乙型肝炎病毒前S2抗原决定簇与乙型肝炎病毒核心抗原的基因融合与表达

乙肝前Ｓ２（ＨＢＶ ＰｒｅＳ２）肽段由５５个氨基酸组成,其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇。我们将化学合成的ＰｒｅＳ２ ｅｐｉｔｏｐｅ（１２０－１４５）基因不同位点进行融合,融合基因在大肠杆菌中获得表达,并对融合蛋白进行了纯化。经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验表明,融合蛋白具有ＰｒｅＳ２和ＨＢｃＡｇ两者的抗原性。此外,研究还表明,强启动子能使表达水平有一定的提高。     

乙肝前S2抗原决定簇串联体与核心抗原的基因融合与表达

乙肝前Ｓ２（ＨＢＶＰｒｅＳ２）肽段由５５个氨基酸组成,其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇,为增强ＰｒｅＳ２的抗原性,本实验采用将化学合成的ＰｒｅＳ２ｅｐｉｔｏｐｅ（１２０－１４５）基因以串联方式与ＨＢｃＡｇ的基因进行了融合,融合基因在大肠杆菌中获得表达,并通过ＥＬＩＳＡ比较研究了融合蛋白中ＰｒｅＳ_２ｅｐｉｔｏｐｅ单体及串联体的抗原性差异。     

乙肝前S2抗原决定簇串联体与核心抗原的基因融合与表达

乙肝前Ｓ２（ＨＢＶＰｅｒＳ２）肽段由５５个氨基酸组成,其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇,为增强ＰｒｅＳ２的抗原性,本实验采用将化学合成的ＰｒｅＳ２ｅｐｉｔｏｐｅ（１２０－１４５）基因以串联方式与ＨＢｃＡｇ的基因进行了融合,融合基因在大肠杆菌中获得表达,并通过ＥＬＩＳＡ比较研究了融合蛋白中ＰｒｅＳ２ｅｐｉｔｏｐｅ单体及串联体的抗原性差异。     

核酸免疫可特异地诱发小鼠对乙型肝炎病毒表面抗原S+PreS1免疫反应

运用改造后的乙型肝炎病毒表面抗原Ｓ＋ＰｒｅＳ１融合基因（将ＰｒｅＳ１区肝细胞受体结合位点（２１－４７ａａ）编码基因融合到Ｓ蛋白Ｃ端２２３位残基下游）,构建了一系列真核表达载体,并在ＣＨＯ细胞上有效表达了分泌型的、具有Ｓ＋ＰｒｅＳ１双重抗原性的融合蛋白颗粒。用上述质粒ＤＮＡ肌肉免疫Ｃ５７ＢＬ／６Ｊ小鼠,成功地检测到了抗－ＨＢｓ和抗－ＰｒｅＳ１抗体,加强免疫能显著改善免疫效果。本研究还建立了检测抗－ＰｒｅＳ１的竞争抑制法和羊抗人ＨＢｓＡｇ封闭法,两种方法符合率达９６％,但后者更为敏感。用羊抗人ＨＢｓＡｇ封闭法测得抗－ＰｒｅＳ１滴度最高可达１：１２８０以上。     

乙肝PreS_2抗原决定簇和霍乱毒素B亚基基因的融合与表达

通过全化学法按大肠杆菌密码偏性合成了乙肝炎病毒（ＨＢＶ）前Ｓ２抗原（ＰｒｅＳ２）抗原决定簇基因,与霍乱毒素Ｂ亚基基因的３’端融合。重组质粒转化大肠杆菌后融合基因得到高效表达,表达量达３０μｇ／ｍＬ,表达产物９５％以上分泌到胞外。表达的融合蛋白能与神经节苷脂ＧＭ１结合,说明融合蛋白保持了霍乱毒素Ｂ亚基（ＣＴＢ）的基本高级结构和生物学功能;酶联免疫吸附实验证明融合蛋白具有ＣＴＢ和ＨＢＶＰｒｅＳ２的抗原性;应用亲和层析纯化后得到了电泳纯融合蛋白制品,为研究融合蛋白免疫原性并进一步构建基因工程肽苗奠定了基础。     

