摘要
几丁质酶3样蛋白1(CHI3L1)在感染甲型流感病毒(IAV)后,表达水平发生显著变化,暗示其可能参与抗病毒免疫反应的调控。然而,当前尚未发现CHI3L1与IAV之间的直接相互作用。由于CHI3L1在免疫过程中可能具有重要作用,研究者推测其可能在调节抗病毒免疫中扮演关键角色。然而,现有的研究工具不足,迫切需要开发一种高效的CHI3L1检测方法,以助力后续的研究工作。因此本研究以CHI3L1为目标蛋白,通过构建CHI3L1真核表达质粒,成功进行了体外表达与纯化,并使用该纯化蛋白免疫羊驼。通过4次免疫后,采集羊驼外周血并分离淋巴细胞,测定抗体水平达到了1∶64000,符合构建噬菌体展示文库的标准。随后,利用巢式PCR扩增了单个重链可变区(VHH)基因,并将该基因克隆至pComb噬菌体载体,成功构建了一个库容量为1.2×10^(8)cfu·mL^(-1)的CHI3L1纳米抗体噬菌体展示文库。通过三轮的免疫亲和筛选和富集,筛选得到四株不同序列的特异性纳米抗体(Nb)。随后,研究进一步构建了CHI3L1特异性纳米抗体的真核表达质粒,并转染293F细胞进行表达,使用Protein A亲和纯化系统纯化了所表达的蛋白。经ELISA测定,Nb-YC8和Nb-YC4显示出较好的亲和力。利用Western blot进一步验证了这些纳米抗体与商品化抗体相比,能够正确识别CHI3L1蛋白。免疫荧光分析显示,Nb-YC4、Nb-YD10和Nb-YF8能够识别293T细胞中表达的CHI3L1蛋白,而Nb-YC8未能成功识别CHI3L1,推测其可能识别的是线性表位。本研究成功筛选并鉴定了4株具有不同亲和力的CHI3L1特异性纳米抗体,这些抗体有望成为研究机体免疫系统中CHI3L1功能的有力工具。通过对CHI3L1的进一步研究,可能有助于揭示其在抗病毒免疫反应中的潜在作用,并为开发基于CHI3L1的诊断或治疗手段提供基础。
Chitinase 3-like protein 1(CHI3L1)expression levels significantly change after infection with influenza A virus(IAV),suggesting that it may play a role in regulating the antiviral immune response.However,no direct interaction between CHI3L1 and IAV has been found so far.Since CHI3L1 may play an important role in the immune process,researchers hypothesized that it may play a key role in regulating the antiviral immune response.However,the current research tools are insufficient.Therefore,there is an urgent need to develop an efficient method for detecting CHI3L1 to facilitate subsequent research.In this study,CHI3L1 was targeted as the protein of interest,and a eukaryotic expression plasmid was constructed for its successful in vitro expression and purification.The purified protein was immunized with alpacas.After four immunizations,peripheral blood was collected from the alpacas and lymphocytes were isolated,and the antibody level was determined to be 1:64000,which met the standard for constructing phage display libraries.Then,a single heavy chain variable region(VHH)gene was amplified using nested PCR and cloned into the pComb phage vector,successfully constructing a library with a capacity of 1.2×10^(8) cfu·mL^(-1)of CHI3L1 nanobody phage display library.Through three rounds of immune affinity selection and enrichment,four specific nanobodies(Nb)were selected.Subsequently,the CHI3L1-specific nanobody expression plasmid was further constructed and transfected into 293F cells for expression,and the protein was purified using the Protein A affinity purification system.The Nb-YC8 and Nb-YC4 showed good binding affinity as determined by ELISA.Western blot was used to further verify that these nanobodies were able to correctly recognize CHI3L1 protein compared to commercial antibodies.Immunofluorescence analysis showed that Nb-YC4,Nb-YD10 and Nb-YF8 could recognize CHI3L1 protein expressed in 293T cells,while Nb-YC8 failed to recognize CHI3L1,suggesting that it may recognize a linear epitope.In this study,four strains of CHI3L1-specific nanobodies with different affinities were successfully screened and identified,which are expected to be powerful tools to study the function of CHI3L1 in the body’s immune system.Further studies of CHI3L1 may help to uncover its potential role in the antiviral immune response and provide a basis for the development of CHI3L1-based diagnostic or therapeutic approaches.
作者
钟梦丹
吉艳红
张吉豫
朱啟超
冯赛祥
廖明
ZHONG Mengdan;JI Yanhong;ZHANG Jiyu;ZHU Qichao;FENG Saixiang;LIAO Ming(College of Veterinary Medicine,South China Agricultural University,Guangzhou 510000,China;Key Laboratory of Animal Disease Prevention and Control,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China)
出处
《畜牧兽医学报》
北大核心
2025年第7期3453-3462,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
甘肃省自然科学基金面上项目(22JR5RA028)
甘肃省重大科技专项(22ZD6NA001)。
