| 猪流行性腹泻病毒S2基因B细胞表位的筛选及其单克隆抗体的制备与鉴定 |
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| 引用本文： | 猪流行性腹泻病毒S基因B细胞表位的筛选及其单克隆抗体的制备与鉴定.猪流行性腹泻病毒S2基因B细胞表位的筛选及其单克隆抗体的制备与鉴定[J].畜牧与饲料科学,2022,43(4):14-18. |
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| 作者姓名： | 猪流行性腹泻病毒S基因B细胞表位的筛选及其单克隆抗体的制备与鉴定 |
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| 作者单位： | 1. 伊金霍洛旗动物疫病预防控制中心,内蒙古 伊金霍洛旗 017200;2. 内蒙古农业大学兽医学院,内蒙古 呼和浩特 010018 |
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| 基金项目： | 农业农村部兽用药物与诊断技术广东科学观测实验站; 广东省畜禽疫病防治研究重点实验室开放课题项目（YDWS202107） |
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| 摘 要： | 目的 应用生物学软件与单克隆抗体技术相结合的方法鉴定PEDV S2基因B细胞表位肽。方法 利用CLC Sequence viewer 6.8软件及在线数据库IEDB筛选出PEDV S2基因B细胞表位,并人工合成表位肽。将表位肽与钥孔血蓝蛋白（KLH）耦联后作为抗原,免疫雌性BALB/c小鼠,通过ELISA法筛选出抗体效价较高的小鼠进行1次加强免疫,3 d后取脾脏制备脾细胞悬液进行细胞融合。经HAT选择培养基培养筛选有效杂交瘤细胞,采用ELISA法筛选阳性克隆继续进行扩大培养。将部分阳性杂交瘤细胞进行小鼠腹腔注射,并收集腹水。通过ELISA方法分别检测小鼠腹水和单克隆细胞株培养上清液抗体效价,确定最高效价作为后备细胞株。结果 筛选出B细胞表位肽序列为:MQYVYTPTYYML;免疫多肽抗原后融合前血清抗体效价达到1:2 000;BALB/c小鼠腹水及单克隆细胞株培养物上清液ELISA检测结果显示抗体效价达到1:4 000。结论 筛选出了PEDV S2基因B细胞表位,为PEDV表位肽疫苗载体构建研究提供参考。
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| 关 键 词： | 猪流行性腹泻病毒 S2基因 B细胞表位 单克隆抗体 |
| 收稿时间： | 2022-04-12 |
Screening of B-cell Epitope of Porcine Epidemic Diarrhea Virus S2 Gene and Preparation and Identification of Its Monoclonal Antibody |
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| Mengke,BAI Wei-qin,Kachula,WU Zhi-yong,Miaomiao,Geriletu.Screening of B-cell Epitope of Porcine Epidemic Diarrhea Virus S2 Gene and Preparation and Identification of Its Monoclonal Antibody[J].Animal Husbandry and Feed Science,2022,43(4):14-18. |
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| Authors: | Mengke BAI Wei-qin Kachula WU Zhi-yong Miaomiao Geriletu |
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| Institution: | 1. Ejin Horo Banner Center for Animal Disease Prevention and Control,Ejin Horo Banner 017200,China;2. College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China |
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| Abstract: | Objective] To identify the B-cell epitope peptide of porcine epidemic diarrhea virus （PEDV） S2 gene by combinative use of bioinformatics software and monoclonal antibody technology. Method] The B-cell epitope of PEDV S2 gene was screened using CLC Sequence viewer 6.8 software and IEDB online database, and the obtained epitope peptide was synthesized artificially. Female BALB/c mice were immunized with the conjugate of epitope peptide and keyhole hemocyanin （KLH） as antigen. Mice with higher antibody titers were identified by ELISA assay and then received an additional immunization. The spleen of the mice was taken 3 days post immunization to prepare the splenocyte suspension for cell fusion. The cells were grown on HAT selective medium to screen for effective hybridoma cells. The positive clones screened by ELISA assay were then used for expanding culture. Positive hybridoma cells were intraperitoneally injected to mice and ascites were collected. ELISA assay was used to determine the antibody titers in mice ascites and in the supernatants of monoclonal cell strains. The cells with the highest antibody titers was used as cell strain for subsequent use. Result] The selected B-cell epitope peptide sequence was MQYVYTPTYYML. Following immunization with the peptide antigen, the serum antibody titer before cell fusion reached 1:2 000. The ELISA assay of ascites from BALB/c mice and the supernatants from monoclonal cell strain cultures demonstrated that the antibody titer reached 1:4 000. Conclusion] The B-cell epitope of PEDV S2 gene was identified, which may be helpful for the vector construction of a epitope based peptide vaccine against PEDV. |
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| Keywords: | porcine epidemic diarrhea virus S2 gene B-cell epitope monoclonal antibody |
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