Ma Lina, Liu Yong-sheng, Ding Yao-zhong, Chen Hao-tai, Zhou Jian-hua, Liu Wen-qian, Wang Meng, Zhang Jie
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
Hybridoma (Larchmt). 2010 Oct;29(5):409-12. doi: 10.1089/hyb.2010.0031.
A short linear peptide was designed according to the antigenic site analysis of VP1 protein of foot-and-mouth virus (FMDV) serotype O and synthesized as the peptide immunogen. The peptide, which covers the region from amino acid 133 to 160 of VP1 of FMDV, was linked to the N-terminal cysteine and conjugated with the carrier protein of keyhole limpet hemocyanin (KLH). Normal 6- to 8-week-old BALB/c mice were immunized with the 20 μg dose conjugated peptide antigen four times. The splenocytes from the immunized mice were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and subcloned four times with limiting dilution. Five stable hybridoma cell lines, designated as 4F9, 1B11, 1E10, 1D4, and 4B8, were obtained. Isotyping of all obtained MAbs indicated that the MAbs of 4F9, 1E10, and 4B8 belonged to IgG2b; the 1B11 and 1D4 belonged to IgG1 and IgM, respectively. The micro-neutralization test indicated that the MAbs of 4F9, 4B8, and 1B11 were capable of neutralizing FMDV serotype O with neutralization indices ranging from 1.81 to 2.11. These results suggest that linear synthetic peptide conjugate can elicit antibodies against native FMDV virus and can be used as an alternative immunogen for production of MAbs with exact epitope.
根据口蹄疫病毒（FMDV）O型VP1蛋白的抗原表位分析设计了一种短线性肽，并将其合成为肽免疫原。该肽覆盖FMDV VP1蛋白第133至160位氨基酸区域，与N端半胱氨酸相连，并与钥孔戚血蓝蛋白（KLH）载体蛋白偶联。用20μg剂量的偶联肽抗原对6至8周龄的正常BALB/c小鼠进行4次免疫。将免疫小鼠的脾细胞与SP2/0小鼠骨髓瘤细胞融合，通过间接酶联免疫吸附测定（ELISA）筛选阳性杂交瘤，并通过有限稀释法进行4次亚克隆。获得了5株稳定的杂交瘤细胞系，分别命名为4F9、1B11、1E10、1D4和4B8。对所有获得的单克隆抗体进行的亚型鉴定表明，4F9、1E10和4B8的单克隆抗体属于IgG2b；1B11和1D4分别属于IgG1和IgM。微量中和试验表明，4F9、4B8和1B11的单克隆抗体能够中和O型FMDV，中和指数在1.81至2.11之间。这些结果表明，线性合成肽偶联物能够引发针对天然FMDV病毒的抗体，可作为一种替代免疫原用于生产具有确切表位的单克隆抗体。
