不同年龄人群血清不定型流感嗜血杆菌外膜蛋白 P6及其 T-B 联合表位肽抗体水平

Serum concentrations of antibodies against outer membrane protein P6 and its T-and B-combined antigenic epitopes of nontypeable Haemophilus influenzae in children and adults of different ages

目的：研究不同年龄人群血清不定型流感嗜血杆菌（ nontypeable Haemophilus influen-za， NTHi）外膜蛋白P6及其T-B联合表位肽抗体水平，为NTHi 外膜蛋白P6多抗原肽疫苗的研究提供科学依据。方法构建NTHi P6原核表达系统，纯化重组蛋白P6，同时采用Epitope prediction 1．0软件和ANTIGENIC程序预测NTHi P6的T表位和B表位，合成T-B联合表位肽。以P6及其T-B联合表位肽为抗原，采用ELISA法检测血清中相应抗体水平。研究对象为605例来我院体检者，年龄范围为1天～103岁，组间抗体水平比较采用Mann-Whitney U检验，相关性采用Pearson相关分析法。结果研究中预测到NTHi P6的T-B联合表位肽有4条，分别是P6-2、P6-61、P6-95和P6-122。＜1个月组血清NTHi P6及其上述表位肽抗体水平均显著低于1～6个月组（ P均＜0．001）和7个月～3岁组（P均＜0．001）。7个月～3岁组 P6及其各表位肽抗体水平最高，显著高于4～6岁组（P 均＜0．01），4～6岁组又显著高于7～14岁组（P均＜0．01），其他各相邻年龄组间5种抗体差异较小。血清表位肽P6-2、P6-61、P6-95和P6-122抗体水平与P6抗体水平均显著相关（ P＜0．0001）。结论本研究预测的NTHi P6的T-B联合表位肽与P6在人血清中的抗体分布高度一致，提示其具有良好的免疫原性；7个月～3岁组儿童血清中P6及其表位肽抗体水平最高，可能与该年龄段NTHi感染率高有关。

Objective To measure the concentrations of antibodies against outer membrane protein P6 and its T-and B-combined antigenic epitopes of nontypeable Haemophilus influenzae （ NTHi） in children and adults of different ages and to evaluate the differences among different subjects for further investigation on NTHi multiple antigenic peptide vaccine .Methods A prokaryotic expression system was established to ex-press the recombinant outer membrane protein P 6 of NTHi.The expressed protein was purified by using Ni-NTA affinity chromatography .T-and B-cell epitopes in protein P6 were predicted with Epitope prediction software 1.0 and ANTIGENIC program and were used to synthesize T-and B-combined antigenic epitopes .A total of 605 subjects aged from 1 day to 103 years old were recruited from October 2013 to March 2014 .Ser-um concentrations of antibodies against protein P 6 and its T-and B-combined antigenic epitopes were meas-ured by using ELISA .Mann-Whitney U test was used to analyze the differences between groups .Pearson product-moment correlation coefficient was used for correlation analysis .Results Four T-and B-combined antigenic epitopes including P 6-2, P6-61, P6-95 and P6-122 were predicted and synthesized .The levels of antibodies against NTHi P6 and P6-2, P6-61, P6-95 and P6-122 were significant lower in the ＆lt;1 months group than those in the 1-6 months group （all P〈0.001） and 7 months-3 years group （all P〈0.001）.Three groups including 7 months-3 years group , 4-6 years group and 7-14 years group showed significant differ-ences regarding to the antibodies levels , among the 7 months-3 years group showed the highest levels , fol-lowed by the 4-6 years group and the 7-14 years group.However, no significant difference was found be-tween other adjacent groups .Concentrations of antibodies against P 6-2, P6-61, P6-95 and P6-122 were pos-itively correlated with the level of antibody against P 6 （P〈0.0001）.Conclusion The distribution of anti-bodies against T-and B-combined antigenic epitopes in P6 was highly in accord with those against P6, which indicated good immunogenicity of those epitopes .The highest antibodies levels were found in subjects aged 7 months to 3 years old , which might correlate with the high risk of NTHi infection at that stage .