【摘要】：利什曼原蟲病是一種由白蛉叮咬傳播的,由利什曼原蟲引起的傳染性寄生蟲病,廣泛分布在亞、非、歐、拉美等洲的熱帶及亞熱帶地區(qū)。主要可以引起三類利什曼病:皮膚利什曼病,皮膚黏膜型利什曼病和內(nèi)臟利什曼病。利什曼病痊愈后能產(chǎn)生穩(wěn)固的免疫力,為用疫苗防治利什曼原蟲病提供了前提條件,因此各國都在不遺余力地研制可以針對利什曼原蟲的有效疫苗,目前研究的疫苗主要有以下幾類:滅活疫苗、基因工程減毒活疫苗、基因工程重組疫苗、裸DNA疫苗等,但是還未獲得一種對各種利什曼原蟲的感染均具有完全免疫力的疫苗。因此目前針對利什曼病的治療,主要是聯(lián)合使用抗生素等。單鏈抗體是基因工程抗體的重要代表類型。本研究從兩株可分泌IgG型抗利什曼原蟲糖蛋白的單克隆細(xì)胞系3BF和WMB與一株可分泌IgM型抗利什曼原蟲LPG的單克隆細(xì)胞系CDA中,提取總RNA,經(jīng)反轉(zhuǎn)錄PCR后,擴(kuò)增每株細(xì)胞系中單克隆抗體的重鏈可變區(qū)(VH)和輕鏈可變區(qū)(VL),然后將VH和VL用 Linker 連接成 scFv,并克隆到載體 pCANTAB-5E,轉(zhuǎn)化Escherichia colf TG1感受態(tài)細(xì)胞,構(gòu)建了單鏈抗體庫。通過輔助噬菌體M13K07感染后,使單鏈抗體展示在噬菌體衣殼蛋白pⅢ的N端,得到噬菌體展示的抗體庫。將抗體庫用于"淘洗-富集-擴(kuò)增"2-4輪以后,得到抗利什曼原蟲糖蛋白和脂磷酸聚糖的噬菌體抗體。經(jīng) Invitrogen Biotechnology Co.Ltd 測序顯示:3BF(IgG),CDA(IgM),WMB(IgG)VH基因序列長度分別為411bp,390bp,393bp;VL基因序列的長度分別為359bp,341bp,359bp。每個scFv均符合小鼠免疫球蛋白可變區(qū)基因特征,含有4個框架區(qū)(FR)、3個抗原互補(bǔ)決定區(qū)(CDR)及抗體特征性的2個半胱氨酸殘基。然后對測序正確的克隆,噬菌體上清侵染E.coli HB2151,并在IPTG誘導(dǎo)下表達(dá)出目的蛋白。從而為進(jìn)一步研究如蛋白生理生化分析,功能鑒定等奠定了基礎(chǔ)。
[Abstract]:Leishmania is an infectious parasitic disease caused by Leishmania spp. It is widely distributed in tropical and subtropical regions of Asia, Africa, Europe and Latin America. There are mainly three types of leishmaniasis: cutaneous leishmaniasis, cutaneous mucosal leishmaniasis and visceral leishmaniasis. Leishmaniasis has a strong immune system after it is cured, which provides a prerequisite for the vaccine against Leishmania, so countries are sparing no effort to develop an effective vaccine against Leishmania. At present, the vaccines studied mainly include inactivated vaccines, live attenuated vaccines, recombinant vaccines and naked DNA vaccines. However, no vaccine has been obtained which has complete immunity to all kinds of Leishmania infection. Therefore, the current treatment for leishmaniasis, mainly combined use of antibiotics and so on. Single chain antibody (scFv) is an important representative type of genetic engineering antibody. In this study, total RNAs were extracted from two monoclonal cell lines, 3BF and WMB, which secrete IgG type anti-Leishmania glycoprotein, and a monoclonal cell line CDA, which secreted IgM type LPG. The heavy chain variable region (VH) and the light chain variable region (VL),) of monoclonal antibody in each cell line were amplified. VH and VL were ligated into scFv by Linker and cloned into vector pCANTAB-5E, then transformed into Escherichia colf TG1 receptive cells, and the single chain antibody library was constructed. The phage display antibody library was obtained by assisting phage M13K07 infection to display scFv in the N terminal of phage capsid protein p 鈪，

本文編號：2150248