摘要
目的利用噬菌体展示技术及重组抗体构建技术筛选出特异性醛固酮(ALD)抗体,为ALD诊断试剂盒的研发提供原材料。方法取5只健康清洁级新西兰大白兔,将抗原ALD-血蓝蛋白(KLH)稀释至2 mg·L^(-1),采用背部多点注射方法对5只新西兰大白兔进行首次免疫,免疫剂量为每只1 mg;每隔2周进行1次免疫,且免疫剂量减少50%;从第3次免疫开始,每次免疫1周后采集大白兔耳缘静脉血,使用包被0.25 mg·L^(-1) ALD-BSA抗原的化学发光板,采用间接法和竞争法测定血清滴度;第5次免疫后选取血清效价高、特异性好的大白兔,取其脾脏和骨髓等组织。将脾脏组织研磨,采用TRIzol试剂一步法提取RNA,获取轻链可变区(VL)和重链可变区(VH)基因序列。通过连接肽连接成单链片段(ScFv)并构建至噬菌粒载体Pcomb3xss中;然后,通过电转化方法将其导入大肠杆菌TG1,完成ALD ScFv噬菌体展示库构建。对文库进行3~5轮富集筛选,并挑取单克隆进行噬菌体上清制备,应用酶联免疫吸附试验(ELISA)竞争法鉴定后送测,获得高竞争性克隆序列;将筛选得到的克隆序列插入至pCMV3表达载体,提取质粒后使用瞬时转染方法进行HEK293细胞转染;1周后,收取上清,应用亲和层析纯化及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳鉴定其纯度及表达量。结果5只大白兔在第5次免疫后,间接法检测其血清效价结果显示,4#、5#大白兔血清在稀释32000倍后,效价仍>10000。竞争法检测结果显示,5#大白兔血浆样本低值与高值的比值为2:1,优于其他大白兔。选取5#大白兔进行噬菌体文库构建。使用常规聚合酶链反应扩增出VL及VH基因片段,经搭桥拼接成ScFv(VL+VH)后,琼脂凝胶电泳分析可看到750 bp左右大小条带,与最初设计的片段大小一致。ScFv经酶切连接电转至大肠杆菌TG1中,构建一个库容为4.73×10^(8) cfu·mL^(-1)的噬菌体文库。经3轮淘洗出库平皿挑取300个
Objective To screen out specific aldosterone(ALD)antibodies using phage display technology and recombinant antibody technology,providing raw materials for the research and development of ALD diagnostic kits.Methods Five healthy and clean New Zealand white rabbits were selected and immunized for the first time against the diluted ALD-keyhole limpet hemocyanin antigen(2 mg·L^(-1))using a multi-point injection method on the back,with a dose of 1 mg per rabbit.Immunization was administered again every 2 weeks,with a 50%reduction in dose.Starting from the third immunization,the ear vein blood of the rabbits was collected one week after each immunization.A chemiluminescent plate coated with 0.25 mg·L^(-1) ALD-bovine serum albumin antigen was used to measure serum titers via indirect and competitive methods.After the 5 th immunization,the rabbit with high serum titers and good specificity was selected,and its spleen and bone marrow were removed.The spleen tissue was grinded,and RNA was extracted using TRIzol reagent in one step to obtain gene sequences in the variable region of light chain(VL)and the variable region of heavy chain(VH).The single-chain variable fragment(ScFv)was connected through the linker and constructed into the bacteriophage vector Pcomb3xss;then,it was carried to Escherichia coli TG1 through electrotransformation,and the ALD ScFv phage display library was constructed accordingly.Three to five rounds of enrichment screening were performed against the library.Monoclonal clones,identified by enzyme-linked immunosorbent assay(ELISA)competitive method,were selected for phage supernatant preparation,and a highly competitive clone sequence was obtained.The screened clone sequence was inserted into the pCMV3 expression vector,and the HEK293 cell was transfected using the transient transfection method after the plasmid was extracted.One week later,the supernatant was collected,and its purity and expression were identified by affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophore
作者
王素娟
田晓平
赵巧辉
王天云
WANG Sujuan;TIAN Xiaoping;ZHAO Qiaohui;WANG Tianyun(Zhengzhou Immuno Biotech Co.,Ltd.,Zhengzhou 450016,Henan Province,China;Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
出处
《新乡医学院学报》
CAS
2024年第3期214-220,共7页
Journal of Xinxiang Medical University
关键词
噬菌体展示
醛固酮抗体
蛋白A亲和纯化
phage display
aldosterone antibodies
protein A affinity purification
