血清4型禽腺病毒Fiber-2蛋白截短表达及单克隆抗体制备

【目的】获得截短表达的血清4型禽腺病毒（FAdV-4） Fiber-2重组蛋白及其单克隆抗体，为建立快速灵敏的病毒检测方法提供基础材料。【方法】对Fiber-2基因序列进行密码子优化，截取Fiber-2蛋白抗原性集中片段， PCR扩增其基因序列，连接至pET-32a（+）构建原核表达载体。将表达的重组蛋白进行纯化，并通过Western blotting验证其在大肠杆菌中的表达情况。将重组蛋白免疫BALB/c小鼠后取脾细胞与SP2/0细胞融合，利用间接酶联免疫吸附试验（ELISA）法筛选阳性细胞株，再利用秋水仙素裂解法、间接ELISA法和间接免疫荧光法（IFA）对其进行鉴定。【结果】成功构建Fiber-2基因截短片段的原核表达系统，获得大小约为67 kD的 Fiber-2截短蛋白，以包涵体的形式出现，经纯化后可获得单一的特异性条带，经Western blotting鉴定证明其可与FAdV-4阳性血清结合从而产生一条特异性条带。经过4次亚克隆后筛选到5株能稳定分泌抗体的杂交瘤细胞株（2C2、 4F6、 5A5、 6G10和10B5），其抗体分泌稳定性、特异性好，诱生的细胞上清液抗体效价为1:1600~1:6400，腹水抗体效价为1:320000~1:1280000，秋水仙素裂解检测染色体数目为94~110条。2C2、 4F6和10B5杂交瘤细胞分泌的抗体重链为IgG2b亚型，轻链为Kappa链； 5A5和6G10杂交瘤细胞分泌的抗体重链为IgG1亚型，轻链为Kappa链。5株杂交瘤细胞株与感染FAdV-4的鸡肝癌上皮细胞系 （LMH）进行IFA检测，结果均为阳性。【结论】制备的Fiber-2截短重组蛋白及单克隆抗体具有良好的反应原性和特异性，可用于FAdV-4病毒检测试剂盒研发。

Expression of truncated fowl adenovirus serotype 4 Fiber-2 protein and the preparation of Anti-FAdV-4 monoclonal antibodies

【Objective】 To obtain the truncated recombinant fowl adenovirus serotype 4（FAdV-4）Fiber-2 protein and its monoclonal antibodies，thereby providing the basic materials for the rapid and sensitive detection of fowl adenoviruses.【Method】 Codon optimization was performed on the Fiber-2 gene sequence，and a sequence fragment encoding a truncated Fiber-2 protein with strong antigenicity was amplified by PCR and cloned into pET-32a（+）to construct the vector for its prokaryotic expression.The recombinant protein was purified，and its expression in Escherichia coli was validated by SDS-PAGE and Western blotting with anti-FAdV-4 antibodies.Following immunization of BALB/c mice with the recombinant protein，splenocytes were harvested and fused with SP2/0 cells，from which the positive cell lines were identified by colchicine lysis and screened using indirect enzyme-linked immunosorbent assay（ELISA）and indirect immunofluorescence assay（IFA）.【Result】 The prokaryotic expression system of the truncated fragment of Fiber-2 gene was successfully constructed，and the truncated Fiber-2 protein of about 67 kD was obtained in the form of inclusion bodies.After purification，a single specific band was obtained in SDS-PAGE.Western blotting results confirmed this single band could bind to FAdV-4 positive serum.5 hybridoma cell lines（2C2，4F6，5A5，6G10 and 10B5）characterized by good stability and specificity in antibody secretion were obtained after four times of subcloning.The antibody titers of the resulting cell supernatant and ascites were 1:1600-1:6400，1:320000-1:1280000，respectively，and the number of chromosomes detected in colchicine lysis was 94-110.In addition，the heavy chain of antibodies secreted by 2C2，4F6 and 10B5 cells was the IgG2b heavy chain，while the light chain was Kappa chain.Likewise，the heavy chain of antibodies secreted by 5A5 and 6G10 was the IgG1 heavy chain，whereas the light chain was Kappa chain.IFA was performed on the 5 hybridoma cell lines and chicken hepatoma cell lines（LMH）infected with FAdV-4，and the results were all positive.【Conclusion】 The truncated recombinant Fiber-2 protein prepared shows good immunogenicity and the monoclonal antibodies prepared can be used in research and the development of a FAdV-4 virus detection kit against fowl.