乙肝病毒核心蛋白病毒样颗粒作为载体在表位疫苗中的应用

乙型肝炎病毒核心抗原(hepatitis B core antigen,HBcAg)是由乙型肝炎病毒(Hepatitis B virus,HBV) C基因编码的病毒蛋白,是HBV的衣壳蛋白,主要存在于HBV核衣壳表面。乙肝病毒核心蛋白(hepatitis B virus core protein,HBc)来源于HBcAg,由183或185个氨基酸组成。由于HBc本身具有高度的免疫原性,并且可携带自身或外源病原体的抗原/表位在体外自组装成病毒样颗粒,20世纪80年代开始HBc就被用于疫苗载体的研究。现对HBc近年来作为载体在表位疫苗中的应用作一概述。     

绿色荧光蛋白分裂肽基因重组乙型肝炎病毒复制子模型的建立

为构建一种重组乙型肝炎病毒(hepatitis B virus,HBV)复制子模型,使其能够在病毒感染的细胞中表达可视化报告基因蛋白,本研究删除HBV基因组核心蛋白(HBV core,HBc)编码区部分序列,构建HBV1.1-ΔHBc113复制子载体.利用内含肽(intein)介导蛋白拼接的特性,选取加强绿色荧光蛋白(...     

乙型肝炎病毒核心蛋白变构调节剂的研究进展

由于乙型肝炎病毒(hepatitis B virus,HBV)的共价闭合环状DNA的持续存在和病毒介导的宿主免疫反应钝化,导致慢性乙型肝炎病毒感染很难治愈。现有的核苷(酸)类似物或聚乙二醇干扰素疗法难以实现高比率的HBV表面抗原清除。目前正在研发的核心蛋白变构调节剂有望大幅度降低血清表面抗原。本文就HBV核心蛋白的结构、功能以及核心蛋白变构调节剂的分类、应用前景等方面进行综述。     

乙型肝炎病毒进入肝细胞机制研究进展

乙型肝炎病毒(hepatitis B virus,HBV)感染早期进入肝细胞机制研究一直是HBV研究领域的热点和难点．简单易得的HBV体外感染细胞模型是HBV感染进入机制研究无法逾越的主要障碍．近年来,随着新型HBV体外感染细胞模型的建立和应用(HepRG细胞和树鼩原代肝细胞),HBV的进入机制研究取得了一系列重大发现．综述了近几年HBV进入肝细胞机制的最新研究进展,主要包括HBV表面蛋白进入相关结构域的鉴定,已发现的候选HBV进入相关分子和尚待解决的问题．     

自噬在HBV感染发病机制中的作用

自噬是一种新陈代谢过程,通过溶酶体降解受损蛋白质和细胞器来维持细胞内环境的稳态.乙型肝炎病毒(hepatitis B virus,HBV)能利用自噬功能增强其复制的能力,引起肝炎、肝硬化以及肝细胞癌(hepatocellular carcinoma,HCC).本文将从HBV相关蛋白、内质网应激、信号通路、细胞因子、微小...     

病毒非编码RNA与肿瘤发生发展

超过12%的人类肿瘤与病毒密切相关,如Epstein-Barr virus(EBV)、high-risk human papillomaviruses(HPV)、hepatitis B virus(HBV)、hepatitis C virus(HCV)、Kaposi’s sarcoma herpesvirus(KSHV)等。传统观点认为病毒调控细胞恶性转化及肿瘤发生主要与病毒编码蛋白相关,但随着组学和测序技术发展,人们开始认识到病毒非编码RNA在肿瘤发生发展中的重要性。这些非编码RNA不翻译为蛋白质,仅在RNA水平参与对宿主细胞和病毒自身生命活动的多方面调控。现将针对长非编码RNA、短非编码RNA及microRNA在肿瘤中的调控进行综述。     

