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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%,0~2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。
In order to investigate the expression and application value of human metapneumovirus (FMP) in prokaryotic expression system, the hMPV F1 subunit protein was expressed in Escherichia coli expression system and the Ni-NTA affinity layer Purification, and use it as antigen, Western Blot detection of human serum antibodies. The F1 subunit was selected as the target region based on the prediction of the hydrophobicity, antigenic site and surface accessibility of the F protein. Different genotype hMPVs with 6 histidine (His) markers were obtained in E. coli BL21 Of F1 subunit protein expression, the target protein size of about 37.0 kD, mainly in the form of inclusion bodies exist. Western Blot showed that the expressed protein could specifically bind to the guinea pig polyclonal antibody to hMPV and to the serum of acute hMPV-infected patients, and to the respiratory syncytial virus (RSV) and parainfluenza virus There was no cross-reaction between the virus (PIV) (type 2 and type 3), showing that the F1 protein expressed was of good specificity and antigenicity. Application of the expressed protein in the detection of some human serum antibodies showed that the total positive rate of serum anti-hMPV-F IgG in the population was about 66% -67%. The positive rate of serum antibody in 0-2 years old group was presented with age Gradually declining trend, the highest positive rate of neonatal antibodies, about 85%,> 1 year old to 2 years old group, the lowest positive rate, suggesting the presence of mother antibodies. Subsequently, the positive rate of serum antibody increased gradually with the increase of age in all age groups, and the level of antibody in the crowd of> 60 years old was the highest. These results suggested that children <2 years old were susceptible to hMPV.