本文選題：仿刺參 + AjC3　； 參考：《大連海洋大學(xué)》2016年碩士論文

【摘要】：補(bǔ)體C3(AjC3)在仿刺參的固有免疫系統(tǒng)中扮演著重要角色,是補(bǔ)體系統(tǒng)中含量最高的組成成分。補(bǔ)體AjC3產(chǎn)生的一系列裂解物C3a,C3b等能調(diào)節(jié)仿刺參免疫系統(tǒng)。補(bǔ)體AjC3還能夠通過形成溶膜復(fù)合物清除靶細(xì)胞。目前還沒有檢測(cè)仿刺參補(bǔ)體AjC3比較靈敏的工具。本實(shí)驗(yàn)的目的是克隆AjC3部分基因片段,原核表達(dá)并純化重組蛋白,制備補(bǔ)體AjC3的多克隆抗體,為進(jìn)一步研究仿刺參補(bǔ)體AjC3的免疫機(jī)制奠定基礎(chǔ)。利用PCR技術(shù)擴(kuò)增AjC3基因片段,將該片段與表達(dá)載體pGS-21a連接,構(gòu)建原核表達(dá)重組質(zhì)粒。將鑒定正確的重組表達(dá)質(zhì)粒轉(zhuǎn)化到Transetta(DE3)中,在不同溫度,時(shí)間,IPTG濃度下誘導(dǎo)表達(dá)。超聲破碎后,SDS-PAGE分析重組蛋白的表達(dá)形式。表達(dá)的重組蛋白經(jīng)鎳柱純化后,作為抗原免疫小鼠制備AjC3多克隆抗體。間接ELISA檢測(cè)抗體的效價(jià),Western blot檢測(cè)抗體的特異性及AjC3蛋白在各組織中的分布。PCR擴(kuò)增得到長(zhǎng)度分別為1185bp(1d),873bp(2d),555bp(3d)的目的片段;基因測(cè)序證明成功的構(gòu)建了pGS-21a-1d,pGS-21a-2d,pGS-21a-3d重組質(zhì)粒;其中pGS-21a-1d未表達(dá)蛋白質(zhì),pGS-21a-2d,pGS-21a-3d最佳誘導(dǎo)條件是30℃,0.2 mmol/L IPTG,誘導(dǎo)6h;SDS-PAGE分析表明pGS-21a-2d,pGS-21a-3d均以包涵體的形式表達(dá)。鎳柱純化后重組蛋白純度達(dá)到90%,pGS-21a-2d表達(dá)的重組蛋白分子量約為67KDa,pGS-21a-3d表達(dá)的重組蛋白分子量約為56KDa;間接ELISA檢測(cè)抗體效價(jià)分別為1:400,1:25600;Western blot結(jié)果顯示多克隆抗體具有良好的特異性,AjC3蛋白在體腔液及體壁中均有分布。構(gòu)建了重組表達(dá)載體pGS-21a-1d,pGS-21a-2d,pGS-21a-3d;純化獲得了重組蛋白;成功制備了具有高效價(jià)和特異性的補(bǔ)體AjC3多克隆抗體;研究了補(bǔ)體AjC3在仿刺參不同組織中的分布。為補(bǔ)體AjC3的進(jìn)一步研究提供了檢測(cè)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，

本文編號(hào)：1989982