高效构建卵清白蛋白scFv噬菌体文库及其筛选

目的:建立一种高效噬菌体文库构建方法,获得抗鸡卵清蛋白(ovalbumin,OVA)的单链抗体(scFv)噬菌体展示文库,筛选鉴定获得OVA单链抗体。方法:用OVA蛋白免疫Balb/C小鼠,选取血清抗体效价高的小鼠提取脾脏RNA,利用RT-PCR方法扩增获得小鼠重链和小鼠轻链基因。通过无缝连接酶一步将小鼠重链基因、轻链基因和linker DNA连接起来,插入噬菌体表达载体中,构建OVA scFv噬菌体展示文库。测定文库容量,对文库进行富集筛选,ELISA鉴定阳性克隆,测序后构建真核表达载体,转入Expi-CHO悬浮细胞进行真核表达,利用Western blot进行鉴定。结果:成功获得库容量为1. 2×10~7cfu的OVA scFv噬菌体展示文库,并从中筛选出8个阳性克隆,选取效价最高的2号克隆,在Expi-CHO悬浮细胞中表达获得可溶性抗体。结论:建立了一种高效构建scFv噬菌体文库的方法,筛选获得高结合活性的OVA单链抗体,并成功进行了真核表达,为OVA ELISA检测试剂盒的研制奠定了基础。

Construction and Screening of a Phage Display Library of Single Chain Fv Antibody Efficiently from Mouse Immunized with Ovalbumin

Objective:to establish an efficient method for construction of a phage display library of single chain Fv (scFv) antibody efficiently from mouse immunized with ovalbumin (OVA) and obtain anti-OVA single chain antibody.Methods:Balb/C mice were immunized with OVA. The heavy chain and light chain variable region gene of immunoglobulin were amplified from mRNA of spleen cells of mice with high serum OVA antibody titer by RT-PCR and joined by a DNA linker with seamless cloning. These fragments were inserted into phage expression vector to construct a phage display library. After measurement of library capacity, affinity selection and ELISA analysis were run to find high affinity scFv. Its protein was expressed by Epxi-CHO cells and identified by Western blot analysis.Results:The OVA scFv phage display library was constructed and its library capacity was 1.2 x 10 ⁷ cfu. Eight high affinity scFv were screened from this library. The 2# clone with the highest affinity was cloned into the eukaryotic expression vector and expressed in expi-CHO cells. The Western blot analysis showed that it is a soluble antibody. Conclusion:A highly efficient method for construction of a scFv phage library and generated a high affinity OVA scFv antibody are established. These laid a good foundation for the development of the research of OVA ELISA analysis kit.