山东某医院患者乙型肝炎核心抗体阳性特征分析

目的 了解2016—2020年山东某医院就诊患者乙型肝炎核心抗体(core antibody against hepatitis B, HBcAb)分布特征及乙型病毒性肝炎(viral hepatitis type B,简称乙型肝炎)感染现状。方法 用电化学发光免疫分析法(electrochemi-luminescence immunoassay)对采集的257 922份血清样本进行乙肝五项检测,并对HBcAb阳性样本的时间、性别、年龄及乙肝五项不同组合模式的特征进行分析。结果 人群中乙型肝炎病毒(hepatitis b virus, HBV)核心抗体(core antibody against hepatitis B, HBcAb)阳性有102 598例,阳性率为39.78%;2020年阳性例数最多而阳性率最低,阳性率总体呈现逐年下降的趋势,时间差异有统计学意义(χ²=122.74,P<0.05);男性阳性率高于女性,性别差异有统计学意义(P<0.05);>70岁组阳性率最高为66.38%,不同年龄组阳性率差异有统计学意义(χ²     

A case of hepatitis B reactivation in an anti-HBs positive, anti-HBc positive non-Hodgkin’s lymphoma patient

Dear Editor, We report a case of HBV reactivation in an anti-HBs positive,anti-HBc positive non-Hodgkin's lymphoma patient.Hepatitis B virus (HBV) reactivation is a well-recognized complication of patients undergoing chemotherapy or immunosuppressive therapy for lymphomas.The presence of antibodies to the hepatitis B surface antigen (anti-HBs) has been identified to be a factor preventing HBV reactivation in patients with occult HBV infection receiving chemotherapy.In this paper,we present a non-Hodgkin Lymphoma patient who,before immunosuppressive therapy,displayed positive anti-HBs and positive antibodies to hepatitis B core antigens (anti-HBc),as markers of resolved HBV infection,and developed hepatitis B surface antigen (HBsAg) and high viraemia with an HBV escape mutant after rituximabbased administration.The sequencing data revealed HBV genotype D with two known escape mutations,P120S.     

CAR-T免疫细胞疗法在抗病毒感染中的研究进展

目前,世界上大多数病毒感染没有针对性的特效药,尤其是具有潜伏感染能力的病毒,如乙型肝炎病毒(hepatitis B virus, HBV)、巨细胞病毒(cytomegalovirus, CMV)、Epstein-Barr病毒(epstein-barr virus,EBV)、人类免疫缺陷病毒(human immunodeficiency virus, HIV)等,严重威胁人类健康。这些病毒感染宿主细胞后,与宿主蛋白互作,导致宿主细胞在其表面表达一些特异性的感染或潜伏标志蛋白,可以成为CAR-T细胞疗法(chimeric antigen receptor T-cell immunotherapy, CAR-T)的靶点,为难治性病毒性疾病的治愈提供了新的方向。     

构建表达Flag-GPI的重组乙型肝炎病毒载体

乙型肝炎病毒(hepatitis B virus,HBV)是小型的嗜肝DNA病毒,能够特异性地感染人肝实质细胞。HBV的易感性可以归于多个方面,包括细胞表面的受体以及细胞内的蛋白或其他因子,目前对HBV易感性的了解还有待深入。本课题利用课题组原有的HBV 5c3c载体和糖基磷脂酰肌醇(glycosylphosphatidylinositol,GPI)的特性,构建了重组HBV载体5c3c-CD59-Flag-GPI和5c3cT-Flag-GPI,转染Huh7细胞后可以将Flag标签锚定在细胞膜上,且可利用Flag抗体通过流式细胞术对其进行分选。在回补HBV包膜蛋白的情况下,5c3cT-Flag-GPI能包装形成完整的重组HBV颗粒。本研究为后续优化重组HBV的包装效率,进而为HBV易感性研究提供工具并奠定基础。     

抗HBsAg人源单链可变区抗体的筛选与可溶性抗体的表达

采用噬菌体表达展示技术,以从乙型肝炎病毒（HBV）表达抗原（HBsAg）阳性血汪有超速离心纯化的HBsAg为固相抗原,从噬菌体单链可变区半合成抗体库中经过5轮“吸附－洗脱－扩增”筛选过程,获得特异性较强的HBsAg人源单链可变区抗体（ScFv）克隆并提取质粒,经SfiⅠ/NotⅠ酶切鉴定后,亚克隆到pCANTAB5E表达载体中,转化大肠杆菌XL1－Blue。经IPTG诱导后,表达的可溶性HBsAg特异性ScFv以50％硫酸胺沉淀,经SDS－PAGE电泳表明,XL1－Blue中表达的HBsAg可溶性ScFv的分子量约28kD。免疫活性检测结果表明,该单链抗体具有较强的抗原结合性和特异性。HBsAg人源单链抗体的筛选和表达成功,为今后HBsAg人源抗体的研究和应用奠定了基础。     