大肠杆菌脂蛋白在CTB—pres2抗原基因的融合及表达

首次采用基因融合方式,在乱毒素Ｂ亚基－乙型肝炎病毒Ｐｒｅｓ２抗原融合基因的５‘端引入编码大肠杆菌脂蛋白信号肽及Ｎ端九个氨基酸的核苷酸序列,分别置于ｃｔｂ及ｌａｃ启动子下在大肠杆菌中获得分泌性表达,表达的融合蛋白均定位于膜上,并且可以和ＧＭ１、抗－ＣＴＢ抗体及ＨＢＶＰｒｅＳ２单克隆抗体结合说明该融合蛋白保留了ＣＴＢ的基本高级结构及ＣＴＢ、ＰｒｅＳ２抗原的抗原性,＾３Ｈ－棕榈酸标记实验证实该融合蛋白发     

用Bac—to—Bac杆状病毒表达系统高效表达绿色荧光蛋白标记的HBVe抗原

通过基因工程操作,使乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）基因与绿色荧光蛋白基因（ＧＦＰ）融合,用新型Ｂａｃ－ｔｏ－Ｂａｃ杆状病毒表达系统在昆虫细胞中高效表达了ＨＢｅＡｇ－ＧＦＰ双功能融合蛋白。经ＥＬＩＳＡ法和荧光显微镜观察证实,表达产物既能发射易于检测的绿色荧光,又具有ＨＢＶ的ｅ抗原活性,为免疫诊断新方法的建立进行了有益的探索。     

用Bac-to-Bac杆状病毒表达系统高效表达绿色荧光蛋白标记的HBVe抗原

通过基因工程操作,使乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）基因与绿色荧光蛋白基因（ＧＦＰ）融合,用新型Ｂａｃ－ｔｏ－Ｂａｃ杆状病毒表达系统在昆虫细胞中高效表达了ＨＢｅＡｇ－ＧＦＰ双功能融合蛋白。经ＥＬＩＳＡ法和荧光显微镜观察证实,表达产物既能发射易于检测的绿色荧光,又具有ＨＢＶ的ｅ抗原活性,为免疫诊断新方法的建立进行了有益的探索     

乙肝病毒preS抗原决定簇与核心抗原的融合表达

将乙肝病毒表面抗原的ｐｒｅＳ抗原决定簇片段与核心抗原进行融合,分别构建了在核心抗原中间对应第７５～８３位氨基酸之间的融合及在核心抗原羧端对应第１５６位氨基酸处的融合,并在ｔａｃ启动子的控制下于大肠杆菌中表达。表达产物经ＥＬＩＳＡ检测和ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ分析,表明融合蛋白均被表达,其单体分子量大小与推算值一致．电镜观察和ＣｓＣｌ密度梯度超离心测定都表明融合蛋白能形成颗粒,其密度略小于天然的ＨＢｃ颗粒。初步纯化的融合蛋白免疫Ｂａｌｂ／ｃ小鼠;能产生高滴度的抗－ｐｒｅＳ１抗体,表明ＰｒｅＳｌ（２１～４７）在核心抗原ｅｌｌｏｏｐ区的融合能大大提高其免疫原性．     

两株乙型肝炎病毒基因组结构分析及其复制和抗原表达特性的比较

为从全基因组水平研究乙型肝炎病毒（ＨＢＶ）的核苷酸结构及复制和抗原表达特性,分别从２例ＨＢｓＡｇ和ＨＢｅＡｇ阳性、ＨＢＶ ＤＮＡ滴度为１０＾１４和１０＾１３拷贝／ｍｌ的慢性乙型肝炎患者（编号为５６和２－１８）血清中扩增、克隆了ＨＢＶ全基因组,并测定了核苷酸序列。两株病毒基因组转染ＨｅｐＧ２细胞的培养上清中ＨＢｓＡｇ水平基本相同,但＃５６毒株表达的ＨＢｅＡｇ约为＃２－１８株的３倍,Ｓｏｕｔｈｅｒｍ印     

