【作者】 王磊；

【导师】 查丽莎； 南雨辰；

【作者基本信息】 安徽农业大学 ， 兽医硕士（专业学位）， 2022， 硕士

【摘要】 猪圆环病毒2型（PCV2）是一个小型的单链环状DNA病毒,会导致仔猪免疫抑制,从而引起多种并发症。PCV2的Cap蛋白是该病毒的唯一结构蛋白,具有多个抗原表位,能够诱导产生中和抗体。单链抗体（Single chain antibody,sc Fv）是免疫学诊断、治疗等领域的重要材料,筛选出PCV2的单链抗体能为该病的检测诊断起到重要的作用。本研究主要通过Novopro蛋白结构预测软件来分析PCV2-Cap蛋白（GenBank登录号:MF981845.1）的二级结构及相应抗原表位,为了提高蛋白的的表达量去除Cap蛋白N端NLS核定位信号序列,把Cap蛋白的50-234氨基酸序列作为目的片段。通过PCR扩增Cap基因,构建重组质粒p ET-28a-PCV2-Cap后进行原核表达,成功表达大小在37 k Da的可溶性蛋白。用成功表达的蛋白免疫兔子后,抗体效价达到8×10⁴。从免疫后的兔子的脾脏提取RNA,再把RNA通过反转录获得c DNA序列,通过PCR扩增抗体重链VH和轻链VL基因,然后将VH与VL使用SOE-PCR拼接成sc Fv基因,将sc Fv基因片段与噬菌体载体p Comb3XSS连接后,电转到ER2738感受态细胞中,再用M13K07辅助噬菌体进行侵染构建噬菌体展示文库。然后以PCV2-Cap为靶蛋白淘选特异性单链抗体,最终成功的淘选到sc Fv。将淘选成功的sc Fv克隆至真核载体p TT5-Fc上进行真核表达,获得重组单链抗体PCV2-sc Fv,而重组抗体的评价中测定EC₅₀为10.68ng/m L。通过噬菌体展示技术,筛选出PCV2相应的sc Fv抗体,为建立快速、准确和及时的检测方法提供了一定可能性,也为该病毒的免疫学检测诊断奠定基础。更多还原

【Abstract】 Porcine circovirus type 2(PCV2)is a small,single-stranded circular DNA virus that causes immunosuppression in piglets,leading to various complications.The Cap protein of PCV2 is the only structural protein of the virus and has multiple epitopes that can induce neutralizing antibodies.Single-chain antibody(sc Fv)is an important material in the fields of immunological diagnosis and treatment.Screening out the single-chain antibody of PCV2can play an important role in the detection and diagnosis of the disease.In this study,Novopro protein structure prediction software was used to analyze the secondary structure and corresponding epitopes of PCV2-Cap protein(Gen Bank accession number:MF981845.1).Sequence,the 50-234 amino acid sequence of Cap protein was used as the target fragment.The Cap gene was amplified by PCR,and the recombinant plasmid p ET-28a-PCV2-Cap was constructed and expressed in prokaryotic cells.The soluble protein with a size of 37 k Da was successfully expressed.After immunizing rabbits with the successfully expressed protein,the antibody titer reached 8×10~4.RNA was extracted from the spleen of the immunized rabbit,and then the RNA was reverse transcribed to obtain the c DNA sequence.The antibody heavy chain VH and light chain VL genes were amplified by PCR,and then the VH and VL were spliced into the sc Fv gene using SOE-PCR.The sc Fv gene fragment was ligated with the phage vector p Comb3XSS,electroporated into ER2738 competent cells,and then infected with M13K07helper phage to construct a phage display library.Then the specific single-chain antibody was panned with PCV2-Cap as the target protein,and finally the sc Fv was successfully panned.The successfully panned sc Fv was cloned into the eukaryotic vector p TT5-Fc for eukaryotic expression,and the recombinant single-chain antibody PCV2-sc Fv was obtained.The EC50 of the recombinant antibody was determined to be 10.68 ng/m L.Through phage display technology,the corresponding sc Fv antibodies of PCV2 were screened out,which provided a certain possibility for the establishment of a rapid,accurate and timely detection method,and also laid a foundation for the immunological detection and diagnosis of the virus.更多还原