抗HBsAg人源单链可变区抗体的筛选与可溶性抗体的表达

采用噬菌体表面展示技术,以从乙型肝炎病毒(HBV)表面抗原(HBsAg)阳性血清超速离心纯化的HBsAg为固相抗原,从噬菌体单链可变区半合成抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得特异性较强的HBsAg人源单链可变区抗体(ScFv)克隆并提取质粒,经SfiⅠ/NotⅠ酶切鉴定后,亚克隆到pCANTAB5E表达载体中,转化大肠杆菌XL1-Blue。经IPTG诱导后,表达的可溶性HBsAg特异性ScFv以50%硫酸胺沉淀,经SDS-PAGE电泳表明,XL1-Blue中表达的HBsAg可溶性ScFv的分子量约28?kD。免疫活性检测结果表明,该单链抗体具有较强的抗原结合活性和特异性。HBsAg人源单链抗体的筛选和表达成功,为今后HBsAg人源抗体的研究和应用奠定了基础。     

Polyhydroxyalkanoate chip for the specific immobilization of recombinant proteins and its applications in immunodiagnostics

In this study, a novel strategy was developed for the highly selective immobilization of proteins, using the polyhydroxyalkanoate (PHA) depolymerase substrate binding domain (SBD) as an active binding domain. In order to determine the appropriacy of this method for immunodiagnostic assays, the single-chain antibody (ScFv) against the hepatitis B virus (HBV) preS2 surface protein and the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVe) were fused to the SBD, then directly immobilized on PHA-coated slides via microspotting. The fluorescence-labeled HBV antigen and the antibody against SCVe were then utilized to examine specific interactions on the PHA-coated surfaces. Fluorescence signals were detected only at the spotted positions, thereby indicating a high degree of affinity and selectivity for their corresponding antigens/antibodies. Furthermore, we detected small amounts of ScFv-SBD (2.7 ng/mL) and SCVe-SBD fusion proteins (0.6 ng/mL). Therefore, this microarray platform technology, using PHA and SBD, appears generally appropriate for immunodiagnosis, with no special requirements with regard to synthetic or chemical modification of the biomolecules or the solid surface.     

Production of human single-chain fragment antibody (ScFv) against human epidermal growth factor receptor-2 (HER-2) by phage display technology

Breast cancer with more than 1.7 million diagnoses per year has been known as one of the most prevalent cancers among women worldwide. Despite the availability of advanced treatment options, cancer progression and metastasis is observed in 20% of patients. Human epidermal growth factor receptor-2 (HER-2) is considered as an important prognostic and diagnostic tumor marker for breast cancer. While HER-2 is expressed on the surface of normal cells, its overexpression occurs in 20–25% on breast cancer tumor cells. This type of tumor which is referred to as HER-2+ is the most aggressive type of breast cancer and shows more resistance to radiotherapy. Single-chain fragment antibodies (ScFvs) offer several advantages in comparison to conventional whole antibodies due to their small size. Particularly, ScFv fragments show improved diffusion and solid tumor penetration. In this study, a human ScFv antibody library was used to isolate anti-HER-2 ScFv antibodies through cell panning and mix antigen-cell panning strategies. Analysis of the binding activity and specificity of isolated ScFv antibodies against HER-2-expressing cell lines and recombinant HER-2 antigen indicated the higher efficiency of the cell panning strategy in isolation of ScFv antibody fragments.     

