[1]孙卫国,孙雯娜,侯江厚,等.人巨细胞病毒pp150-gp52蛋白原核可溶性表达与IgM捕获ELISA方法建立和应用[J].现代检验医学杂志,2015,30(05):1-3.[doi:10.3969/j.issn.1671-7414.2015.05.001]
 SUN Wei-guo,SUN Wen-na,HOU Jiang-hou,et al.Soluble Expression of HCMV pp150-gp52 Protein in Prokaryotic System and Establishment of IgM Capture-ELISA[J].Journal of Modern Laboratory Medicine,2015,30(05):1-3.[doi:10.3969/j.issn.1671-7414.2015.05.001]
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人巨细胞病毒pp150-gp52蛋白原核可溶性表达与IgM捕获ELISA方法建立和应用()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第30卷
期数:
2015年05期
页码:
1-3
栏目:
论著
出版日期:
2015-12-10

文章信息/Info

Title:
Soluble Expression of HCMV pp150-gp52 Protein in Prokaryotic System and Establishment of IgM Capture-ELISA
作者:
孙卫国孙雯娜侯江厚杨秉芬张灵霞
解放军第309医院,结核病研究所,全军结核病防治重点实验室,北京100091
Author(s):
SUN Wei-guoSUN Wen-naHOU Jiang-houYANG Bing-fenZHANG Ling-xia
Army Tuberculosis Prevention and Control Key Laboratory, Institute for Tuberculosis Research,the 309 Hospital of PLA,Beijing 100091,China
关键词:
巨细胞病毒pp150蛋白gp52蛋白可溶性表达
分类号:
R373;Q503
DOI:
10.3969/j.issn.1671-7414.2015.05.001
文献标志码:
A
摘要:
目的原核系统内表达并纯化巨细胞病毒pp150-gp52融合蛋白优势抗原表位,建立IgM捕获ELISA方法并应用。方法通过重叠PCR技术扩增获得巨细胞病毒pp150-gp52优势片段核酸序列,在原核系统内可溶性表达DsbC-pp150-gp52融合蛋白并纯化,用Western blot和ELISA检测融合蛋白的特异性和应用价值。结果纯化获得的融合蛋白DsbC-pp150-gp52经酶标记建立IgM捕获ELISA方法,检测60份临床阳性血清和60份健康人血清。其中以酶标记DsbC-pp150-gp52蛋白建立的捕获ELISA法阳性检出率96.7% ,阴性检出率100%,初步验证DsbC-pp150-gp52融合肽具有非常好的抗原特异性。结论融合蛋白DsbC-pp150-gp52在大肠埃希菌中以可溶性表达形式存在,获得的高纯度重组融合蛋白具有抗原性和特异性强的特点,采用IgM捕获ELISA的实验方法,可开发检测试剂盒用于风疹病毒的早期检测。
Abstract:
ObjectiveTo obtain epitopes of pp150-gp52 fusion protein of HCMV expression in solubility in prokaryotic system and to establish IgM antibody capture-ELISA test.MethodsThe gene sequence of antigen superiority segment of HCMV pp150-gp52 was obtained by overlap PCR from HCMV genome,which was cloned into pET-DsbC vector.After expression and purification,the antigenicity and specificity of the fusion protein were examined by Western Blot and ELISA.ResultsThe fusion protein DsbC-pp150-gp52 showed a strong antigenicity when examined by Western Blot.Detecting 60 HCMV positive sera and 60 negative sera by IgM capture ELISA assay showed that the positive rate was 98.3% and negative rate was 100%.ConclusionThe results suggested that the fusion protein DsbC-pp150-gp52 could express solubly in Escherichia coli.The recombinant protein with high purity may be a potential candidate of serodiagnostic reagent for early clinical detection of HCMV infection.

参考文献/References:

[1]Onno M,Pangault C,Le Friec G,et al.Modulation of HLA-G antigens expression by human cytomegalovirus:specific induction in activated macrophages harboring human cytomegalovirus infoetion[J].J Immunol,2000,164(12):6426-6434.
[2]Revello MG,Gerna G.Diagnosis and management of human cytomegalovirus infection in the mother,fetus,and newborn infant[J].Clin Microbiolgy,2002,15(45):680-715.
[3]Greijer AE,VanDe Crommert JG,Stevens SC,et al.Molecular finespecificity analysis of antibody responses to human cytomegalovirus and design of novel synthetic-peptide-based serodiagnostic assays[J].J Clin Microbiol,1999,37(1):179-188.
[4]Vornhagen R,Plachter B,Hinderer W,et al.Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens[J].J Clin Microbiol,1994,32(4):981-985.
[5]孙卫国,金程燕,李邦印,等.巨细胞病毒gp52蛋白原核可溶性表达与应用[J].实用预防医学,2013,20(12):1419-1422. Sun WG,Jin CY,Li BY,et al.Soluble expression and application of HCMY gp52 protein in prokaryotic system[J].Practical Preventive Medicine,2013,20(12):1419-1422.
[6]姜永玮,吴英松,徐伟文,等.人巨细胞病毒部分抗原基因的克隆及表达[J].热带医学杂志,2007,7(3):218-221. Jiang YW,Wu YS,Xu WW,et al.Cloning and expression of HCMV genes[J].J Tropical Medicine,2007,7(3):218-221.
[7]La Rosa C,Wang Z,Lacey SF,et al.Characterization of host immunity to cytomegalovirus pp150(UL32)[J].Human Immunology,2005,66(2):116-126.
[8]Schlapschy M,Skerra A.Periplasmic chaperones use-d to enhance functional secretion of proteins in E.coli[J].Methods Mol Biol,2011(705):211-224.

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备注/Memo

备注/Memo:
基金项目:国家传染病重大项目(2013ZX-10003-006)。 作者简介:孙卫国(1973-),男,博士,助理研究员,主要研究方向:传染性疾病的诊断与治疗,E-mail:sunwg2009@163.com。 通讯作者:张灵霞,Tel:010-66775969,E-mail邮箱:zhanglingxia7@aliyun.com。
更新日期/Last Update: 1900-01-01