抗布鲁氏杆菌Ⅳ型分泌系统表面蛋白VirB12纳米抗体的制备  

Preparation of anti-VirB12 nanoantibodies,a surface protein of typeⅣsecretory system of Brucella

作　　者：熊婧璠 谢媛 张鑫 王飞 陈媛媛 李江伟[1] Xiong Jingfan;Xie Yuan;Zhang Xin;Wang Fei;Chen Yuanyuan;Li Jiangwei(College of Life Science and Technology,Xinjiang University,Urumqi 830046,China;Xinjiang UniqueMab Biotech Co.,Ltd.,Urumqi 830026,China)

机构地区：[1]新疆大学生命科学与技术学院,乌鲁木齐830046 [2]新疆优迈生物技术有限公司,乌鲁木齐830026

出　　处：《国际生物医学工程杂志》2022年第4期287-293,共7页International Journal of Biomedical Engineering

基　　金：国家自然科学基金面上项目(31570935)。

摘　　要：目的制备能与VirB12抗原高亲和力结合的新疆双峰驼纳米抗体,为后续研究奠定基础。方法采用VirB12重组蛋白6次免疫新疆双峰驼,从外周血中分离得到淋巴细胞后提取总RNA,通过巢式PCR扩增VHH基因片段构建噬菌体VHH展示文库。采用酶联免疫吸附测定(ELISA)固相亲和富集方法进行筛选。进行3轮亲和筛选后随机挑取出第2、3轮富集的克隆,ELISA分析可溶性表达的纳米抗体与VirB12蛋白的结合情况。经过序列测定和多重比对去除重复序列,最后得到个5非冗余序列,分别命名为D1、E6、H8、H9和H10。将筛选鉴定的5个纳米抗体基因转化WK6菌株,在16℃下进行胞间质可溶性表达,经Ni亲和柱纯化表达后,Western Blot和ELISA法检测纳米抗体与VirB12抗原的结合能力与热稳定性。结果经VirB12蛋白免疫后的血清抗体效价最少为1∶24000。从VirB12抗原免疫的新疆双峰驼淋巴细胞中获得了滴度为2.8×108 cfu/ml的VHH噬菌体展示文库。5株纳米抗体在WK6菌中以可溶形式表达。结果表明,5株抗VirB12纳米抗体质量分数接近90%,且具有较高的抗原结合活性与明显的抗原-抗体浓度相关性;4株纳米抗体均表现出较高的热稳定性,在90℃处理后,仍能保留60%以上的结合活性。结论通过固相筛选富集和可溶性单克隆检测鉴定获得了5株具有较高亲和力和热稳定性的抗VirB12纳米抗体,为VirB12纳米抗体的进一步开发奠定了基础。Objective To prepare camel-derived nanoantibodies that can bind to the recombinant protein VirB12 antigen with high affinity and lay the foundation for further research.Methods Xinjiang Bactrian camels were immunized six times with VirB12 recombinant protein,total RNA was extracted from lymphocytes isolated from peripheral blood,and the VHH gene fragment was amplified by nested PCR to construct a phage VHH display library.ELISA solid-phase affinity and enrichment methods were used for screening.After three rounds of affinity screening,the clones enriched in the second and third rounds were randomly picked out,and the binding of a nanoantibody with soluble expression to VirB12 was analyzed by ELISA.After sequence determination and multiple alignment,repetitive sequences were removed,and finally five non-redundant sequences were obtained,which were named D1,E6,H8,H9,and H10.The five identified nanoantibody genes were transformed into the WK6 strain,and the soluble expression of an intercellular substance was carried out at 16°C.After purified expression of Ni-NTA,the binding ability and thermal stability of nanoantibodies and the antigen VirB12 protein were detected by Western Blot and ELISA.Results Five strains of nanoantibodies were expressed in WK6 bacteria in soluble form.SDS-PAGE showed that the purity of five anti-VirB12 nanoantibodies was close to 90%,and they had high antigen-binding activity and obvious antigen-antibody concentration dependence.All four strains of nanoantibodies showed high thermal stability,and after being treated at 90℃,they could still retain more than 60%binding activity.Conclusions In the study,a VHH phage display library with a capacity of 2.8×108 cfu/ml was constructed from Xinjiang Bactrian camel lymphocytes immunized with VirB12 recombinant protein.Five anti-VirB12 nanoantibodies with high affinity and thermal stability were obtained through solid-phase screening and enrichment and soluble monoclonal ELISA detection.These results laid the foundation for further development of

关 键 词：布鲁氏杆菌 VirB12 纳米抗体 噬菌体展示文库 热稳定性 

分 类 号：R392-33[医药卫生—免疫学]