乙肝表面抗原专一的单链嵌合抗体在大肠杆菌中的表达

利用重组ＰＣＲ技术将乙肝表面抗原专一单抗的Ｖｋ与人源的Ｃ基因拼接成轻链嵌合抗体Ｖ－Ｃ,再通过编码柔性肽段（Ｇｌｙ４Ｓｅｒ）的Ｌｉｎｋｅｒ与Ｖ连接成单链嵌合抗体ＳｃＦＶ－Ｃ,并分别在大肠杆菌的热敏与分泌型表达系统中进行表达。表达产物的Ｗｅｓｔｅｒｎ－ｂｌｏｔ与间接ＥＬＩＳＡ检测结果表明,ＳＣＦｖ－Ｃ产两种系统中的表达产物均具有抗原结合活性,并已在分泌肽的指导下ＳｃＦｖ－Ｃｋ能被Ｅ．ｃｏｌｉ分泌出胞外。     

乙肝病毒表面抗原的抗体双阳性者中病毒S区基因序列分析

为了解乙肝病毒（HBV）表面抗原和抗体双阳性患者中病毒的基因型及其HVB S区是否有变异。用放射免疫试剂检测HBsAg阳性样品中的抗－HBs抗体,用聚合酶链反应法检测双阳性样品中的HBV DNA,然后对阳性样品进行克隆和基因序列分析,并将所得序列与HBV不同基因型的代表株进行比较分析。结果显示389例HBsAg阳性样品中有10例为抗HBs抗体阳性;该10例双阳性样品中有5例为HBV DNA阳性;序列分析显示该5株HBV均为B基因型,其中4株为adw亚型,1株为adr亚型;其中有2株在S区的“a”决定簇的氨基酸发生了变异。     

乙肝阳性血浆中HBsAg的滴度与保存温度和疫苗收量关系

本文报导了乙型肝炎阳性血浆中ＨＢｓＡｇ的滴度与保存温度和疫苗收量的关系。证明：１．４─５年于─２０℃保存该血浆对ＨＢｓＡｇ滴度无明显影响;２．于─２０℃和３０℃反复冻融八次以上ＨＢｓＡｇ滴度开始下降。３．于２５℃保存５６天后,该血浆中的ＨＢｓＡｇ可下降一个滴度,提示要尽可能避免反复冻融,缩短室温保存时间。用乙肝阳性血浆制备疫苗时疫苗的收量与血浆中ＨＢｓＡｇ的滴度密切相关,当ＲＰＨＡ滴度为１：５１２时,收量明显偏低,ＲＰＨＡ滴度为１：２０４８以上时疫苗收量较高。     

乙肝病毒表面抗原抗体可变区基因的克隆及人－鼠嵌合抗体重链基因的表达

本文采用反转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术,从鼠抗乙肝病毒表面抗原（ＨＢｓＡｇ）单克隆细胞中克隆到了该抗体重、轻链可变区（Ⅴ区）基因,并分别将其与人的恒定区基因Ｃγ３,Ｃｋ相拼接,构建人－鼠嵌合抗体基因。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ－Ｂｌｏｔ分析结果证实嵌合抗体重链基因在Ｅ．ｃｏｌｉ中得到了表达。间接ＥＬＩＳＡ法免疫测定的结果表明该表达产物具有与乙肝表面抗原结合的能力。     

4例乙肝表面抗原阴性乙肝病毒DNA阳性标本结果分析

目的 探讨4例乙肝病毒HBV-DNA阳性标本乙肝表面抗原(HbsAg)阴性的原因.方法 4例HbsAg阴性HBV-DNA阳性标本和1例HbsAg阳性HBV-DNA阳性标本,碱裂解法提取乙肝病毒基因组,采用巢式PCR扩增s基因全长片段,T-A克隆后测序,根据s基因序列推导氨基酸序列,分析乙肝表面抗原α决定簇区域的氨基酸变异情况.结果 与参考株比较,5例血清中HBV-DNAs基因均出现不同位点碱基突变,导致乙肝表面抗原α决定簇区域的氨基酸突变主要为:1号标本R122K、I126A、S143T;2号标本R122K、I126N、Q129N;3号标本R122K、I126S、T131N、M133T;4号标本R122K、I126S、T131N、M133T及5号标本的R122K.结论 乙肝表面抗原α决定簇区域126位氨基酸突变可能会影响HbsAg的检测结果.     

