摘要
为获得抗牛结核分枝杆菌的VHH纳米抗体,本研究利用牛结核分枝杆菌Ag85B蛋白免疫双峰驼,免疫效价达到1∶8 000后采外周血分离淋巴细胞,提取RNA反转录成cDNA,利用巢式PCR方法扩增出编码单域抗体VHH的400 bp基因片段。将目的片段用限制性内切酶EcoRⅠ和HindⅢ双酶切,与T7噬菌体载体臂T7Select 10-3RI Arms连接,再通过体外重新包装含目的片段的噬菌体。对建立的VHH噬菌体抗体库进行鉴定,原始文库库容为5.7×10~8 PFU,库阳性率为85.7%。通过4轮生物亲和淘选,筛选并表达出2株高特异性的VHH抗体,均对牛结核分枝杆菌Ag85B蛋白具有特异性。本研究为进一步探讨VHH抗体在牛结核病的诊断和治疗奠定了基础。
To obtain the VHH antibody against bovine Mycobacterium tuberculosis ,the bovine My-cobacterium tuberculosis Ag85B protein was used to immunize Bactrian camel. When the immune titer reached 1: 8 000, lymphocytes were isolated from peripheral blood. Then the RNA was ex-tracted from lymphocytes, it was reverse transcribed into cDNA and amplified by nested PCR. A 400 bp fragment encoding the single domain antibody VHH was generated.The fragment of was digested with restriction endonucleases EcoR Ⅰ and Hind Ⅲ, then ligated with T7Select 10-3RI Arms,and the phage containing the fragment of interest was assembled by in vitro repackaging. The VHH antibody library was identified and the library capacity was 3.1 × 10^13 PFU.The results showed that the positive rate of the library was 85.7%.Two high-specificity VHH antibodies were screened and expressed from the phage library by four rounds of biological affinity panning, and the two VHH antibodies were specific to the bovine Mycobacterium tuberculosis Ag85B protein.This study laid the foundation for further research on the diagnosis and treatment of tuberculosis with VHH antibodies.
作者
杨艳丽
张齐
张星星
许龙
黄新
吴桐忠
韩猛立
钟发刚
张小莺
YANG Yan-li;ZHANG Qi;ZHANG Xing-xing;XU Long;HUANG Xin;WU Tong-zhong;HAN Meng-li;ZHONG Fa-gang;ZHANG Xiao-ying(College of Veterinary Medicine ,Xin-jiang Agricultural University ,Urumqi 830000, China;State Key Laboratory for Sheep Genetic Improvement and Healthy Production ,Xinjiang Academy of Agricultural and Reclamation Science , Shihezi, Xinjiang 832000 ,China;College of Veterinary Medicine,Northwest A & F University,Yangling,Shaanxi 712100 ,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2019年第10期1987-1993,共7页
Chinese Journal of Veterinary Science
基金
乌鲁木齐市科学技术计划资助项目(P161310001)
关键词
牛结核分枝杆菌
Ag85B蛋白
VHH抗体
T7噬菌体展示
bovine Mycobacterium tuberculosis
protein Ag85B
variable domain of heavy-chain antibodies ( VHH )
T7 phage display
