[1]王素娟,田晓平,赵巧辉,等.基于噬菌体展示技术醛固酮特异性抗体的筛选[J].新乡医学院学报,2024,(3):214-220.[doi:10.7683/xxyxyxb.2024.03.004]
　WANG Sujuan,TIAN Xiaoping,ZHAO Qiaohui,et al.Screening of aldosterone-specific antibodies based on phage display technology[J].Journal of Xinxiang Medical University,2024,(3):214-220.[doi:10.7683/xxyxyxb.2024.03.004]
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基于噬菌体展示技术醛固酮特异性抗体的筛选
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
期数:
2024年3
页码:
214-220
栏目:
基础研究
出版日期:
2024-03-05

文章信息/Info

Title:
Screening of aldosterone-specific antibodies based on phage display technology
作者:
王素娟1; 田晓平1; 赵巧辉1; 王天云2
（1.郑州伊美诺生物技术有限公司，河南　郑州　450016；2.新乡医学院，河南　新乡　453003）
Author(s):
WANG Sujuan1; TIAN Xiaoping1; ZHAO Qiaohui1; WANG Tianyun2
(1.Zhengzhou Immuno Biotech Co.,Ltd.,Zhengzhou 450016,Henan Province,China;2.Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
关键词:
噬菌体展示; 醛固酮抗体; 蛋白A亲和纯化
Keywords:
phage display; aldosterone antibodies; protein A affinity purification
分类号:
R392.3
DOI:
10.7683/xxyxyxb.2024.03.004
文献标志码:
A
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-1ALD-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32 000倍后，效价仍>10 000。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4.73×108 cfu·mL-1的噬菌体文库。经3轮淘洗出库平皿挑取300个单克隆，制备单克隆噬菌体，ELISA结果显示，300个克隆中阳性率达100%，校准品竞争检测阳性克隆42个，筛出率14%；42个阳性克隆进一步进行临床样本竞争检测，筛出16株单克隆符合要求，将16株进行复测，2次检测结果一致；测序后优选6条抗体序列进行构建表达，纯化后SDS-PAGE还原胶电泳结果显示，在重链50 000及轻链25 000位置均有条带，获得6株高亲和力、竞争性的兔ALD单抗。
结论　通过噬菌体展示技术成功筛选出6株高亲和力、竞争性的兔ALD抗体，这为小分子抗体的发现提供了一种参考方法。筛选出的AD185、AD277抗体在校准品及临床样本竞争中，均表现出比阳性对照高出2倍的竞争优势，这为ALD检测试剂盒原材料的开发提供了可能性。
Abstract:
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 5th 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 electrophoresis (SDS-PAGE).
Results　After the 5th immunization,the serum titers of 5 rabbits were indirectly tested,and the results showed that the serum titers of 4# and 5# white rabbits were still greater than 10,000 after being diluted by 32,000 times.The test results based on the competitive method showed that the ratio of low to high values in the plasma sample of 5# white rabbit was 2∶1,superior to that of other white rabbits.The 5# white rabbit was selected for phage library construction.The VL and VH gene fragments were amplified by conventional polymerase chain reaction,and then bridged into ScFv (VL+VH).The agar gel electrophoresis analysis showed that the size of the band was about 750 bp,which was consistent with the size of the originally designed fragment.ScFv was cleaved and electroporated into Escherichia coli TG1 to construct a phage library with a storage capacity of 4.73 × 108 cfu·mL-1.After 3 rounds of washing,300 monoclonal clones were selected from the outbound petri dishes to prepare monoclonal bacteriophages.The ELISA results showed a positive rate of 100% among the 300 clones,and 42 clones were tested positive for calibration competition,with a screening rate of 14%.The 42 positive clones were further subjected to clinical sample competition testing,and 16 monoclonal strains that met the requirements were screened.The 16 strains were retested,and the results of the two tests were consistent.After sequencing,6 antibody sequences were selected for construction and expression.After purification,SDS-PAGE reduced gel electrophoresis results showed that there were bands at positions 50,000 on the heavy chain and 25,000 on the light chain.Six highly affinitive and competitive rabbit ALD monoclonal antibodies were obtained.
Conclusion　Six highly affinitive and competitive rabbit ALD antibodies are successfully screened using phage display technology,which provides a reference method for the discovery of small molecule antibodies.The screened AD185 and AD277 antibodies show a competitive advantage twice that of the positive control in the competition of calibration and clinical samples,providing a possibility for the development of raw materials for ALD detection kits.