2007～2012年中国大学生乙肝表面抗原阳性检出率的meta分析

目的 评价2007～ 2012年中国大学生乙肝表面抗原阳性的总检出率.方法 通过检索Pub Med、重庆维普(VIP)、清华同方(CNKI)、万方等全文数据库,搜索中国大学生乙肝表面抗原阳性检出率的相关文献,采用Meta Analyst Beta3.13软件对检出率进行Meta分析,评估中国大学生乙肝表面抗原携带状况.结果 根据入选标准,共筛出文献35篇,总样本量为437 290人,检出乙肝表面抗原阳性27 895人,Meta分析显示,中国大学生乙肝表面抗原阳性总检出率为6.38％.结论 我国大学生乙肝表面抗原阳性率低于一般人群,但是仍然需要加大对乙肝健康教育的力度,防止乙型肝炎在大学校园里的传播.     

乙型肝炎病毒表面抗原-抗体复合物治疗慢性乙型肝炎患者病毒S基因变异研究

目的 研究应用乙型肝炎病毒表面抗原(HBsAg)-抗体复合物治疗性疫苗(YIC)治疗慢性乙型肝炎患者是否会诱生S基因免疫逃逸变异株的出现。方法 选取5例用30μg或60μgYIC治疗后血清乙型肝炎病毒(HBV)DNA水平下降>2log10、伴有乙型肝炎病毒e抗原(HBeAg)转阴应答,但在随访6个月后病毒DNA水平重复升高的患者作为研究对象,另选取1例对YIC治疗始终无应答患者和1例注射安慰剂患者作为对照,用聚合酶链反应(PCR)方法扩增治疗前(0周)及治疗后(44周)血清中HBV DNA的S基因、前-核心基因、核心基因启动子片段,并进行序列比对分析。结果 S基因“a”决定簇及前-核心基因均未发生变异,但YIC治疗后有3例HBV的核心基因启动子1762/1764位点序列有变异,另有2例在核心基因启动子的其他位点有核苷酸变异。结论 研究显示5例出现病毒重新复制的患者并非由于发生了病毒S基因逃逸变异所致。     

分子筛层析法分析重组乙肝表面抗原聚集物的形成

分子筛层析作为分析蛋白质颗粒聚集物的一种有力工具,被用于研究重组乙肝表面抗原聚集物的形成。已去除聚集物的表面抗原放置在不同的理化条件下或经过不同的纯化方法处理后,应用HPLC分析其聚集物的形成。为研究发酵过程中是否形成表面抗原聚集物,酵母细胞破碎后立即用Sepharose 4 FF层析柱分离为不同的组分,并分别进行HPLC分析。结果发现,在纯化过程和酵母发酵阶段都有表面抗原聚集物的产生。     

2022年重庆市巴南区3～18岁学生人群乙肝表面抗体水平调查研究

目的 了解2022年重庆市巴南区3～18岁学生人群的乙肝表面抗体（hepatitis B surface antibody, HBsAb）水平。方法 用非比例分层抽样方法选取中心城区、乡镇两层3～18岁学生人群111 208人,使用免疫层析法检测样本静脉血中的HBsAb,分析其HBsAb阳性数和阳性率。结果 HBsAb阳性数有48 385例,阳性率为43.51%,95%CI为43.20%～43.80%。中心城区阳性数35 753例,阳性率为44.03%;乡镇阳性数12 632例,阳性率为42.10%。不同区域学生阳性率差异有统计学意义（χ²=33.19,P<0.05）。3～6岁组阳性数5 025例,阳性率为71.19%,年龄组阳性率差异有统计学意义（χ²=6289.67,P<0.05）。性别比为1.01∶1,阳性率性别差异无统计学意义（χ²=2.55、P=0.11,P>0.05）。幼儿园阳性数2 318例,阳性率高达77.01%,各类学校阳性率差异有统计学意义（χ²=5913.42,P<0.01）。中心城区各类学校学生阳性数有35 753例,阳性率为44.03%,高于乡镇各类学校学生。结论 巴南区3～18岁学生人群HBsAb阳性率总体不高,中心城区人群阳性率高于乡镇,4类学校学生阳性率随年龄呈现降低趋势。     

