Epstein-Barr病毒壳抗原gp125的纯化和应用

目的研制ｅｐｓｔｅｉｎ－Ｂａｒ（ＥＢ）病毒诊断试剂。方法将重组痘苗病毒表达的Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）壳抗原（ＶＣＡ）主要多肽ｇｐ１２５纯化，作为诊断抗原建立了酶联免疫吸附试验（ＥＬＩＳＡ），检测了４８份鼻咽癌（ＮＰＣ）病人血清及１０份正常人血清中的ＶＣＡ／ＩｇＡ抗体。结果该方法与免疫荧光（ＩＦ）检测结果一致，但ＥＬＩＳＡ的平均几何滴度（ＧＭＴ）是ＩＦ的１２倍。结论以纯化的ＥＢ病毒壳抗原主要多肽ｇｐ１２５作为诊断抗原建立的检测方法，更适合于ＥＢＶ相关疾病的血清学诊断和血清流行病学调查。     

用重组抗原检测鼻咽癌病人血清IgA／gp125抗体

用重组抗原检测鼻咽癌病人血清ＩｇＡ／ｇｐ１２５抗体薛绍礼１皮国华２谷淑燕２李平１抗Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）壳抗原（ＶＣＡ）ＩｇＡ抗体，是鼻咽癌（ＮＰＣ）早期诊断的一个重要血清学指标。曾先后用免疫荧光法（ＩＦ）和免疫酶法（ＩＥ）进行检测［１...     

基因重组Epstein—Barr病毒壳抗原gp125的免疫原性

天然的Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒ｇｐ１２５蛋白有极强的免疫原性，是ＥＢＶ初次感染的主要免疫原，也是诊断ＥＢＶ既往感染和新近感染最有价值的抗原。文章在用重组痘苗病毒表达ｇｐ１２５的基础上，研究了表达产物的特异性，并用活的重组痘苗病毒免疫中国本兔，用表达产物免疫ＢＡＬＢ／Ｃ小鼠，研究了其免疫原性和免疫抗体的特异性。结果显示：重组痘苗病毒在感染细胞内特异性地表达ｇｐ１２５，活病毒及表达产物能诱导动物产     

Epstein-Barr病毒壳抗原的纯化及其在检查血清抗体中的应用

为了建立纯化Ｅｐｓｔｅｉｎ－Ｂａｒｒ（ＥＢ）病毒壳抗原（ＥＢＶ－ＶＣＡ）的方法，用于酶联免疫吸附试验（ＥＬＩＳＡ）检测人血清中的相关抗体，我们用重组昆虫病毒在感染的ｓｆ９细胞中表达ＥＢＶ－ＶＣＡ。感染的细胞经裂解后，层析纯化表达产物。用纯化的ＶＣＡ作为抗原包被ＥＬＩＳＡ板或硝基纤维膜，检查血清中ＶＣＡ／ＩｇＡ和ＶＣＡ／ＩｇＧ抗体，为ＥＢＶ感染的检测和ＮＰＣ的诊断发展了一个敏感、特异和简便的方法。     

Epstein—Barr病毒壳抗原的纯化及其在检查血清抗体…

为了建立纯化Ｅｐｓｔｅｉｎ－Ｂａｒｒ（ＥＢ）病毒壳抗原（ＥＢＶ－ＶＣＡ）的方法，用于酶联免疫吸附试验（ＥＬＩＳＡ）检测人因清中的相关抗体，我们用重组昆虫病毒在感染的ｓｆ９细胞中表达ＥＢＶ－ＶＣＡ。感染的细胞经裂解后，层析纯化表达产物。用经的ＶＣＡ作为抗原包虫被ＥＬＩＳＡ板或硝基纤维膜，检查血清中ＶＣＡ／ＩｇＡ和ＶＣＡ／ｇＧ抗体，为ＥＢＶ感染的的检测和ＮＰＣ的诊断发展了一个敏感、特异和简便的方法。     

