【作者】 李新军；

【导师】 胡川闽；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2011， 硕士

【摘要】 乙型肝炎病毒的致病性来源于包封有病毒DNA的直径约42nm的球形病毒颗粒。乙肝病毒基因组有四个开放阅读框,由于翻译起点不同,前S区/S区基因编码病毒表面三种抗原分子,分别是前S1蛋白(preS1),前S2蛋白(preS2)和S蛋白。乙肝表面抗原由三种被膜糖蛋白组成,分别是乙肝病毒大蛋白(LHBs),中蛋白(MHBs)和主蛋白(SHBs)。从最上游启动密码子开始翻译产生的乙肝病毒大蛋白(LHBs)被认为在病毒与宿主细胞受体的结合以及病毒颗粒的聚集和从细胞中释放等过程中发挥重要作用。与其它表面膜蛋白不同的是,LHBs在其N末端拥有preS1区域。因此,研究该区域的生物学特性和免疫原性非常重要。preS1分子位于LHBs的表面,受宿主免疫应答调节,具有很强的免疫原性,所以preS1区域也反映了HBV的基本生物学特性。研究发现,在乙肝表面抗原阴性患者的血清及肝脏中出现preS1蛋白已经被认为是HBV感染的标志物,代表着病毒的复制。因为LHBs大多位于感染性病毒颗粒的表面,所以对LHBs的检测显得尤为重要,而检测的关键问题在于如何获得针对preS1区域的特异性抗体,那么预测preS区域的B细胞免疫表位很有必要。在我们的研究中,我们利用生物信息学软件在线预测出了preS区域的免疫表位并将表位肽段合成且与BSA交联。将交联好的表位肽段按照一种新型免疫程序免疫Balb/c小鼠制备单克隆抗体。与此同时,我们制备了可用于preS1结构和功能分析以及筛选单克隆抗体的重组preS1蛋白。在获得的单克隆抗体的基础上,我们建立了检测血清中HBV LHBs的ELISA分析方法,并对413例临床血清样本进行了检测,以验证所获抗体的特异性及高效性。上述方法有助于更高效的检测乙肝病毒感染。主要研究内容和结果:1.preS1高效原核表达基因载体的构建及免疫检测标准品的制备我们从GENBANK获得了由357个碱基组成的编码preS1(1-119aa)蛋白的基因序列(B基因型,adr血清亚型)。将此序列递交于Graphical codon usage analyzer (http://guca.schoedl.del)分析发现该基因序列中有部分编码氨基酸的密码子在E.coli BL21中使用的频率较低,因此可能导致构建的重组质粒无法在大肠杆菌中顺利表达。根据大肠杆菌偏嗜的密码子,通过同义置换的方式对preS1基因序列进行偏嗜性改造,改造后的基因序列编码的氨基酸序列同改造前一致。接下来我们构建了preS1原核表达载体。将大肠杆菌偏好的编码preS1氨基酸序列同天然preS1分子(1~119aa)相同的DNA片段合成并克隆入质粒pET32a,再将重组质粒转化大肠杆菌E.coli。经1.0mmol/L IPTG 37℃诱导表达5h,pET32a-preS1在BL21中表达出了融合蛋白,分子量约为30.5kD,表达量占菌体总蛋白的30 %以上。经鉴定表达蛋白破菌后主要在上清中,为可溶性表达。采用离子交换、His亲和层析柱、EK酶切及过分子筛等技术建立了preS1重组蛋白的纯化方法,获得了高纯度的preS1蛋白。经HPLC检测所获纯化蛋白纯度可达98%,证明其可用于筛选抗preS1单克隆抗体及分析preS1分子的结构和功能。2.抗preS1单克隆抗体的制备与鉴定利用B细胞表位在线预测软件,综合分析蛋白结构β-转角,蛋白抗原表面可及性,蛋白柔韧性,蛋白抗原性,疏水性及线性抗原表位,我们选择了具有较强抗原特异性的区段(36-58aa)作为免疫原免疫小鼠。将该肽段合成并与BSA交联后免疫Balb/ c小鼠,免疫脾细胞与骨髓瘤细胞(SP2/0)在50%聚乙二醇条件下融合,获得了7株分泌抗preS1单克隆抗体的杂交瘤细胞株。通过对所获抗体进行亚类分析,我们得到了1-E6(IgG1)和2-G5(IgG2a)两株抗体,通过ELISA分析、Western blotting、抗体亲和力测定以及免疫细胞化学方法鉴定,证实了其对preS1蛋白的免疫特异性,可以被用来检测血清中的乙肝大蛋白。3.抗preS1单克隆抗体在检测血清LHBs中的应用在研究中,我们把特异性的抗preS1单克隆抗体作为捕获抗体包被在ELISA微孔板孔中,同时用辣根过氧化物酶标记的抗HBsAg兔多克隆抗体作为二抗即检测抗体,建立了检测LHBs的双抗体夹心ELISA方法。我们搜集了2009年9月到2010年8月期间第三军医大学西南医院感染科门诊或住院的413例乙肝感染者血清标本以及100例体检正常者血清标本作为检测对象。为了比较不同抗体对血清LHBs检出阳性率的高低,我们同时用市面上商业化的抗preS1单克隆抗体作为捕获抗体进行平行检测。另外我们用同样的方法对100例体检正常的血清标本进行检测,以证实前述检测结果并非假阳性。研究证实,单克隆抗体1-E6比商业化抗体以及2-G5更能特异结合preS1,作为捕获抗体时对LHBs的检出率也高于商业化抗体以及2-G5作为捕获抗体时对LHBs的检出率。因此,所获得的高特异性抗体1-E6将会为今后LHBs的检测以及preS1免疫检测试剂盒的研发奠定重要的基础。更多还原

