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为了探讨人偏肺病毒(hMPV)融合蛋白(F)在原核表达系统中的表达效果及其应用价值,用大肠杆菌表达系统表达hMPV F1亚单位蛋白并用镍柱(Ni-NTA)进行亲和层析纯化,并以其为抗原,用Western Blot方法进行人血清抗体检测的探索。根据F蛋白的疏水性、抗原位点和表面可及性的预测结果选择F1亚单位为目标区域,在大肠杆菌BL21中分别得到了带有6个组氨酸(His)标记的不同基因型hMPV的F1亚单位蛋白的高效表达,目的蛋白大小约37.0 kD,主要以包涵体形式存在。Western Blot检测显示表达的目的蛋白可以特异性地结合抗hMPV的豚鼠多克隆抗体以及确定为hMPV急性感染患者的血清,而且与副粘病毒科肺病毒属的呼吸道合胞病毒(RSV)和副流感病毒(PIV)(2和3型)之间没有交叉反应,显示表达的F1蛋白的特异性和抗原性良好。应用该表达蛋白进行部分人群血清抗体检测的探索,结果显示人群血清抗hMPV-F蛋白的IgG抗体总阳性率约为66%~67%,02岁组的血清抗体阳性率随年龄增长呈现逐渐下降趋势,新生儿的抗体阳性率最高,达85%左右,>1岁2岁组幼儿阳性率最低,提示母传抗体的存在。随后各年龄组人群随年龄的增长血清抗体阳性率逐渐增高,>60岁年龄组人群抗体水平最高,上述结果提示<2岁儿童为hMPV的易感人群。
Abstract：To understand the effectiveness of prokaryotic expression of fusion protein(F) of human metapneumovirus(hMPV) and its application as antigen,F proteins from different genotypes of hMPV were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column.According to the hydrophobicity,antigen index and surface probability of F protein,the subunit 1(F1) region of F protein was generated and expressed in E.Coil.BL21(DE3).The 6-His-F1 proteins with molecular weight of approximately 37 kD generated from hMPV of two genotypes were expressed efficiently mainly in inclusion body.The antigenicity and specificity of the expressed proteins were tested and confirmed by Western Blot using polyclonal antibody against hMPV and one serum specimen from a patient with confirmed hMPV acute infection,and polyclonal antibodies against human respiratory syncytial virus and parainfluenza virus 2 and 3.The results of preliminary use of the expressed proteins for detecting antibodies against hMPV in 457 serum specimens collected from different age groups in Beijing indicated that 66%-67% of sera in all age groups were positive.The positive rate of antibodies declined in children in age groups from birth to 2 year-old and then rose along with the increase in age,in which the lowest was in age group from 1 to 2 year-old and the highest in newborn and people older than 60 years.The data indicated the existence of maternal transferred antibodies against hMPV in infants and the risk of hMPV infections in children younger than 2 years old.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002156
中图分类号:R373
引用信息:
[1]朱汝南,钱渊,赵林清,等.人偏肺病毒融合蛋白F的原核表达及初步应用[J].病毒学报,2011,27(02):144-150.DOI:10.13242/j.cnki.bingduxuebao.002156.
基金信息:
国家自然科学基金(No.30570080No.30872153);; 北京市新星计划(2004B34);; 北京市优秀人才项目(20071D0303200120)
2011-03-15
2011-03-15