抗人类免疫缺陷病毒gp41和丙型肝炎病毒NS3杂交瘤细胞株的建立

目的为检测血清中人类免疫缺陷病毒（ＨＩＶ）和丙型肝炎病毒（ＨＣＶ）抗原提供血清学指标。方法用纯化的重组ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原蛋白作为联合免疫原，免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与Ｓｐ２／０小鼠骨髓瘤细胞融合，并采用挑单个细胞的方法进行一次克隆。结果分别获得４株和６株能稳定分泌高效价抗ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原蛋白单克隆抗体杂交瘤细胞株。初步建立了双抗体夹心法检测ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原的酶联免疫吸附试验。结论本方法是建立单抗杂交瘤细胞株的快速方法，所建杂交瘤细胞株特异性强，效价高，分泌抗体性能稳定，有推广价值。     

人乳头瘤病毒16E6蛋白单克隆抗体的研制

运用杂交瘤技术，我们成功地建立了两株能稳定分泌小鼠抗人乳头瘤病毒１６Ｅ６蛋白单克隆抗体的杂交瘤细胞，经鉴定单克隆抗体属ＩｇＧ１ｋ。试验结果表明，所得单克隆抗体仅１与ＰＶ１６Ｅ６融合蛋白反应。不与ＨＰＶ１６Ｅ７、Ｌ１、Ｌ２融合蛋白以及Ｌ１－Ｌ２真核表达蛋白反尖，也只与Ｃａｓｋｉ细胞反应，不与Ｈｅｌａ细胞反应，初步结果说明，该抗体是ＨＰＶ１６Ｅ１６蛋白特异性的ＭｃＡｂ。     

人乳头瘤病毒16 E6蛋白单克隆抗体的研制

运用杂交瘤技术，我们成功地建立了两株能稳定分泌小鼠抗人乳头瘤病毒１６Ｅ６蛋白单克隆抗体的杂交瘤细胞。经鉴定单克隆抗体均属ＩｇＧ１ｋ。试验结果表明，所得单克隆抗体仅与ＨＰＶ１６Ｅ６融合蛋白反应，不与ＨＰＶ１６Ｅ７、Ｌ１、Ｌ２融合蛋白以及Ｌ１－Ｌ２真核表达蛋白反应，也只与Ｃａｓｋｉ细胞反应，不与Ｈｅｌａ细胞反应。初步结果说明，该抗体是ＨＰＶ１６Ｅ１６蛋白特异性的ＭｃＡｂ。     

EB病毒膜蛋白在CHO细胞中的表达

目的研制ＥＢ（Ｅｐｓｔｅｉｎ－Ｂａｒ）病毒基因工程疫苗。方法构建了含有Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）膜蛋白（ＭＡ）基因的重组表达质粒ｐＣＭＶ／ＭＡ，转染ＣＨＯ细胞，研究表达产物的生物学性状，及培养液中不同血清含量、冻存、Ｇ４１８对ＣＨＯ细胞分泌ＭＡ的影响。结果获得两个稳定表达ＭＡ的细胞株。免疫印迹检测表达产物的分子量约为３４０ｋＤ和２２０ｋＤ，间接免疫荧光和免疫斑点确定表达产物能与抗ＭＡ的单克隆抗体特异性结合，用薄层扫描和Ｌｏｗｒｙ法计算ＭＡ的表达量为每天１９μｇ／ｍｌ。经纯化的ＭＡ免疫小鼠，在血清中检测到抗ＭＡ的特异性抗体。结论为开发有效的表达系统用于ＥＢ病毒基因工程疫苗的生产提供有利的实验基础。     

Epstein－Barr病毒膜抗原gp250／350在CHO细胞中高表达株的初筛

Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒膜抗原ｇｐ２５０／３５０在ＣＨＯ细胞中高表达株的初筛周玲，曾毅Ｈ．Ｗｏｌｆ使用Ｈ．Ｗｏｌｆ教授研究所以前构建的Ｅｐｓｔｅｉｎ－Ｂａｒｓ病毒（ＥＢＶ）重组ＤＮＡ质粒ＰＭＤⅢＧＰＴＲ（移去穿膜序列），用此质粒转化ＣＨＯ细胞。经...     

