jì)數(shù)法驗(yàn)證其對成肌細(xì)胞C2C12無毒性作用。實(shí)驗(yàn)分組對小白鼠肌肉定量注射納米抗體,比較各試驗(yàn)組小白鼠體重增長變化,對試驗(yàn)小白鼠肌肉組織切片和免疫熒光觀察其肌肉形態(tài)學(xué)變化和MSTN蛋白表達(dá)量變化情況。結(jié)果表明納米抗體可以明顯促進(jìn)小白鼠的體重增長,注射納米抗體的小白鼠肌纖維橫截面積高于未注射納米抗體組和對照組,表明MSTN納米抗體對肌肉生長具有明顯促進(jìn)作用。綜上所述,本研究通過四輪親和淘選成功篩選到駝源綿羊MSTN納米抗體,并利用大腸桿菌成功表達(dá)重組MSTN納米抗體,其對肌肉細(xì)胞無毒性,能夠有效促進(jìn)小白鼠肌肉生長。該項(xiàng)研究為研發(fā)抗體促生長生物制劑提供了候選材料。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826

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本文編號：1903720