丙型肝炎病毒结构蛋白在痘苗病毒中的表达

为研究中国丙型肝炎病毒（ＨＣＶ）的抗原性及在细胞内的加工，将丙型肝炎病毒（ＨＣＶ）５’非编码区（ＮＴＲ）和结构基因（Ｃｏｒｅ＋Ｅ１＋Ｅ２／ＮＳ１）插入痘苗病毒表达载体ｐＪＳＡ１１７５中，转染ＴＫ－１４３细胞，经纯化得到丙型肝炎（ＨＣＶ）重组痘苗病毒ｖＪＳＡ１１７５ＣＥ株。Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交表明，ＨＣＶ结构基因存在于痘苗病毒之中。Ｗｅｓｔｅｒｎｂｌｏｔ分析发现，ｖＪＳＡ１１７５ＣＥ表达蛋白带位于９０ｋＤａ，为一多聚蛋白；此蛋白为分泌型，分泌量与细胞裂解物内量大致相同     

用PCR组建巨细胞病毒抗原决定簇基因的表达克隆

巨细胞病毒感染的血清学诊断方法是目前临床诊断中应用最广泛的方法之一。但由于缺少高效价、高纯度和高特异性的抗原，使各种血清学方法在其敏感性和特异性方面均不能令人满意。文献报道，通过分子生物学得到的或化学合成的单一病毒蛋白或部分成份可作为血清学诊断的良好抗原。本文报道用聚合酶链反应扩增了巨细胞病毒主要磷蛋白ｐＰＵＬ３２的一个强抗原决定簇基因，并同时在扩增产物的两端引入了相应的限制性内切酶识别位点序列，经与表达载体连接后转入相应的细菌中，得到了能够表达此抗原决定簇的克隆，其表达的融合蛋白在免疫转印检测中与人巨细胞病毒阳性血清有强特异性反应。     

丙型肝炎病毒核心蛋白基因重组质粒的构建及其在真核细胞中的表达

目的：构建包含丙型肝炎病毒（ＨＣＶ）核心蛋白（Ｃ）基因片段的重组真核表达载体，并在肝细胞癌细胞株７７２１细胞中表达。方法：将从ｐＢＲＴＭＨＣＶ１－３０１１质粒切下的ＨＣＶＣ基因片段插入ｐｃＤＮＡ３质粒的ＣＭＶ启动子下游，构建真核表达质粒ｐｃＤＮＡＨＣＶ－Ｃ，然后，采用脂质体转染技术，转染７７２１细胞进行瞬时表达，转染细胞裂解煮沸后，通过ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ检测表达的核心抗原。结果：用限制性内切酶酶切后，片段大小与计算值相符。Ｗｅｓｔｅｒｎｂｌｏｔ证实，表达抗原的Ｍｒ约为２２０００。结论：ＨＣＶＣ基因能够插入ｐｃＤＮＡ３真核表达载体，并使其在真核细胞中表达，为进一步ＨＣＶ基因疫苗的研制和探讨抗ＨＣＶ感染打下了基础。     

人钙调素基因表达载体的构建及在大肠杆菌DH5α中的表达

目的;构建人钙调素基因表达载体并在大肠杆菌ＤＨ５α中高效表达人ＣａＭ蛋白。方法：用ＰＣＲ方法获得人钙调素基因Ⅲ（ｈＣａＭⅢｃ ＤＮＡ）,将其插入表达载体ｐＢＶ２２０,构建重组表达质粒ｈＣａＭα／ｐＢＶ２２０,用酶切,ＤＮＡ测序,ＰＣＲ扩增鉴定阳性克隆。结果：阳性重组子在大肠杆菌ＤＨ５α中经温度诱导可高效表达ＣａＭ蛋白,经１５％ＳＤＳ－ＰＡＧＥ分析,可观察到一分子量与理论值相符的诱导表达条带,其表达     

