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人巨细胞病毒PP65抗原单克隆抗体的制备及鉴定
Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen
- 第三军医大学学报 2020年42卷第12期 页码：1182-1187
- 作者机构：
030001 太原，山西医科大学微生物学与免疫学教研室
- 作者简介：
[ 樊卫平，E-mail：fanweiping26418@126.com FAN Weiping, E-mail:fanweiping26418@126.com
- 基金信息：山西省专利推广实施资助专项(20171009);山西省科技开发基金(033068)
DOI：10.16016/j.1000-5404.202002059
中图分类号： R373.9; R392-33; R392.11收稿：2020-01-22，
修回：2020-03-08，
纸质出版：2020-06-30
- 稿件说明：
移动端阅览
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.
Shuting ZHANG, Haixia TIAN, Qingqing LIU, et al. Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen[J]. Journal of Third Military Medical University, 2020, 42(12): 1182-1187.
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. DOI： 10.16016/j.1000-5404.202002059.
Shuting ZHANG, Haixia TIAN, Qingqing LIU, et al. Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen[J]. Journal of Third Military Medical University, 2020, 42(12): 1182-1187. DOI： 10.16016/j.1000-5404.202002059.
摘要
目的
2
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
2
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
2
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
2
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
Objective
2
To prepare monoclonal antibodies (mAbs) against human cytomegalovirus (HCMV) PP65 antigen and characterize their reactivity to HCMV PP65 antigen by immunohistochemistry.
Methods
2
The 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 an
tigen
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.
Results
2
Eight 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.
Conclusion
2
We successfully prepared HCMV PP65 mAbs
and 6 of these mAbs have good reactivity with HCMV PP65 antigen in leukocytes.
关键词
Keywords
references
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