抗CD5单链抗体基因克隆和表达

以分泌抗CD5单克隆抗体的杂交瘤细胞poly(A) mRNA为模板,通过RTPCR扩增出抗CD5单克隆抗体的重链可变区(VH)和轻链可变区(VL) cDNA片段组装出抗CD5单链抗体(ScFv) cDNA片段。该ScFv片段被克隆到pCANTAB 5E载体上,以E.coli TG1为宿主,进行噬菌体表面呈现。通过Molt4细胞表面分子CD抗原,对噬菌体表面呈现的ScFv进行免疫亲和富集筛选。经细胞ELISA鉴定,得到4株高亲和力克隆。DNA序列分析得知,单链抗体全长732碱基,其中VH为339碱基,VL为300碱基。抗CD5 ScFv在E.coli HB2151中以可溶形式分泌表达,产物主要分布于周质之中,占周质中总蛋白的20%。     

抗人CD19单链抗体基因的构建、表达及功能测定

采用RT-PCR方法从分泌抗人类白细胞表面分化抗原CD19单克隆抗体的杂交瘤细胞中克隆出V_H和V_L可变区基因,再通过重叠延伸拼接（spliceoverlap extension）PCR方法在V_H和V_L可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗人CD19单链抗体（抗CD19-ScFv）基因。将其克隆至表达载体PET28a并在大肠杆菌中表达。SDS-PAGE和Westernblot分析结果表明,抗CD19-ScFv在BL21（DE3）菌中获得表达,重组蛋白的相对分子量为27kD,表达产物以不溶性包涵体形式存在,经过溶解包涵体,镍柱亲和层析纯化和体外复性过程,获得了高纯度的单链抗体片段。流式细胞分析结果证实抗CD19ScFv可与人类白细胞表面的分化抗原CD19结合,保留了鼠源性单抗与CD19结合活性。抗人CD19-ScFv的构建与表达,为下一步针对B淋巴系统恶性肿瘤的靶向治疗奠定了基础。     

人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。     

靶向人源BCMA的嵌合抗原受体T细胞构建及体外抗肿瘤活性研究

摘要 目的：通过对CAR结构的ScFv单链可变区进行改造,构建并筛选具有更强杀伤肿瘤细胞功能的新型靶向人源B细胞成熟抗原（BCMA）的嵌合抗原受体 (CAR)-T细胞。方法：构建靶向人源BCMA的CAR分子,用逆转录病毒载体包装成功后转导健康志愿者的T细胞,制备Anti-BCMA-CAR-T细胞。将Anti-BCMA-CAR-T细胞作为观察组,普通T细胞作为对照组,将其与RPMI-8226细胞共培养,采用CFSE染色的T细胞增殖实验观察两组体外增殖能力。采用荧光素酶化学发光实验检测两组细胞在不同效靶比（1:8、1:4、1:2、1:1、2:1、4:1）对RPMI-8226细胞的杀伤效率,采用流式细胞术检测两组细胞在不同效靶比（1:4、1:2、1:1、2:1、4:1）对RPMI-8226细胞的杀伤效率。结果：CFSE检测结果显示,与对照组比较,观察组FITC信号明显左移,表明T细胞增殖能力越强。流式细胞术检测结果显示,相同效靶比时,观察组对RPMI-8226细胞的杀伤效率均高于对照组（P均<0.05）;荧光素酶化学发光实验结果显示,相同效靶比时,观察组对RPMI-8226细胞的杀伤效率均高于对照组（P均<0.05）。在效靶比为4:1时,CAR170-T（未经改造的传统的ScFv）细胞和CAR174-T（经改造的ScFv）细胞的杀伤效率分别达到了88.5±0.3 %和98.5±0.7 %。结论：通过对CAR结构的ScFv单链可变区进行改造后成功构建出的新型靶向BCMA的CAR-T细胞,它能保持较强的增殖活性且具有更强的杀伤肿瘤细胞的能力。     

Production and characterization of the single chain antibodies against human alpha2b-interferon

A combinatorial library of single-chain antibodies (ScFv) from mice immunized with human alpha2b interferon (hIFN-alpha2b) was constructed. For obtaining of phage display antibodies the DNA fragments of ScFv were cloned into phagemid vector pCANTAB-5E and rescued from Escherichia coli cells by infection with bacteriophage M13. Bacterial clones synthesizing specific ScFv against hIFN-alpha2b were isolated by several rounds of affinity selection of phage library. After isolation and affinity purification of ScFv-IFN from bacteria cells a high ability to binding of the hIFN-alpha2b has been shown. The sequencing of isolated ScFv DNA and analysis of the data obtained have been carried out. The data of expression stability of obtained E. coli producers such as some features of ScFv expression are also discussed.