7型腺病毒疫苗株载体的构建及β-半乳糖苷酶基因的表达

目的构建Ｅ３区缺失的７型腺病毒疫苗株（Ａｄ７ｖ）载体并表达β半乳糖苷酶基因。方法从人二倍体细胞Ｗ１３８培养的Ａｄ７ｖ中分离病毒ＤＮＡ，利用Ａｄ７ｖＤＮＡ天然的酶切位点，经过多步亚克隆，克隆的同时将Ｅ３区７８８８７ｍｕ片段缺失，并将多克隆酶切位点带入Ａｄ７ｖ载体。为了验证载体的功能，将带有巨细胞病毒（ＣＭＶ）早期启动子β半乳糖苷酶基因插入缺失的Ｅ３区。将这一重组质粒和ＥｃｏＲⅠ酶切Ａｄ７ｖＤＮＡ共转染２９３细胞，获得表达β半乳糖苷酶重组病毒。结果构建了缺失部分Ｅ３区Ａｄ７ｖ载体，在ＣＭＶ启动子的作用下该载体能有效地表达外源基因。结论Ａｄ７ｖ载体的构建并成功表达外源基因为开发和应用这一载体打下了重要的基础     

中国株HGV NS3 区基因的原核表达及其产物的抗原性分析

目的研究ＨＧＶＮＳ３区基因产物的抗原性,并探讨ＮＳ３蛋白在血清学检测中的应用。方法将中国株ＨＧＶＮＥ３区的３个基因片段分别克隆到ｐＲＳＥＴＢ和ｐＲＳＥＴＣ质粒载体中,构建成原核表达载体。ＩＰＴＧ诱导下,在大肠杆菌ＢＬ２１中高效表达,获得３个重组蛋白,用Ｗｅｓｔｅｒｎｂｌｏｔ和ＥＬＩＳＡ法分别对表达产物进行分析。结果所构建的表达载体均得到高效表达,得到的重组蛋白ＰＡ、Ｐ３和Ｐ４的分子量分别为４２０００,３００００和２４０００,在Ｗｅｓｔｅｒｎｂｌｏｔ和ＥＬＩＳＡ反应中均可被ＨＧＶ阳性血清识别,其中ＨＧＶＮＳ３区Ｎ端的基因产物抗原性较强。结论中国株ＨＧＶＮＳ３区的Ｎ端存在优势的抗原决定簇,其基因产物有较好的抗原性。     

从ＨＣＶ核心蛋白噬菌体随机展示肽库中筛选抗原表位

目的 探讨病毒抗原序列研究的新方法。方法 应用噬菌体展示技术,筛选ＨＣＶ核心蛋白的抗原序列,用抗－ＨＣＶ核心抗体单独阳性的血清,对已构建的ＨＣＶ核心蛋折扣长菌体随机展示肽库进行４轮筛选,检测筛选阳性的克隆数、插入阳性率和杂交阳笥率来评价筛选的效果。测定和分析了７个序列中,６个为ＨＣＶ核心蛋白序列,其中５个被正常地展示于噬菌体表面,１个可能被正确展示。这些序列均含有重要的ＨＣＶ核心抗原表位。另１个为大肠杆菌nrfa基因。结论 噬菌体展示技术完全可应用于病毒蛋白抗原序列的筛选,并具有简便、快速、准确的优点。     

恶性疟原虫疫苗研究VIPfCMR基因与人白细胞介素-2基因的连接与克隆

用限制性内切酶ＥｃｏＲＩ和ＳａｌＩ将恶性疟原虫复合抗原基因ＰｆＣＭＲ从质粒ｐＷＲ４５０－１／ＰｆＣＭＲ中切下，插入质粒ｐＢＶ２２０／ＩＬ－２中人白细胞介素－２（ＩＬ－２）基因的ＥｃｏＲＩ位点。重组质粒转化大肠杆菌ＤＨ５ａ，通过ＰＣＲ扩增和酶切鉴定，筛选出正向插入的重组载体ｐＢＶ２２０／ＰｆＣＭＲ－ＩＬ－２。为表达ＰｆＣＭＲ－ＩＬ－２融合蛋白打下基础。     