Serum immunoglobulin A (IgA)-mediated immunity in human immunodeficiency virus type 2 (HIV-2) infection

In the present study, we sought to define the importance of serum IgA (sIgA)-mediated immunity in HIV-2 infection. Serum samples from a total of 29 HIV-2-infected patients from Guinea-Bissau (n = 20) and Portugal (n = 9) were studied. Samples from seronegative individuals were used as controls. Antibody reactivity to native and recombinant envelope glycoproteins as well as peptides representing various regions of the envelope glycoproteins was investigated. Furthermore, the capacity of purified IgA to neutralize the HIV-2(SBL6669) strain was tested. All serum samples showed IgA reactivity against whole HIV-2 antigen. Twenty-eight out of 29 IgA samples (96%) reacted with native HIV-2 gp125, 26/29 (90%) with recombinant gp105, and 29/29 (100%) with recombinant gp36. When using peptides, the most prominent IgA reactivity was seen against the peptide representing aa 644-658 of the transmembranous protein gp36, to which 72% of the sera reacted. Purified sIgA antibodies showed neutralizing effects against HIV-2(SBL6669) in 17/29 cases (59%). This work describes the HIV-2-specific sIgA antigenic response. Moreover, our findings show, for the first time, that sIgA may play a role in the in vitro neutralization of HIV-2.     

抗人IgA单克隆抗体的特性分析和初步应用

用常规免疫法和脾内直接注射抗原(SIgA)法免疫小鼠,平行作细胞融合实验,建立4株分泌抗人IgA McAb杂交瘤细胞株,所得McAb对人IgA有较高特异性和抗体效价,经免疫印染法表明对α重链呈特异性。用抗原竞争抑制ELISA和ELISA添加试验分析McAb的抗原结合位点,均表明McAb A4与A1b、A3b、A9针对不同的抗原位点。用于检测EB病毒VCA和EA的IgA抗体,显示满意的特异性和敏感性,这将为有关试剂的生产提供稳定的抗体来源。     

抗牛型结核杆菌单克隆抗体的制备、鉴定及其纯化抗原的研究

用牛结核杆菌超声抗原及纯蛋白衍生物(PPD)免疫BALB/c小鼠,经融合、筛选和克隆化得到9株稳定分泌单克隆抗体(McAb)的杂交瘤细胞株,应用抗原蛋白酶消化试验、抗原过碘酸氧化试验、加成试验及免疫印迹分析等,表明9株McAbs所识别抗原决定簇均对蛋白酶敏感,其中2株的抗原决定簇既存在于蛋白质中,又存在于脂类中。9株McAbs所识别的抗原分子量均为30kDa。4株均作用于同一抗原决定簇,其它McAb各作用于另外互不相同的抗原决定簇。免疫电镜观察,McAb 1A3所作用的蛋白质抗原主要分布在细胞内,少量分布于细菌胞壁。9株McAbs除1株外均显示出很强的特异性,其中4株仅与牛型结核茵反应阳性。利用McAb 1A3通过亲和层析柱纯化牛型结核杆菌PPD后,得到亲和层析纯抗原Ag 1A3,它仅与牛型结核杆菌免疫血清反应阳性,与其它分枝杆菌免疫血清反应阴性;对牛型结核杆菌致敏的豚鼠皮肤反应阳性,而与人型及BCG致敏豚鼠皮肤反应阴性。     

Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1

Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.     

Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH₂-terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.     

Expression of the bovine leukemia virus envelope glycoprotein (gp51) by recombinant baculovirus and its use in an enzyme-linked immunosorbent assay

The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH2-terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.     