参考文献/References:

［1］　JIN Y J,YU D,TIAN X L,et al.A novel and effective approach to generate germLine-like monoclonal antibodies by integration of phage and mammalian cell display platforms［J］.Acta Pharmacol Sin,2022,43(4):954-962
［2］　郎巧利,余琳,何麒麟，等.高效构建卵清白蛋白scFv噬菌体文库及其筛选［J］.中国生物工程杂志,2018,38(11):25-31.
LIANG Q L,YU L,HE Q L,et al.Construction and screening of a phage display library of single chain Fv antibody efficiently from mouse immunized with ovalbumin［J］.Chin Biotechnol,2018,38(11):25-31.
［3］　BAURAND P E,BALLAND J,REYNAS C,et al.Development of anti-LRRC15 small fragments for imaging purposes using a phage-display ScFv approach［J］.Int J Mol Sci,2022,23(20):12677.
［4］　DU J,XIANG D,LIU F,et al.Hijacking the self-replicating machine of bacteriophage for PCR-based cascade signal amplification in detecting SARS-CoV-2 viral marker protein in serum［J］. Sens Actuators B Chem,2023,374:132780.
［5］　PEISSERT F,PLSS L,GIUDICE A M,et al.Selection of a PD-1 blocking antibody from a novel fully human phage display library［J］.Protein Sci,2022,31(12):e4486.
［6］　TAKAKUSAGI Y,TAKAKUSAGI K,SAKAGUCHI K,et al.Phage display technology for target determination of small-molecule therapeutics:an update［J］.Expert Opin Drug Discov,2020,15(10):1199-1211.
［7］　MENDOZA-SALAZAR I,GMEZ-CASTELLANO K M,GONZ-LEZ-GONZLEZ E,et al.Anti-SARS-CoV-2 Omicron antibodies isolated from a SARS-CoV-2 delta semi-immune phage display library［J］.Antibodies,2022,11(1):13.
［8］　陈婷,贾秋蕾,褚瑜光,等.盐敏感性高血压收缩压负荷与肾素-血管紧张素-醛固酮系统、皮质醇相关性研究［J］.中国循证心血管医学杂志,2022,14(1):84-86.
CHEN T,JIA Q L,CHU Y G,et al.Correlation among systolic blood pressure load,renin-angiotensin-aldosterone system and cortisol in patients with salt-sensitive hypertensive［J］.Chin J Evid Based Cardiovasc Med,2022,14(1):84-86.
［9］　COLUSSI G,CATENA C,LAPENNA R,et al.Insulin resistance and hyperinsulinemia are related to plasma aldosterone levels in hypertensive patients［J］.Diabetes Care,2007,30(9):2349-2354.
［10］　张念荣,李文歌.糖尿病患者高血压的治疗［J］.中国临床医生杂志,2021,49(12):1399-1402.
ZHANG N R,LI W G.Treatment of hypertension in diabetic patients［J］.Chin J Clin,2021,49(12):1399-1402.
［11］　MORIMOTO R,OMATA K,ITO S,et al.Progress in the management of primary aldosteronism［J］.Am J Hypertens,2018,31(5):522-531.
［12］　VAN KLEEF M E A M,VISSEREN F L J,WESTERINK J,et al.Development of a clinical decision tool to reduce diagnostic testing for primary aldosteronism in patients with difficult-to-control hypertension［J］.BMC Endocr Disord,2020,20(1):56.
［13］　CHEN F,CHENG Z,PENG Y,et al.A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for simultaneous quantification of aldosterone,renin activity,and angiotensin II in human plasma［J］.J Chromatogr B Analyt Technol Biomed Life Sci,2021,1179:122740.