恶性疟原虫疫苗研究VⅡ.PfCMR基因与人白细胞介素—2基因…

用限制性内切酶ＥｃｏＲＩ和Ｓａｌｌ将恶性疟原虫复合抗原基因ＰｆＣＭＲ从质粒ＰＷＲ４５０－１／ＰｆＣＭＲ中切下，插入质粒ＰＢＶ２２０／ＩＬ－２中人白细胞介素－２（ＩＬ－２）基因的ＥｃｏＲＩ位点，重组质粒转化大肠杆菌ＤＨ５ａ，通过ＰＣＲ扩增和酶切鉴定，筛选出自向插入的重组载体ＰＢＶ２２０／ＰｆＣＭＲ－ＩＬ－２。为表达ＰｆＣＭＲ－ＩＬ－２融合蛋白打下基础。     

重组HBsAg免疫逃逸突变体的表达及其抗原性分析

为了深入研究乙型肝炎病毒S基因变异所致的包膜抗原抗原性的改变，从含ＨＢＶＤＮＡ双拷贝的质粒载体ｐＥｃｏｂ６，获得一个８３７ｂｐ的ＨＢＶ－Ｓ基因片段，将其插入至载体ｐＢｌｕｅｓｃｒｉｐｔ ＫＳ~＋的ＳｍａⅠ位点，通过体外寡核苷酸介导的人工定点突变分别获得第１４５位、１２６位和第１４５位＋１２６位氨基酸三种Ｓ基因变异型。然后将这些Ｓ基因变异片段克隆到真核表达载体ｐＭＥｐ４上，从而构建了含ＨＢＶ－Ｓ基因及其突变型的表达载体ＰＭＥｐ４ＨＢＶＳＭ。用其转染人肝癌细胞系ＨｅｐＧ２，获得稳定分泌ＨＢｓＡｇ及其变异体的抗性细胞系。经体外初步研究表明，ＨＢｓＡｇ １４５位氨基酸变异体可影响ＨＢｓＡｇ的“ａ”抗原决定簇的结构。     

Production of mouse monoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library

Lpp20, an outer membrane protein of Helicobacter pylori (H. pylori), has been identified as an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against it and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, the Lpp20 gene was obtained from H. pylori genomic DNA by PCR (GenBank accession no. DQ106902), cloned into pGEX-4T-1 vector and expressed in Escherichia coli (E. coli) as a recombinant fusion protein with glutathione-S-transferase (GST), which was purified by GST-affinity chromatography. mAbs were produced by the hybridoma technique using Lpp20-GST as the immunogen. Using mAb as the target molecule and immunoscreening phage-displayed random dodecapeptide library (Ph.D.-12), the positive phage clones were sequenced and analyzed. Phage clones were chosen to immunize mice to evaluate the potential of phagotopes as effective vaccines. One mimotope (SWPLYSDASGLG) showed a good match with the Lpp20 proteins at 114-117aa (DASG) and the serum of mice induced by the phage clone clearly recognized Lpp20 protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Lpp20 providing an alternative approach for the diagnosis and development of a vaccine for H. pylori.     

旋毛虫Ts87抗原单克隆抗体的制备及其识别肽段的研究

将原核系统成功表达的Ts87重组蛋白纯化后免疫动物,利用淋巴细胞杂交瘤技术制备抗Ts87抗原的单克隆抗体.通过ElISA、免疫印迹、免疫组化等方法筛选出能分泌抗Ts87御抗原的抗体的阳性克隆,在1000个融合的杂交瘤细胞中,有3株融合细胞分泌的单克隆抗体(2A2,5A3,6G12)鉴定结果为阳性.这3株融合细胞分泌的抗体均能识别Ts87重组蛋白、旋毛虫成虫和幼虫的Ts87抗原以及旋毛虫幼虫组织切片.获得的抗Ts87抗原的单克隆抗体在将来的研究中可作为旋毛虫病的诊断试剂.为了进一步鉴定抗体识别的相应抗原表位和模拟抗原表位,选择了其中一株单克隆抗体5A3筛选噬菌体十二肽库.噬菌体M7展示的肽段是抗体识别Ts87抗原的线性表位,其他的噬菌体克隆展示的肽段是Ts87抗原的模拟表位.该技术为旋毛虫病多表位疫苗的构建奠定了基础.     

Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay.

Serological detection of human cytomegalovirus (HCMV)-specific antibody varies greatly because of antigen composition and the lack of antigen standardization. Antigenic materials composed of single well-characterized viral proteins or portions of them, produced via molecular biology, have proven to be promising tools in improving serodiagnosis. We constructed a recombinant protein containing two regions of ppUL32 (p150) and half of ppUL44 (p52) and compared the immunoglobulin M (IgM) reactivity of this triple-antigen fusion protein with that of a double-antigen fusion protein containing the two ppUL32 fragments and that of a monoantigen fusion protein containing half of ppUL44. We also constructed and tested two other monoantigen fusion proteins containing a large fraction of ppUL80a and a fraction of ppUL83. More than 700 serum samples from different groups of immunocompetent and immunosuppressed subjects were tested for the presence of HCMV IgM by recombinant enzyme immunoassay (rec-EIA) and by a commercially available EIA. Western blotting (immunoblotting) and (in the case of immunosuppressed individuals) antigenemia tests by immunofluorescence and PCR of polymorphonuclear leukocytes were also carried out. The results obtained demonstrate that (i) the triple-antigen fusion protein can replace the individual proteins; (ii) the triple-antigen fusion protein cannot be used alone to replace the virus or infected cells in the serological detection of anti-CMV IgM; (iii) the addition of the fusion proteins containing portions of ppUL83 and ppUL80a is essential for the formation of an antigenic mixture that can replace the virus for the search of HCMV-specific IgM; (iv) rec-EIA is very specific and is more sensitive than the commercially available EIA, and the results obtained are consistent with those obtained by Western blotting; and (v) rec-EIA can reliably be used to detect HCMV-specific IgM in different groups of patients with active HCMV infection.     

Demonstration of an amino terminal La epitope recognized by human anti-La sera

In order to study the antigenic properties of the La protein we have isolated a 1650 base pair (bp)-long human cDNA encoding an anti-La reactive protein. Restriction enzyme analysis and DNA sequencing was used to compare this clone with two published but inconsistent partial sequences. Our clone extends about 220 bp further towards the 5' end than the two clones previously studied and includes a putative initiation codon. When introduced into an expression vector, stable fusion proteins were made both from the initial clone and from two deletion clones. The recombinant proteins were tested by immunoblotting against a panel of anti-La sera. All reacted with the fusion protein produced by the 1650-bp clone. About half of the anti-La sera showed reactivity against the recombinant protein from the shortest deletion clone. This indicates the presence of an epitope in the amino terminal part of the La protein, encoded by sequences not present in previously published clones.     

Cloning of cDNAs encoding a 28 kilodalton antigen of Toxoplasma gondii

By screening cDNA libraries in lambda gt11 with antibodies raised against the previously described protective F3G3 antigen of Toxoplasma gondii, and subsequently screening with nucleic acid probes, we have isolated cDNA clones that encode a 28 kDa antigen of T. gondii that is likely one of the two antigenic components of the F3G3 antigen. The gene apparently exists as a single copy in the tachyzoite haploid genome of the three strains of T. gondii examined. Northern blot analyses revealed that the cDNAs hybridize with a major T. gondii RNA species of 1.1 kb. Together the cDNAs encompass 1051 bp of cDNA sequence containing an open reading frame with the capacity to encode a 28 kDa protein. Antibodies that were affinity purified using recombinant fusion proteins produced by two of the clones reacted on protein blots of whole T. gondii lysate with a single antigen having an apparent molecular mass of 28 kDa. Both recombinant fusion proteins reacted with IgG antibodies in sera of mice and humans infected with T. gondii and therefore might be useful for the development of diagnostic assays for T. gondii infection.     

