摘要
从未经主动免疫的BALB/c小鼠脾脏淋巴细胞中提取总RNA,用RT-PCR扩增鼠抗体重链（VH）和轻链（VL）可变区基因,将轻链和重链可变区基因经重叠延伸拼接PCR反应,构建成单链抗体（ScFv）基因。将ScFv基因克隆到噬菌体载体PCANTAB-5E,转化入大肠杆菌TG1中,筛选出阳性克隆,接种于LB培养基,经辅助噬菌体M13KO7拯救,构建天然鼠源噬菌体抗体库,并进行抗体库滴度测定,通过DNA finger printing技术及单链抗体基因序列分析鉴定抗体库的多样性。以堆型艾美耳球虫裂殖子为靶抗原,对构建的噬菌体抗体库进行亲和筛选,将强阳性重组噬菌体克隆感染大肠杆菌HB2151,经IPTG诱导表达可溶性ScFv抗体,并对表达产物进行SDS-PAGE和Western blot鉴定,用ELISA法鉴定其对堆型艾美耳球虫裂殖子抗原的结合活性。结果表明,天然鼠源噬菌体抗体库成功构建,库容量约为2.50×10^（11） CFU/mol,单链抗体库具有一定的多样性。经过4轮亲和筛选,携带抗鸡堆型艾美耳球虫裂殖子表面抗原的特异性噬菌体抗体得到了富集。特异性ScFv抗体在E.coli中分泌表达,表达产物具有免疫活性。筛选出5个与鸡堆型艾美耳球虫裂殖子抗原结合活性较高的克隆,构建的单链噬菌体抗体库的有效表位具有多样性。本研究为进一步认识鸡球虫的各个发育阶段奠定理论基础,为鸡球虫病免疫控制研究提供参考。
The experiment was conducted to construct the naive mouse phage antibody library and screen the single-chain variable fragment （scFv） antibodies against the Eimeria acervulina （E. acervulina） merozoite of chicken. After extracting the total RNA from the fresh spleens of naive mice, the gene fragments encoding immunoglobulin heavy-chain variable （VH） and light-chain var- iable region （VL） genes were amplified by RT-PCR and inserted into scFv antibody genes using a DNA linker encoding a polypeptide of 15 amino acid residues through splicing overlapping exten- sion-PCR （SOE-PCR）. The scFv gene repertoires were cloned into the phage vector pCANTAB5E and transformed into Escherichia. coli （E. coli） TG1. Those ampicillin resistant bacteria colonies were scraped into Lysogeny Broth （LB） medium and superinfected by M13 KO7 helper phage to construct the phage antibody library. The diversity of the scFv phage was detected and analyzed by DNA fingerprinting. The scFv phage antibody library was screened with the E. acervulina merozo- ite of chicken as the target antigen. Recombinant phage clones with strong positive binding to the E. acervulina merozoite were used to infect log-phage E. coli HB2151. The expression of the solu- ble scFv antibodies was induced by isopropyl-β-D-thiogalactopyranoside（IPTG） and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis （SDS-PAGE）, Western blot and en- zyme-linked immunosorbent assay （ELISA）. The results showed that the naive mouse phage anti- body library of 2.5 × 10-11CFU/mol was successfully constructed and the scFv genes had certain di- versity. After four rounds of panning, the phage display carrying scFv gene expression antibodies against E. acervulina merozoite surface antigen was enriched. The expressed products of ScFv had immunological activity by Western blot. There were five scFv phages with specific binding ability against E. acervulina merozoite antigen detected by ELISA. The results indicated that antibodies agai
作者
康茜
任志军
唐欣浩
王雪伟
陈颖彬
刘立元
秦建华
赵月兰
KANG Qian, REN Zhi-jun, TANG Xin-hao, WANG Xue-wei, CHEN Ying-bin, LIU Li-yuan, QIN Jian- hua , ZHAO Yue-lan(College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei 071001, China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第11期2114-2124,共11页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(30571394)
河北省自然科学基金资助项目(C2007000523)