［14］　SHIDLOVSKYI V O,SHIDLOVSKYI O V,TOVKAI O A,et al.Topical diagnosis and determination of the primary hyperaldosteronism variant［J］.J Med Life,2019,12(4):322-328.
［15］　WANNACHALEE T,TURCU A F.Primary aldosteronism:a continuum from normotension to hypertension［J］.Curr Cardiol Rep,2021,23(8):105.
［16］　赖凤华,曹筱佩,林慧美,等.两种血浆肾素、醛固酮检测方法对原发性醛固酮增多症筛查效率的评价［J］.中华高血压杂志,2015,23(2):150-153.
LAI F H,CAO X P,LIN H M,et al.Comparison of plasma renin and aldosterone measured by radioimmunoassay and chemiluminescence immunoassay in screening primary aldosteronism［J］.Chin J Hypertens,2015,23(2):150-153.
［17］　PILZ S,KEPPEL M H,TRUMMER C,et al.Diagnostic accuracy of the aldosterone-to-active renin ratio for detecting primary aldosteronism［J］.J Endocr Soc,2019,3(9):1748-1758.
［18］　李亚楠,张赛春,臧丽,等.甲状旁腺激素水平在原发性醛固酮增多症及肾上腺无功能瘤患者鉴别诊断中的应用价值［J］.解放军医学院学报,2021,42(9):913-917.
LI Y N,ZHANG S C,ZANG L,et al.Value of parathyroid hormone level in differential diagnosis of patients with primary aldosteronism and nonfunctional adrenal tumors［J］.Acad J Chin PLA Med Sch,2021,42(9):913-917.
［19］　YANAI K,ISHIBASHI K,MORISHITA Y.Systematic review and meta-analysis of renin-angiotensin-aldosterone system blocker effects on the development of cardiovascular disease in patients with chronic kidney disease［J］.Front Pharmacol,2021,12:662544.
［20］　WANG Y,LI C X,LIN Y N,et al.The role of aldosterone in OSA and OSA-related hypertension［J］.Front Endocrinol,2021,12:801689.
［21］　WANG P,LUO C,ZHU D,et al.Pericardial adipose tissue-derived leptin promotes myocardial apoptosis in high-fat diet-induced obese rats through Janus kinase 2/reactive oxygen species/Na+/K+-ATPase signaling pathway［J］.J Am Heart Assoc,2021,10(18):e021369.
［22］　BOTHOU C,BEUSCHLEIN F,SPYROGLOU A.Links between aldosterone excess and metabolic complications:a comprehensive review［J］.Diabetes Metab,2020,46(1):1-7.
［23］　STEWART COATS A J,SHEWAN L.Eplerenone′s role in the management of complex cardiovascular disorders［J］.Int J Cardiol,2015,200:1-2.
［24］　KALIL G Z,HAYNES W G.Sympathetic nervous system in obesity-related hypertension:mechanisms and clinical implications［J］.Hypertens Res,2012,35(1):4-16.
［25］　LIN X,ULLAH M H E,WU X,et al.Cerebro-cardiovascular risk,target organ damage,and treatment outcomes in primary aldosteronism［J］.Front Cardiovasc Med,2021,8:798364.
［26］　TOMILIN V N,PYRSHEV K,KHAYYAT N H,et al.With-No-lysine kinase 1 (WNK1) augments TRPV4 function in the aldosterone-sensitive distal nephron［J］.Cells,2021,10(6):1482.
［27］　JONES J C,CARTER G D,MACGREGOR G A.Interference by polar metabolites in a direct radioimmunoassay for plasma aldosterone［J］.Ann Clin Biochem,1981,18(Pt 1):54-59.

更新日期/Last Update: 2024-03-05