EB病毒潜伏膜蛋白2A抗原表位的融合表达及其意义

为了探讨含有EB病毒潜伏膜蛋白2A(LMP2A)抗原表位片断表达的融合蛋白在鼻咽癌血清学检测中的应用意义,通过重叠延伸PCR方法,合成了3对相互重叠的寡核苷酸引物,涵盖LMP2A的主要的4个抗原表位,将它们拼接在一起构建一个多肽融合基因,克隆到PGEX-4T-2载体中表达融合蛋白,以GST亲和层析柱法纯化融合蛋白,鉴定后以此为抗原检测鼻咽癌患者的血清。结果表明,获得了含EB病毒LMP2A主要的4个抗原表位的融合蛋白(EC2A),蛋白纯度达90%以上,ELISA结果显示鼻咽癌患者血清的检出率为77.9%,正常人群血清为阴性,与常规的VCA-IgA法进行比较,有9份(13.3%)血清VCA-IgA为阴性而EC2A-IgG检出阳性,为鼻咽癌的临床检测提供了新思路,也为后续的单克隆抗体制备奠定了基础。     

The properties of monoclonal antibodies to HIV gag gene proteins]

Immunization of BALB/c mice with natural purified HIV antigen, fusion of spleen cells with myeloma cells and subsequent selection of hybrid clones using recombinant gag antigen of HIV gave hybridomas producing monoclonal antibodies (MCA) to HIV. The immune blotting method demonstrated that 3 clones interacted with protein p24 and 4 clones with protein p17 of HIV. Competitive EIA led to a conclusion that the resulting MCA detected at least 3 antigenic determinants in proteins, products of gag gene of HIV. The potentials of using these MCA for the detection of viral antigen in HIV-infected continuous cell lines were demonstrated.     

Epitope Detection in the Envelope of Intracellular Naked Orthopox Viruses and Identification of Encoding Genes

Monoclonal antibodies (MAbs) were generated against vaccinia virus, cowpox virus KR2 Brighton, monkeypox virus Copenhagen, or ectromelia virus. Pairwise epitope specificity studies by competition ELISAs identified 23 distinct antigenic sites in 19 different orthopox virus strains. Six epitopes were completely independent of each other, and 17 closely related antigenic sites formed three separate epitope complexes. As shown by immunogold electron microscopy (ELMI), all MAbs reacted with epitopes in the envelope of intracellular naked virus, 16 MAbs recognized proteins of 32, 30, 16 or 14 kDa in Western blotting (WB), and 9 MAbs neutralized virus infectivity. In rabbitpox virus (RPV) 18 epitopes were detected. A λgt11 expression library of RPV DNA was screened with the corresponding 18 MAbs. Fourteen recombinant bacteriophage clones (ph) were isolated. Cross-hybridizations of phage and RPV DNA demonstrated a reaction with the HindIII A, HindIII D, or HindIII H fragments, respectively. DNA of ph3D was related to the A25L gene, which corresponds to the A-type inclusion body gene of cowpox virus. Two phage clones contained sequences of the 14-kDa fusion protein gene (A27L gene). Ph1A contained nearly the entire 14-kDa gene encoding 4 neutralizing (neutr) and 2 nonneutr epitopes. Ph5, expressing only half of this gene product, encoded 1 nonneutr epitope. The fusion protein of vaccinia virus MVA was isolated by immune-affinity chromatography with a neutr. catching MAb. The protein formed hollow rods (ELMI) and the 6 antigenic sites that were present were identical to those expressed by Escherichia coli infected with ph1A. WB detection with a polyclonal hyperimmune serum detected protein bands of 54, 32, 30, 16, and 14 kDa. The catching MAb bound only to a 16-kDa band. The purified fusion protein induced neutralizing antibodies in mice and rabbits.