利用病毒载体在烟草中瞬时表达融合HBsAg基因

利用马铃薯PVX病毒载体构建了外源人工融合乙肝表面抗原HBsAg基因的表达载体,在烟草中利用农杆菌介导进行瞬时表达,以快速鉴定外源基因瞬时表达的状况以及重组蛋白的免疫活性。利用PCR技术从含有人工融合HBsAg基因的表达载体中分别扩增出LP PreS1 PreS2 S、PreS1 PreS2 S、PreS2 S序列,将其分别与PVX病毒载体pgR106连接,构建成PVX-LP、PVX-S1和PVX-S2等3个转化载体,并将此载体导入农杆菌菌株GV3101中用于侵染烟草植株叶片。感染植株经RT-PCR、RNA Dot blotting和HBsAg蛋白的ELISA检测显示,3个人工融合的HBsAg基因均可在植物体内得到转录,翻译成具有活性的蛋白。结果表明,外源融合HB-sAg基因经过植物病毒载体瞬时表达系统可以在植物系统中正常转录和翻译。     

HBcAg表达菌株的筛选、初步鉴定与DNA顺序分析

为提高HBcAg(乙型肝炎核心抗原)在大肠杆菌体内表达水平,将有HBcAg的基因片段用核酸外切酶从两端消化,插入载体质粒pUR222的EcoRI酶切位点,转化Ecoli BMH71—18,用菌落原位酶联免疫法由275个转化子筛选到7株阳性克隆,用ELISA比较了它们的表达水平,表达水平最高者为M2066菌株,P/N=2.0时菌体裂解液的稀泽度为1:33000’比表达最低者M2098高8,000倍,比第一代菌株M206高15,000倍,DNA顺序分析结果表明与mRNA起始密码上游的发卡结构去除有关。用SDS-PAGE和聚乙烯簿膜复印法检测菌体裂解液中HBcAg的分子量为21000,42000及63000,呈单体和聚合物形式存在,比由病人肝脏和血液中提取的HBcAg(19000)分子量大,为融合蛋白。经琼脂免疫双扩散与ELISA阻断试验未发现与β半乳糖苷酶有免疫学交叉反应。用ELIDA法,M2066-HBcAg与肝-HBcAg同时检测40份血清标本的抗-HBc,二者符合率95％。     

Identification and characterization of peptides that interact with hepatitis B virus via the putative receptor binding site

A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein gIV in transfected bovine cells.

The gene encoding bovine herpesvirus 1 (BHV-1) glycoprotein gIV was mapped, cloned, and sequenced. The gene is situated between map units 0.892 and 0.902 and encodes a predicted protein of 417 amino acids with a signal sequence cleavage site between amino acids 18 and 19. Comparison of the BHV-1 amino acid sequence with the homologous glycoproteins of other alphaherpesviruses, including herpes simplex virus type 1 glycoprotein gD, revealed significant homology in the amino-terminal half of the molecules, including six invariant cysteine residues. The identity of the open reading frame was verified by expression of the authentic recombinant BHV-1 gIV in bovine cells by using eucaryotic expression vectors pRSDneo (strong, constitutive promoter) and pMSG (weak, dexamethasone-inducible promoter). Constitutive expression of gIV proved toxic to cells, since stable cell lines could only be established when the gIV gene was placed under the control of an inducible promoter. Expression of gIV was cell associated and localized predominantly in the perinuclear region, although nuclear and plasma membrane staining was also observed. Radioimmunoprecipitation revealed that the recombinant glycoprotein was efficiently processed and had a molecular weight similar to that of the native form of gIV expressed in BHV-1-infected bovine cells. Recombinant gIV produced in the transfected bovine cells induced cell fusion, polykaryon formation, and nuclear fusion. In addition, expression of gIV interfered with BHV-1 replication in the transfected bovine cells.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and l-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed inE. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCI density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBcAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B144C191. Using those fusion proteins, ELISA for screening of antibodies against both HBV and HCV in human sera was also established.