鱼山镇1岁以上人群不同年龄组乙肝表面抗原携带率与乙肝疫苗接种关系的调查

研究鱼山镇乙肝疫苗接种率不同人群的乙肝病毒表面抗原(HBsAg)携带率的差异。将该镇所有户籍登记在册人员按出生日期分成3组,再按随机抽样法对每个年龄组抽取一定数量的人组成样本,调查乙肝疫苗接种史;对每位参加者采集静脉血5ml,无菌分离血清,使用固相放射免疫试剂和酶联免疫试剂检测乙肝病毒表面抗原和抗乙肝病毒表面抗原抗体(HBsAb,抗-HBs)。小年龄组、中年龄组、大年龄组乙肝疫苗接种率依次为80.00%、50.46%、25.36%,差异具有统计学意义(χ2=262.24,P<0.005);小年龄组、中年龄组、大年龄组HBsAg携带率依次为2.07%、11.93%、17.87%,差异具有统计学意义(χ2=77.48,P<0.005);小年龄组、中年龄组、大年龄组HBsAb依次为43.38%、24.77%、10.95%,差异具有统计学意义(χ2=107.28,P<0.005)。乙肝疫苗接种率在小年龄组、中年龄组、大年龄组依次降低,HBsAg携带率依次增高,HBsAb阳性率依次降低。因此,接种乙肝疫苗对人群有较好的保护作用,人群乙肝疫苗接种率越高,HBsAb阳性率越高,HBsAg携带率越低。     

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.     

Size-exclusion chromatographic study of the reduction of recombinant hepatitis B surface antigen

The reduction of the P. pastoris-derived hepatitis B surface antigen (HBsAg) has been investigated by size exclusion chromatography performed in a detergent solution containing 0.3% sodium dodecyl sulfate (SDS) and 0.1 M Tris–HCl, pH 7.0. The HBsAg, reduced under different conditions and passed through the TSK G4000 SW column (600×7.5 mm I.D.) at 0.9 ml min⁻¹, was resolved into two peaks corresponding to the reduced, monomeric, and non-reduced forms, respectively. Under these conditions, the antigen fraction corresponding to the HBsAg dimer can be separated and completely reduced to monomers by repeated reductive treatment with simultaneous lipid removal. The efficiency of reduction was maximal after sample treatment with an equal volume of a solution containing 417 mM dithiothreitol, 4.2% (w/v) SDS and 16% (v/v) 2-mercaptoethanol. In conclusion, complete reduction of recombinant HBsAg to monomer subunits is possible and depends on the efficiency of lipid removal during the reductive treatment.     

Sodium dodecylsulfate polyacrylamide gel electrophoresis of recombinant hepatitis B surface antigen particles

To investigate the factors leading to broadening of the recombinant hepatitis B surface antigen (HBsAg) peak in size-exclusion chromatography, the HBsAg particles eluting in different regions of the peak were subjected here to electrophoretic analysis. In nonreduced samples, the 24-kD band corresponding to the S monomer was detected when excessively large amounts of HBsAg were loaded onto the gel. Hence, some monomers are not disulfide-crosslinked in assembled particles. On the other hand, the results of alkylation experiments indicated the presence of free sulfhydryl group(s) in a little portion of freshly-purified HBsAg which was retarded on the size-exclusion chromatographic column and had significant antigenicity. This fraction of HBsAg was shown to be oligomeric and capable of spontaneous assembly into higher-order structures during aging.     

