张荣红,贾铁文,何志旭,刘杰麟
ZHANG Ronghong,JIA Tiewen,HE Zhixu,LIU Jielin

• 1:贵州医科大学组织工程与干细胞中心
• 2:贵州医科大学免疫学教研室

摘要(Abstract)：

目的:通过有限稀释法制备抗RecQ单克隆抗体(m Ab),鉴定其生物学特性。方法:以纯化鉴定后的重组大肠杆菌RecQ解旋酶免疫Balb/c小鼠,融合免疫小鼠的脾细胞及SP2/0骨髓瘤细胞,有限稀释法筛选杂交瘤细胞,制备m Ab;使用间接ELISA法鉴定单克隆抗体亚型、效价,Western-Blot法测定单克隆抗体特异性;将m Ab与RecQ解旋酶结合后,观察m Ab对RecQ解旋酶活性的阻断作用。结果:获得1株能稳定分泌抗RecQ解旋酶单克隆抗体的杂交瘤细胞株1C1,该杂交瘤细胞染色体数目在94～104; 1C1分泌的单克隆抗体类型为Ig G1型,1C1株所得上清液和腹水中抗体效价分别为1×10-3和1×10-5,该单克隆抗体可特异性结合RecQ,并可抑制RecQ解旋酶的结合DNA活性。结论:成功制备1株抗RecQ m Ab,效价高、特异性强。
Objective: To prepare monoclonal antibodies( m Ab) against RecQ helicase,and characterize their biological properties. Methods: By limiting dilution method,the m Ab was generated by immunizing BALB/c mice with purified recombinant RecQ helicase from E. coli. Splenocytes derived from immunized mice were fused with SP2/0 myeloma cells to generate and screen hybridoma. Colchicine blocking was used to analyze the chromosome karyotype of hybridoma named by 1 C1 cells. 1 C1 hybridoma cells were intraperitoneally injected to BALB/C mice for producing ascites which are in rich m Ab against RecQ helicase. Indirect ELISA methods were used to determine Ig subtype and titer m Ab in ascitic fluid. Western blotting was used to determine the biological sizes of m Ab. Fluorescence polarization immunoassay( FPIA) was used to detect the interactions of m Ab with RecQ helicase.Results: 1 C1 hybridoma stably expressing anti-RecQ m Ab was obtained. 1 C1 hybridoma has chromosomes ranging from from 94 to 104,and its Ig subtype was IgG1. The titer of 1 C1 in supernatant and ascitic fluid was 1 × 10-3 and 1 × 10-5,respectively. The m Ab could specifically recognize E. coli RecQ helicase and thus inhibit its DNA binding activity. Conclusion: The m Ab against RecQ helicase was successfully prepared with high titer and specificity.

关键词(KeyWords)： RecQ解旋酶;单克隆抗体;制备;鉴定;有限稀释法
RecQ helicase;monoclonal antibody;preparation;identification;limiting dilution analysis

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(81360349);; 贵州省优秀科技教育人才省长专项资金项目[黔省专合字(2006)57];; 贵州省国际科技合作计划[黔科合外G字(2011)7012];; 贵州省应用基础研究计划重大专项子课题[黔科合J重大字(2015)2003];; 贵州省教育厅青年科技人才成长项目[黔教合KY字(2018)174]