【Abstract】 Hepatitis B virus (HBV) particle is about 42 nm diameter spherical DNA envelope virus particles. HBV genome has four open reading frames, and the preS/S region of the HBV genome encodes three viral surface antigens named preS1, preS2 and S protein by differential initiation of translation at each of three in-frame initiation codons. HBsAg consists of three related envelope glycoprotein that named large protein (LHBs), Middle protein (MHBs) and the main protein (SHBs). Initiation at the most upstream start codon yields the L (or preS1)protein(LHBs), which is thought to play key roles in the binding of the virus to host-cell receptors and in the assembly of the virion and its release from the cell. The difference between LHBs and other surface envelope proteins is that it has preS1 region on the N terminal. Therefore, the study of biological function and immunogenicity of this region has great significance. PreS1 antigen in the surface of LHBs, is highly immunogenic, mediated by the host immune response. So preS1 domain is responsible for the essential biological properties of HBV. In previous study, the presence of preS1 proteins in serum and liver of HBsAg-positive patients has been considered as a marker for HBV infection. Because LHBs preferentially localizes on infectious viral particles,and the LHBs was speculated to have a pivotal function in viral infectivity and assembly, so LHBs detection is particularly important and the key problem is how to obtain the specific antibodies against preS1. Therefore, predicting the B cell immune epitope within preS1 region is very necessary. In our study, we predicted the B cell epitope of preS1 by bioinformatics software. The epitope peptide was synthesized and cross-linked with BSA. Balb/c mice were immunized with this cross-linked peptide through a novel immunization procedure to produce monoclonal antibodies. At the same time, the recombinant protein preS1 suitable for standard substance and screening monoclonal antibodies was produced. Based on mAbs we obtained, an ELISA analysis system was established to detect HBV LHBs in serum. 413 cases of serum were tested to prove the specificity and efficiency of the mAbs we newly made. This approach is helpful for detection of hepatitis B virus infection more efficiently.Primary research and results as follows:1.Construction of high efficiency prokaryotic expression vector of preS1 and preparation of immunology standardAccording to the preS1 gene sequence (genetypeB,serotype adr) offered by GENBANK, we obtained sequence containing 357 base coding amino acids from aa1 to aa119 of preS1. We used Graphical codon usage analyzer (http://guca.schoedl.del) to analyze this coding sequence and found that a part of codon in the gene sequence encoding amino acids occur seldomly in E.coli BL21,so it is possible that the constructed recombinant plasmid will not expressed in E.coli BL21perfectly. According to the frequency of E. coli codons, preS1 gene sequence was reconstructed by synonymous substitutions. After transformation,the gene encoding the same amino acid sequence as the former.Nextly,we constructed preS1 prokaryotic expression plasmid. An E.coli favorite DNA fragment,encoding amino acids sequence identical to that of the wild type preS1(1-119aa)(genetype B,serotype adr)was synthesized and cloned into plasmid pET32a, and the recombinant plasmid was transformed into E.coli . After induced 5h by induction with 1.0 mmol/L IPTG at 37℃, pET32a-preS1 expressed fusion protein weighting about 30.5kD. The amounts of expressing protein occupy more than 30 percent of the total protein in E.coli. After breaking bacterium, expressing protein was soluble and presenting in supernatant. After isolated by cation exchange, his-tag affinity column, and enterokinase (EK) digestion, the purified preS1 protein were obtained. Especially, the purity of preS1 was 98% by HPLC detection, which proves to be suitable for immunology detection standard and for screening anti-preS1 monoclonal antibody.2. Preparation and identification of monoclonal antibody against preS1By use of B cell epitope prediction software online, we analyzedβ-turn structure,surface antigen accessibility, protein flexibility, protein antigens, hydrophobicity and the linear epitope of preS1. The region(36-58aa) with strong antigen specificity was chosen to be used for immunization of mice. The peptide was synthesized and cross-linked with BSA,Balb/ c mice were immunized and immune spleen cells were fused with the myeloma cell line (SP2/0) using 50% polyethylene glycol (Sigma, USA), hybridomas were obtained and 7 strains of monoclonal antibody against preS1 were produced.Among these mAbs 2 strains of IgG antibody(1-E6 and 2-G5) were obtained for diagnostic reagent by subclass identification. By using ELISA, Western blotting, antibody affinity determination and immunocytochemistry methods, the monoclonal antibody obtained in our experiment proved to be specific to preS1 protein, which can be used in detecting LHBs in human serum.3.Application of anti-preS1 monoclonal antibody in detecting LHBs in serum In our study, specific anti-preS1 mAb was used as capture antibody which was coated in microtiter plate wells. HRP-conjugated anti-HBsAg polyclonal antibody was used as detect antibody. Thus the two-antibody sandwich ELISA was established for LHBs detection .413 cases of HBV-infected serum and 100 cases of normal human serum were collected from the Department of Infectious Diseases, Southwest Hospital, Third Military Medical University, China, between September 2009 and August 2010. In order to compare the detection rate of LHBs, we also used commercial mAb against preS1 as capture antibody. We found that mAb 1-E6 was more specific to preS1 than commercial mAb and 2-G5, and the detection efficiency when 1-E6 was used as capture antibody is higher than that when commercial mAb or 2-G5 was used as capture antibody. So, the mAb 1-E6 obtained in our experiment is proved to be more specific to preS1 protein, which will be the important foundation of LHBs detection and developing new HBV immunodiagnostic kit.更多还原