抗利什曼原虫表面糖蛋白和脂磷酸聚糖单链抗体库的构建及其应用
发布时间：2018-07-28 13:09
【摘要】：利什曼原虫病是一种由白蛉叮咬传播的,由利什曼原虫引起的传染性寄生虫病,广泛分布在亚、非、欧、拉美等洲的热带及亚热带地区。主要可以引起三类利什曼病:皮肤利什曼病,皮肤黏膜型利什曼病和内脏利什曼病。利什曼病痊愈后能产生稳固的免疫力,为用疫苗防治利什曼原虫病提供了前提条件,因此各国都在不遗余力地研制可以针对利什曼原虫的有效疫苗,目前研究的疫苗主要有以下几类:灭活疫苗、基因工程减毒活疫苗、基因工程重组疫苗、裸DNA疫苗等,但是还未获得一种对各种利什曼原虫的感染均具有完全免疫力的疫苗。因此目前针对利什曼病的治疗,主要是联合使用抗生素等。单链抗体是基因工程抗体的重要代表类型。本研究从两株可分泌IgG型抗利什曼原虫糖蛋白的单克隆细胞系3BF和WMB与一株可分泌IgM型抗利什曼原虫LPG的单克隆细胞系CDA中,提取总RNA,经反转录PCR后,扩增每株细胞系中单克隆抗体的重链可变区(VH)和轻链可变区(VL),然后将VH和VL用 Linker 连接成 scFv,并克隆到载体 pCANTAB-5E,转化Escherichia colf TG1感受态细胞,构建了单链抗体库。通过辅助噬菌体M13K07感染后,使单链抗体展示在噬菌体衣壳蛋白pⅢ的N端,得到噬菌体展示的抗体库。将抗体库用于"淘洗-富集-扩增"2-4轮以后,得到抗利什曼原虫糖蛋白和脂磷酸聚糖的噬菌体抗体。经 Invitrogen Biotechnology Co.Ltd 测序显示:3BF(IgG),CDA(IgM),WMB(IgG)VH基因序列长度分别为411bp,390bp,393bp;VL基因序列的长度分别为359bp,341bp,359bp。每个scFv均符合小鼠免疫球蛋白可变区基因特征,含有4个框架区(FR)、3个抗原互补决定区(CDR)及抗体特征性的2个半胱氨酸残基。然后对测序正确的克隆,噬菌体上清侵染E.coli HB2151,并在IPTG诱导下表达出目的蛋白。从而为进一步研究如蛋白生理生化分析,功能鉴定等奠定了基础。
[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，
本文编号：2150248
[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，
本文编号：2150248
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