多种单抗联合检测HIV抗原

目的 建立多种单抗联合早期检测HIV抗原的夹心ELISA方法.方法 以SAS盐析沉淀法和亲和层析法纯化抗HIV-1 p24、gp41、gp120及抗HIV-2 gp36的腹水型单克隆抗体(McAb),用高碘酸钠法将纯化的McAb以HRP进行标记.建立针对单个抗原的双抗体夹心ELISA法,对其灵敏度及特异性进行检测.将筛选得到的4株捕获McAb按比例混合作为捕获抗体,4株酶标McAb按比例混合作为检测抗体,建立多种单抗联合检测HIV抗原的夹心ELISA方法,检测混合HIV抗原.结果 按确定的最优反应条件建立的多种McAb联合夹心ELISA方法,检测到的最高稀释度的HIV混合抗原中各抗原的终浓度分别为:重组HIV-1 p24:0.625 pg/ml,gp41:6.25 ng/ml,gp120:6.25 ng/ml;HIV-2 gp36:9.25 ng/ml.结论 建立了具有高度敏感性的鸡尾酒式多种单抗联合检测HIV抗原的夹心ELISA法,为早期榆测HIV抗原提供了新的思路,为后续的研究奠定了一定基础.     

多种单抗联合检测HIV抗原

目的 建立多种单抗联合早期检测HIV抗原的夹心ELISA方法.方法 以SAS盐析沉淀法和亲和层析法纯化抗HIV-1 p24、gp41、gp120及抗HIV-2 gp36的腹水型单克隆抗体(McAb),用高碘酸钠法将纯化的McAb以HRP进行标记.建立针对单个抗原的双抗体夹心ELISA法,对其灵敏度及特异性进行检测.将筛选得到的4株捕获McAb按比例混合作为捕获抗体,4株酶标McAb按比例混合作为检测抗体,建立多种单抗联合检测HIV抗原的夹心ELISA方法,检测混合HIV抗原.结果 按确定的最优反应条件建立的多种McAb联合夹心ELISA方法,检测到的最高稀释度的HIV混合抗原中各抗原的终浓度分别为:重组HIV-1 p24:0.625 pg/ml,gp41:6.25 ng/ml,gp120:6.25 ng/ml;HIV-2 gp36:9.25 ng/ml.结论 建立了具有高度敏感性的鸡尾酒式多种单抗联合检测HIV抗原的夹心ELISA法,为早期榆测HIV抗原提供了新的思路,为后续的研究奠定了一定基础.     

广州管圆线虫单抗结合抗原定位分布及在免疫诊断上的应用

目的 研制广州管圆线虫单克隆抗体诊断循环抗原提高诊断的特异性.方法 将广州管圆线虫分泌性抗原免疫小鼠,免疫鼠脾细胞与骨髓瘤细胞融合为杂交瘤细胞,用广州管圆线虫阳性患者血清筛选阳性杂交瘤细胞,培养阳性的杂交瘤细胞分离制备单克隆抗体命名为12D5和21B7,用免疫组织化学的方法分析12D5和21B7单抗结合抗原在广州管圆线虫体内的分布,并用筛选的双12D5和21B7单抗进行抗体夹心ELISA检测实验感染广州管圆线虫的大鼠、广州管圆线虫感染病人血清循环抗原(CAg),用其他寄生虫抗原鉴别单抗的特异性,并与抗体检测比较其敏感性和特异性.结果 经鉴定单抗12D5为IgG1,21 B7为IgM,两株单抗同时识别广州管圆线虫成虫相对分子质量为55 × 103的蛋白,两个单抗针对的抗原分布在虫体肠表面,12D5和21 B7双抗体夹心ELISA法对实验感染的广州管圆线虫的大鼠血清中CAg检出率为100%(48/48),广州管圆线虫感染病人血清CAg检出率为100%(32/32),与日本血吸虫、肝吸虫、肺吸虫、旋毛虫、蛔虫、包虫病人血清无交叉反应,与健康人血清无反应;而用抗原检测32个广州管圆线虫感染病人的抗体检出率为75%(24/32),同时抗体检测与其他寄生虫出现一定的交叉反应.结论 12D5和21 B7单抗结合的抗原为肠相关抗原,双抗体夹心ELISA法对感染广州管圆线虫人和动物血清中CAg检测的特异性强,敏感性高,优于抗体检测试剂,并能够确定现症感染.

Abstract：

Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.