乙型肝炎表面抗原结合人乳中乳铁蛋白的筛选与鉴定  

Screening and identification of hepatitis B surface antigen binding to lactoferrin in human milk

作　　者：张昭萍[1] 刘景丽[2] 冯静[3] 戴毅敏[3] 胡娅莉[3] 周乙华[1,2] Zhang Zhaoping;Liu Jingli;Feng Jing;Dai Yimin;Hu Yali;Zhou Yihua(Department of Infectious Diseases,Nanjing Drum Tower Hospital,the Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,China;Department of Experimental Medicine,Nanjing Drum Tower Hospital,the Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,China;Department of Obstetrics and Gynecology,Nanjing Drum Tower Hospital,the Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,China)

机构地区：[1]南京大学医学院附属鼓楼医院感染科,210008 [2]南京大学医学院附属鼓楼医院科研部,210008 [3]南京大学医学院附属鼓楼医院妇产科,210008

出　　处：《中华围产医学杂志》2019年第7期457-460,共4页Chinese Journal of Perinatal Medicine

基　　金：国家自然科学基金(81672002);江苏省自然科学基金(BK20161105);江苏省卫生健康委员会科技项目(H201537).

摘　　要：目的乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)阳性产妇乳汁中含有乙型肝炎病毒(hepatitis B virus,HBV),但母乳喂养不增加母婴传播风险。既往研究显示母乳具有与HBsAg结合的特点,本研究旨在确定母乳中能与HBsAg结合的成分。方法本研究于2015年6月至2017年2月在南京大学医学院附属鼓楼医院完成。取2例HBV血清学标志均阴性的健康产妇的乳汁,同时用牛乳和山羊乳为对照。通过间接免疫印迹技术,观察高纯度重组酵母HBsAg与乳清蛋白结合情况,结合蛋白质谱分析结果,进一步通过竞争抑制实验,确定母乳中能与HBsAg结合的蛋白成分。结果间接免疫印迹显示,2例母乳乳清泳道均可在分子量约80 000处发现明显的反应条带,而牛乳和羊乳无相应条带。该反应条带处的蛋白质经蛋白质谱分析,提示与5种蛋白质序列同源性为28.4%~93.4%,其中同源性最高(93.4%)的蛋白为人乳铁蛋白。使用纯化的重组人乳铁蛋白,进一步经间接免疫印迹验证,显示人乳铁蛋白能与重组HBsAg结合。竞争抑制实验证明,纯化的重组人乳铁蛋白能显著抑制HBsAg与乙肝表面抗体的结合,且随乳铁蛋白浓度降低,抑制率降低。结论母乳中人乳铁蛋白能与HBV结合。进一步研究乳铁蛋白能否抑制HBV的感染性,对阐明母乳喂养不增加HBV母婴传播具有重要意义。Objective Human milk of mothers with positive hepatitis B surface antigen (HBsAg) contains hepatitis B virus (HBV).However,breastfeeding does not increase the risk of mother-to-infant transmission of HBV.Previous investigations demonstrated that breast milk has a property of binding with HBsAg.This study aimed to identify the component in human milk that can bind to HBsAg.Methods This study was performed in Nanjing Drum Tower Hospital,the Affiliated Hospital of Nanjing University Medical School,from June 2015 to February 2017.Human milk samples from two postpartum women with negative HBV markers and two control samples of cow milk and goat milk were analyzed by Far-Western blot,in which highly purified recombinant yeast HBsAg was used to bind with whey proteins.Based on the results of mass-spectrum analysis,competition inhibition test was used to confirm the functioning component.Results Far-Western blot showed remarkable protein bands at the relative molecular weight of about 80 000 in both lanes of human milk,but none in the lane of cow or goat milk.Mass-spectrum analysis of the protein band indicated there were proteins sharing 28.4%-93.4% homology in amino acid sequences with five proteins with the highest homology to lactoferrin (93.4%).Further Far-Western blot with purified recombinant lactoferrin showed that lactoferrin could bind to the recombinant HBsAg.Competition inhibition test suggested that the purified recombinant lactoferrin inhibited the binding of HBsAg to its antibody in a dose-dependent manner.Conclusions This study confirms the capability of lactoferrin in human milk to combine with HBsAg,suggesting that lactoferrin can bind to HBV.Further study on whether lactoferrin can inhibit the infectivity of HBV would be valuable to clarify the reason for not increasing the risk of mother-to-infant transmission of HBV by breastfeeding.

关 键 词：乳 人 乳铁蛋白 乙型肝炎表面抗原 

分 类 号：R392[医药卫生—免疫学]