抗非洲猪瘟病毒单链抗体的构建、表达及活性鉴定

制备抗非洲猪瘟病毒（ASFV）猪源单链抗体（ScFv），并对其生物学活性进行鉴定，筛选出具有ASFV反应活性的猪源单链抗体（ScFv），为ASFV的诊断提供新的材料。采集ASFV感染康复猪的外周血，分离淋巴细胞，提取淋巴细胞总RNA，以此为模板通过PCR扩增得到猪源IgG重链可变区与轻链可变区基因，利用SOE-PCR技术扩增拼接得到猪源ScFv基因；构建pET-30a-ScFv表达载体，进行蛋白的表达与纯化，用ELISA和IFA鉴定ScFv抗体的反应活性。结果显示，成功扩增出猪源ScFv（VH-VLκ、VH-VLλ）基因，鉴定到1株与ASFV存在反应活性的ScFv（VH-VLλ₁₁）抗体。结果表明，成功筛选到1株与ASFV存在反应活性的ScFv（VH-VLλ₁₁）抗体，为ASFV诊断和防控提供了新型原材料。

Construction,Expression and Activity Identification of Anti-ASFV Single-Chain Antibody

In this study, we aimed to prepare anti-ASFV pig-derived single-chain antibody (ScFv), and identify its biological activity, screen out the pig-derived ScFv with ASFV reactivity, and provide a new diagnosis for ASFV material. the peripheral blood of ASFV-infected and recovered pigs were collected to separate lymphocytes, then lymphocyte total RNAs were extracted and used as a template to obtain pig-derived IgG heavy chain variable region and light chain variable region genes by PCR amplification, using SOE-PCR technology to expand. The pig-derived ScFv gene was obtained by splicing; the pET-30a-ScFv expression vector was constructed, and the protein was expressed and purified. The reactivity of the ScFv antibody was identified by ELISA and IFA. The results showed that the pig-derived ScFv (VH-VLκ, VH-VLλ) genes were successfully amplified, and an scFv (VH-VLλ₁₁) antibody that was reactive with ASFV was identified. The results showed that an ScFv (VH-VLλ₁₁) antibody that was reactive with ASFV was successfully screened, providing new raw material for ASFV diagnosis, prevention and control.