[1]张淑婷,田海霞,刘青青,等.人巨细胞病毒PP65抗原单克隆抗体的制备及鉴定[J].第三军医大学学报,2020,42(12):1182-1187.
 ZHANG Shuting,TIAN Haixia,LIU Qingqing,et al.Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen [J].J Third Mil Med Univ,2020,42(12):1182-1187.
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人巨细胞病毒PP65抗原单克隆抗体的制备及鉴定(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第12期
页码:
1182-1187
栏目:
基础医学
出版日期:
2020-06-30

文章信息/Info

Title:
Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen
 
作者:
张淑婷田海霞刘青青康行樊卫平
山西医科大学微生物学与免疫学教研室
Author(s):
ZHANG Shuting TIAN Haixia LIU Qingqing KANG Xing FAN Weiping

epartment of Microbiology and Immunology, Shanxi Medical University, Taiyuan, Shanxi Province, 030001, China
 

关键词:
人巨细胞病毒PP65抗原单克隆抗体免疫组化
Keywords:
human cytomegalovirus PP65 antigen monoclonal antibodies immunohistochemistry
分类号:
R373.9;R392-33;R392.11
文献标志码:
A
摘要:

目的制备人巨细胞病毒(human cytomegalic virus, HCMV)PP65抗原的单克隆抗体,并通过免疫组化鉴定单克隆抗体与HCMV PP65抗原的反应性。方法诱导PQE30-PP65重组质粒表达PP65蛋白并Ni柱亲和层析纯化。PP65抗原免疫BALB/c小鼠,将血清抗体阳性小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,经HTS筛选,扩大培养获取单克隆抗体。经Protein A亲和层析纯化,并通过间接ELISA鉴定其特异性、效价及亚型。将制备的单克隆抗体应用于临床样本的玻片免疫组化检测。结果获得8株高特异性抗HCMV PP65抗原的单克隆抗体,其亚型均为IgG1型,轻链均为κ链,效价均在1∶2 048 000以上。其中6株单克隆抗体能够通过玻片免疫组化法准确鉴定HCMV激活性感染。结论成功制备了HCMV PP65单克隆抗体。单抗与白细胞中HCMV PP65抗原反应良好。
 

Abstract:

ObjectiveTo prepare monoclonal antibodies (mAbs) against human cytomegalovirus (HCMV) PP65 antigen and characterize their reactivity to HCMV PP65 antigen by immunohistochemistry. MethodsThe recombinant plasmid PQE30-PP65 was transformed into competent E.coli M15 cells to obtain the recombinant PP65 antigen, which was purified with Ni column affinity chromatography. BALB/c mice were immunized with the purified antigen, and the spleen cells of the mice were isolated and fused with myeloma cells (SP2/0). The mAbs were obtained by HTS screening and purified by protein A affinity chromatography, and their specificity, titer and subtype were identified by indirect ELISA. The performance of the mAbs in immunohistochemical detection of HCMV PP65 antigen was tested on slides of clinical samples. ResultsEight highly specific mAbs against HCMV PP65 antigen were obtained. All these mAbs were identified to be IgG1 subtype containing κ light chains, with titers all above 1∶2 048 000. Six of the mAbs were shown to accurately identify an active infection of HCMV by slide immunohistochemistry. ConclusionWe successfully prepared HCMV PP65 mAbs, and 6 of these mAbs have good reactivity with HCMV PP65 antigen in leukocytes.

参考文献/References:

[1]赵捷, 闻良珍, 张桂梅. 人巨细胞病毒蛋白pp65诱导特异性免疫应答的研究[J]. 医学分子生物学杂志, 2008, 5(2):  137-140. DOI: 10.3870/j.issn.1672-8009.2008.02.010.

ZHAO J, WEN L Z, ZHANG G M. Human cytomegalovirus pp65 protein induces specific immune response[J]. J Med Mol Biol, 2008, 5(2):  137-140. DOI: 10.3870/j.issn.1672-8009.2008.02.010.

[2]GRIFFITHS P, BARANIAK I, REEVES M. The pathogenesis of human cytomegalovirus[J]. J Pathol, 2015, 235(2):  288-297. DOI: 10.1002/path.4437.

[3]STARAS S A S, DOLLARD S C, RADFORD K W, et al. Seroprevalence of cytomegalovirus infection in the United States, 1988-1994[J]. Clin Infect Dis, 2006, 43(9):  1143-1151. DOI: 10.1086/508173.

[4]DIEJOMAOH M F E, AL-AZEMI M, JIROUS J, et al. The aetiology and pattern of recurrent pregnancy loss[J]. J Obstet Gynaecol, 2002, 22(1):  62-67. DOI: 10.1080/01443610120101754.

[5]BIOLATTI M, DELL’OSTE V, DE ANDREA M, et al. The human cytomegalovirus tegument protein pp65 (pUL83):  a key player in innate immune evasion[J]. New Microbiol, 2018, 41(2):  87-94.

[6]GERNA G, LILLERI D. Huma cytomegalovirus (HCMV) infection/re-infection:  development of a protective HCMV vaccine[J]. New Microbiol, 2019, 42(1):  1-20.

[7]PANDE H, CAMPO K, TANAMACHI B, et al. Human cytomegalovirus strain Towne pp28 gene:  sequence comparison to pp28 of HCMV AD169 and stable expression in Chinese hamster ovary cells[J]. Virology, 1991, 184(2):  762-767. DOI: 10.1016/0042-6822(91)90448-k.

[8]SUN Z R, CENG X S, MAO Z Q, et al. Diagnostic value of HCMV pp65 antigen detection by FCA for symptomatic and asymptomatic infection:  compared to quantification of HCMV DNA and detection of IgM antibody in infants[J]. Med Microbiol Immunol, 2009, 198(2):  107-112. DOI: 10.1007/s00430-009-0112-y.

[9]刘玲玲, 王亮, 李琳, 等. 人巨细胞病毒PP65抗原ELISA检测方法的建立[J]. 免疫学杂志, 2014, 30(6):  545-549. DOI: 10.13431/j.cnki.immunol.j.20140119.

LIU L L, WANG L, LI L, et al. The establishment of ELISA for HCMV-PP65 antigen[J]. Immunol J, 2014, 30(6):  545-549. DOI: 10.13431/j.cnki.immunol.j.20140119.

[10]王亮, 潘丽, 邓智毅, 等. 凝血与抗凝血中T细胞活性的分析[J]. 免疫学杂志, 2014, 30(11):  1007-1010. DOI: 10.19405/j.cnki.issn/1000-1492.2011.03.023.

WANG L, PAN L, DENG Z Y, et al. A comparative analysis of T-lymphocyte activity in blood clot and anticoagulated blood[J]. Immunol J, 2014, 30(11):  1007-1010. DOI: 10.19405/j.cnki.issn/1000-1492.2011.03.023.

[11]金艳. 人巨细胞病毒先天性感染实验室诊断的研究进展[J]. 安徽医科大学学报, 2011, 46(3):  282-285. DOI: 10.19405/j.cnki.issn.1000-1492.2011.03.023.

JIN Y.Progress in laboratory diagnosis of congenital infection of human cytomegalovirus [J]. Acta Univ Med Anhui, 2011, 46(3):  282-285. DOI:  10.19405/j.cnki.issn.1000-1492.2011.03.023.

[12]HAKKI M, GEBALLE A P. Double-stranded RNA binding by humanCytomegalovirus pTRS1[J]. J Virol, 2005, 79(12):  7311-7318. DOI: 10.1128/jvi.79.12.7311-7318.2005.

更新日期/Last Update: 2020-06-19