作者(Author): 张荣红,贾铁文,何志旭,刘杰麟
ZHANG Ronghong,JIA Tiewen,HE Zhixu,LIU Jielin

DOI: 10.19367/j.cnki.1000-2707.2019.02.001

参考文献(References)：

• [1]GUO R,RIGOLET P,ZARGARIAN L,et al. Structuraland functional characterizations reveal the importance of azinc binding domain in Bloom's syndrome helicase[J].Nucleic Acids Research,2005,33(10):3109-3124.
• [2] CHU W K,HICKSON I D. Rec Q helicases:multifunc-tional genome caretakers[J]. Nature Reviews Cancer,2009,9(9):644.
• [3]CROTEAU D L,POPURI V,OPRESKO P L,et al. Hu-man Rec Q helicases in DNA repair,recombination,andreplication[J]. Annual Review of Biochemistry,2014,83:519-552.
• [4]MO D,ZHAO Y,BALAJEE A S. Human Rec QL4 heli-case plays multifaceted roles in the genomic stability ofnormal and cancer cells[J]. Cancer Letters,2018,413:1-10.
• [5]DE RENTY C,ELLIS N A. Bloom’s syndrome:Why notpremature agin:A comparison of the BLM and WRN heli-cases[J]. Ageing Research Reviews,2017,33:36-51.
• [6]OSHIMA J,SIDOROVA J M,MONNATJR R J. Wernersyndrome:clinical features,pathogenesis and potentialtherapeutic interventions[J]. Ageing Research Reviews,2017,33:105-114.
• [7]HUANG L,HU C,DI BENEDETTO M,et al. Inductionof multiple drug resistance in HMEC-I endothelial cellsafter long-term exposure to sunitinib[J]. Onco Targets T-her,2014,7(12):2249-2255.
• [8]LU L,JIN W,WANG L L. Aging in Rothmund-Thomsonsyndrome and related RECQL4 genetic disorders[J].Ageing Res Rev,2017,33:30-35.
• [9]LI L,GAO M,SONG B,et al. Effects of RECQ1 heli-case silencing on non-small cell lung cancer cells[J]. Bi-omed Pharmacother,2016,83:1227-1232.
• [10]VIZITEU E,KLEIN B,BASBOUS J,et al. RECQ1 he-licase is involved in replication stress survival and drugresistance in multiple myeloma[J]. Leukemia,2017,31(10):2104-2113.
• [11]ARORA A,PARVATHANENI S,ALESKANDARANYM A,et al. Clinicopathological and functional signifi-cance of RECQ1 helicase in sporadic breast cancers[J].Mol Cancer Ther,2017,16(1):239-250.
• [12]MO D,ZHAO Y,BALAJEE A S. Human Rec QL4 heli-case plays multifaceted roles in the genomic stability ofnormal and cancer cells[J]. Cancer Lett,2018,413:1-10.
• [13]MAO F J,SIDOROVA J M,LAUPER J M,et al. Thehuman WRN and BLM Rec Q helicases differentially regu-late cell proliferation and survival after chemotherapeuticDNA damage[J]. Cancer Research,2010,8:5467-5472.
• [14]WANG X,HU L. Protein expression of BLM gene and itsapoptosis sensitivity in hematopoietic tumor cell strains[J].华中科技大学学报(医学英文版),2008,28(1):46-48.
• [15]孟惠惠,许厚强,刘金河,等.三种癌细胞株中Bloom综合征解旋酶(BLM)的表达水平高于正常细胞[J].细胞与分子免疫学杂志,2014,30(6):649-651.
• [16]段丽霞,许厚强,陈祥,等.丙酸睾酮对大肠杆菌Rec Q解旋酶结构和功能的影响[J].中国药理学通报,2011,27(4):467-472.
• [17]骆衡,陈祥,段丽霞,等.大肠杆菌Rec Q解旋酶的生物学活性分析[J].中国生物化学与分子生物学报,2010,26(12):1143-1150.
• [18]黄振蓉,吴海丽,张三军,等. E. coli Rec Q解旋酶克隆表达纯化及生物学活性检测[J].中国生物工程杂志,2013,33(3):21-27.
• [19]RAYNARD S,BUSSEN W,SUNG P. A double hollidayjunction dissolvasome comprising BLM, topoisomeraseIIIα,and BLAP75[J]. Journal of Biological Chemistry,2006,281(20):13861-13864.
• [20]张荣红,贾铁文,廉芳,等.抗Rec Q解旋酶单克隆抗体的制备和鉴定及应用[J].细胞与分子免疫学杂志,2018,34(2):169-174.
• [21]张越,屈会化,吴婷婷,等.甘草酸单克隆抗体的制备与鉴定[J].药物分析杂志,2013,33(5):770-774.
• [22]HANACK K,MESSERSCHMIDT K,LISTEK M. Anti-bodies and selection of monoclonal antibodies[M]. Ber-lin:Springer,2016:11-22.
• [23]BLDICKE,T. Protein targeting compounds:predic-tion,selection and activity of specific inhibitors[M].Berlin:Springer,2016.
• [24]GAO Y,HUANG X,ZHU Y,et al. A brief review ofmonoclonal antibody technology and its representative ap-plications in immunoassays[J]. Journal of Immunoassayand Immunochemistry,2018,39(4):351-364.
• [25]苏玉虹,刘玲.用甲基纤维素半固体培养基筛选杂交瘤细胞[J].中国免疫学杂志,1999,15(7):311-311.
• [26]司徒镇强.细胞培养[M]. 2版.北京:世界图书出版公司,2008:249-261.

文章评论(Comment)：