风疹病毒E1抗原特异性片段在酵母系统中重组表达及应用
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摘要
目的 利用真核系统对E1蛋白进行表达并纯化,建立一种以重组E1蛋白为包被抗原的风疹病毒抗体ELISA检测方法。方法 通过生物信息学预测E1优势抗原表位,根据真核系统表达的特点进行序列优化,全基因合成获得风疹病毒E1优势片段核酸序列,构建真核表达载体,在真核系统内进行诱导表达并纯化,用Western blot证实E1蛋白的抗原性。建立ELISA检测系统,并初步检测100份临床血清标本。结果 SDS-PAGE证实重组E1蛋白在酵母中高表达,Western blot证实该E1蛋白具有良好的抗原性,成功建立了检测IgM间接ELISA方法,对临床血清标本的初步检测显示该试剂盒具有较好的敏感性和特异性。结论 本ELISA检测试剂有较高敏感性和较强的特异性,可进一步开发用于检测风疹病毒的早期感染的试剂盒。
Objective To express and purify E1 protein by eukaryotic system, and to establish the ELISA detection method for rubella virus antibody with recombinant E1 protein as coating antigen. Methods The E1 dominant antigen epitopes were predicted by bioinformatics. The nucleic acid sequence of the rubella virus E1 dominant fragment was obtained by whole gene synthesis according to the expression characteristics of eukaryotic system, and the eukaryotic expression vector was constructed. The expression of E1 protein was induced in eukaryotic system and purified. The antigenicity of E1 protein was confirmed by Western blot. ELISA detection system was established, and 100 clinical serum samples were preliminarily detected. Results SDS-PAGE results confirmed that the recombinant E1 protein was highly expressed in yeast. Western blot results confirmed that the E1 protein had good antigenicity. A method for detecting IgM by indirect ELISA was successfully established. The kit had both the good sensitivity and specificity showed by the results of preliminary detection of clinical serum samples. Conclusion Our self-built ELISA detection system has the high sensitivity and specificity, and it can be used to detect the early infection of rubella virus.
Objective To express and purify E1 protein by eukaryotic system, and to establish the ELISA detection method for rubella virus antibody with recombinant E1 protein as coating antigen. Methods The E1 dominant antigen epitopes were predicted by bioinformatics. The nucleic acid sequence of the rubella virus E1 dominant fragment was obtained by whole gene synthesis according to the expression characteristics of eukaryotic system, and the eukaryotic expression vector was constructed. The expression of E1 protein was induced in eukaryotic system and purified. The antigenicity of E1 protein was confirmed by Western blot. ELISA detection system was established, and 100 clinical serum samples were preliminarily detected. Results SDS-PAGE results confirmed that the recombinant E1 protein was highly expressed in yeast. Western blot results confirmed that the E1 protein had good antigenicity. A method for detecting IgM by indirect ELISA was successfully established. The kit had both the good sensitivity and specificity showed by the results of preliminary detection of clinical serum samples. Conclusion Our self-built ELISA detection system has the high sensitivity and specificity, and it can be used to detect the early infection of rubella virus.
引文
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[12] 陈其霞,王婷婷,安静娜,等.1种新的国产风疹病毒IgG抗体化学发光检测试剂的性能评价[J].现代预防医学,2015,42(14):2608-10.
[13] Helden J V,Grangeot-Keros L,Vauloup-Fellous C,et al.Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys (?),immunoassay system[J].J Virol Methods,2014,199:108-15.
[14] 陈其霞,罗岚,王婷婷,等.国产风疹病毒免疫球蛋白M抗体检测试剂与进口试剂的性能比较[J].华西医学,2017(1):67-9.
[15] 孙卫国,杨栗坤,刘艳华,等.风疹病毒糖蛋白2-核蛋白优势抗原表位原核可溶性融合表达与血清学诊断研究[J].中国卫生检验杂志,2016(16):2351-4.
[16] Nedeljkovic J,Jovanovic T,Oker-Blom C.Maturation of IgG avidity to individual rubella virus structural proteins [J].J Clin Virol,2001,22 (1):47-54.
[2] Cozza V,Martinelli D,Cappelli M G,et al.Further efforts in the achievement of congenital rubella syndrome/rubella elimination[J].Hum Vaccin Immunother,2015,11(1):220-4.
[3] Khandaker G,Zurynski Y,Jones C.Surveillance for congenital rubella in Australia since 1993:Cases reported between 2004 and 2013[J].Vaccine,2014,32(50):6746-51.
[4] Zhu Z,Cui A,Wang H,et al.Emergence and continuous evolution of genotype 1E rubella viruses in China[J].J Clin Microbiol,2012,50(2):353-63.
[5] Chen M H,Frolov I,Icenogle J,et al.Analysis of the 3' cis-acting elements of rubella virus by using replicons expressing a puromycin resistance gene[J].J Virol,2004,78(5):2553-61.
[6] Bosma T J,Best J M,Corbett K M,et al.Nucleotide sequence analysis of a major antigenic domain of the E1 glycoprotein of 22 rubella virus isolates[J].J GenVirol,1996,77(Pt10):2523-30.
[7] Starkey W G,Newcombe J,Corbett K M,et al.Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies[J].J Clin Microbiol,1995,33(2):270-4.
[8] 苏秋东,郭敏卓,邱丰,等.风疹病毒多表位诊断抗原的制备与评价[J].病毒学报,2016(3):249-55.
[9] Zhu Z,Rivailler P,Abernathy E,et al.Evolutionary analysis of rubella viruses in mainland China during 2010-2012:endemic circulation of genotype 1E and introductions of genotype 2B[J].Sci Rep,2015,5:7999.
[10] Enders M,Bartelt U,Knotek F,et al.Performance of the elecsys rubella IgG assay in the diagnostic laboratory setting for assessment of immune Status[J].Clin Vaccine Immunol,2013,20(3):420-6.
[11] 谭玉华,于婷,李奕辉,等.时间分辨荧光免疫法风疹病毒IgG抗体定量测定试剂盒的研制与性能评价[J].国际检验医学杂志,2014,35(4):472-4.
[12] 陈其霞,王婷婷,安静娜,等.1种新的国产风疹病毒IgG抗体化学发光检测试剂的性能评价[J].现代预防医学,2015,42(14):2608-10.
[13] Helden J V,Grangeot-Keros L,Vauloup-Fellous C,et al.Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys (?),immunoassay system[J].J Virol Methods,2014,199:108-15.
[14] 陈其霞,罗岚,王婷婷,等.国产风疹病毒免疫球蛋白M抗体检测试剂与进口试剂的性能比较[J].华西医学,2017(1):67-9.
[15] 孙卫国,杨栗坤,刘艳华,等.风疹病毒糖蛋白2-核蛋白优势抗原表位原核可溶性融合表达与血清学诊断研究[J].中国卫生检验杂志,2016(16):2351-4.
[16] Nedeljkovic J,Jovanovic T,Oker-Blom C.Maturation of IgG avidity to individual rubella virus structural proteins [J].J Clin Virol,2001,22 (1):47-54.