抗日本血吸虫SEA纳米抗体制备与分析

PREPARATION AND ANALYSIS OF ANTI-SOLUBLE EGG ANTIGEN NANOBODY AGAINST

利用制备的日本血吸虫虫卵抗原（SEA），免疫健康双峰驼Bactrian camel，分离其外周血淋巴细胞提取总RNA，利用RT—PCR扩增骆驼重链抗体IgG可变区（VHH）基因片段，将VHH片段与载体pMECS连接后电转入大肠杆菌TG1构建纳米抗体文库。经亲和筛选、phage—ELISA以及测序分析后，挑取阳性克隆，抽提质粒，电转入大肠杆菌WK6诱导表达纳米抗体。通过间接ELISA分析纳米抗体VHH-1的敏感性和特异性。结果显示，纳米抗体文库容量为2．3×10^8，测序分析显示，所获得的纳米抗体文库具有良好的多态性。经NI—NTA和AKTA纯化后获得高纯度纳米抗体。ELISA分析表明所获得的纳米抗体VHH-1具有较好的敏感性和特异性。为进一步开展纳米抗体在日本血吸虫诊治研究中的应用奠定一定基础。

The soluble egg antigen （SEA） was prepared and the healthy Bactrian camel was selected for immunization with the SEA. Total RNA was extracted from peripheral lymphocytes for amplification of variable domain of the heavy chain of HCAb, （VHH） fragments by RT-PCR. VHH fragments were then purified and inserted into vector pMECS. The VHH antibody gene library was obtained by electroporating recombinant pMECS VHH vectors into E. coli TG1 cells. After affinity screening, phage-ELISA and sequencing analysis, positive clones were selected. Plasmids from these clones were extracted and electro-transformed into E. coli WK6 for nanobodies expression. Subsequently, the sensitivity and specificity of VHH- 1 were analyzed by indirect ELISA. The result showed that the capacity of the nanobodies libraiy was 2.3 × 10^8 , and possessed high diversity in the sequence analysis. High purity nanobodies were obtained by NI-NTA and AKTA. Indirect ELISA analysis showed that the VHH-1 had significant sensitivity and specificity to Schistosomiasis japonicum. In conclusion, this study laid a foundation for further researches on the application of nanobody in the field of diagnosis and treatment of Schistosomiasis japoaicum.