阳江市健康人群乙型肝炎血清流行病学调查

目的了解阳江市健康人群乙型肝炎表面抗体(抗-HBs)水平,评价乙型肝炎疫苗免疫效果。方法采用整群随机抽样方法,在阳江市辖区内随机抽取20个乡镇的常住健康人群为调查对象。采用电化学发光免疫法定量检测抗-HBs水平,运用SPSS18.0和Excel 2007软件进行数据整理和分析。结果共采集1 054人份血清进行检测,人群中乙肝疫苗接种率为94.88%,抗-HBs阳性率为63.47%、抗-HBs高应答率及GMT分别为29.51%和2 3.47 m IU/m L。乙肝疫苗接种率显示,随着年龄的增长疫苗接种率越低;20岁以下和20岁以上人群疫苗接种率分别为98.35%和54.22%,且男性高于女性;各地区间疫苗接种率差异无统计学意义(χ2=5.99,P=0.11)。抗-HBs阳性率显示,1~14岁儿童抗-HBs阳性率为63.00%。其中,1~岁组儿童最高为87.20%,7~岁组最低为54.56%;江城区抗-HBs阳性率最高(70.61%),阳西县最低(56.60%)。1岁以下儿童的抗-HBs应答率和GMT均最高,分别为61.22%和(91.56±2.15)m IU/m L。5~岁组最低,分别为13.74%和(12.44±1.46)m IU/m L,差异均有显著的统计学意义(χ2=57.31,P=0.00,F=5.77,P=0.00)。结论阳江市健康人群乙肝疫苗接种率高,初步形成免疫屏障。积极开展20岁以下人群的加强免疫和成人的乙肝疫苗的接种工作。     

Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.     

Evidence for the denaturation of recombinant hepatitis B surface antigen on aluminium hydroxide gel

Despite the complexity of the subject of protein–alum interactions, a valuable information can be obtained by analyzing the adsorbed and desorbed protein by common physico–chemical methods. In the present work, to approach the adsorption of hepatitis B surface antigen (HBsAg) on alum, the experimental data were supported by complementary analyses of the adsorbed protein by immunoelectron microscopy and the desorbed protein by denaturing size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. First, the depletion of HBsAg was investigated. The aspects assessed were the conditions, recovery and chromatographic performance of the desorbed protein. The results obtained strongly suggested the loss of particulate structure of HBsAg after adsorption on alum. This conclusion was further reinforced by direct immunoelectron microscopic visualization of HBsAg in the adsorbed state.     

Standardization and validation of an alkaline phosphatase-linked immunoassay to quantify a plant-derived antibody directed against the hepatitis B virus surface antigen

Immunopurification is one of the most effective chromatography steps to purify the hepatitis B surface antigen, which have successfully been used as an active pharmaceutical ingredient of hepatitis B vaccines. Plant-derived antibodies could be an appropriated ligand for such purposes because plants are the most cost-effective production systems and have the additional advantage that plant viruses cannot infect humans. In this work, a polyclonal antibody alkaline phosphatase-linked immunoassay was standardized and validated to quantify a plant-derived antibody directed against the HBsAg. The validation of an immunoassay to quantify plantibodies is a relatively complex task due to the complexity of the plant extract, the low level of expression of this molecule, and the potential interferences of endogenous peroxidases contributed by plants. These results allow estimating the plant-derived antibody concentration up to 3.81 ng/mL with high specificity, precision, and repeatability. The working range of the standard curve was between 3.81 and 60 ng/mL, and the intra- and inter-variation coefficients were between 10% and 20% in a production process's sample dependent way. This enzyme-linked immunosorbent assay is considered valuable to improve the design of the purification process and also to obtain a better estimation of the antibody expression level and process's recovery.     

HBsAg阴性母亲的新生儿乙型肝炎疫苗免疫后抗-HBs的动态研究

HBsAg阴性母亲的新生儿,按0、1、6个月程序接种3剂10μg乙型肝炎血源疫苗,在第一针免疫后9～48个月观察抗-HBs阳转率,发现免后12个月阳转率最高达94.23％;至48个月为81.62％。48个月后抗-HBs GMT仍有263.5mIU/ml,提示此期间不必加强免疫。抗-HBs滴度(mIU/ml)的动态规律是由高滴度(>1000mIU/ml)向低滴度推移。