点突变p53及其抗原肽重组痘苗病毒抗肿瘤效应？…

目的 比较点突变Ｐ５３及其抗原肽重组痘苗诱导的抗瘤免疫反应。为重组抗原轩用于肿瘤免疫治疗提供实验依据。方法 以人１３５位Ｃｙｓ→Ｔｙｒ为Ｐ５３为肿瘤相关抗原模型,观察以表达该点突变Ｐ５３蛋白的重组痘苗病毒ｒＶＶ－ｐ５３ＦＬ与表达包含该点突变抗原肽：５３１２５－１４５的重组痘苗病毒ｒＶＶ－ｐ５３Ｍ所诱导包含该突变抗原肽Ｐ５３１２５－１４５重组的痘苗病毒ｒＶＶ－ｐ５３Ｍ所诱导的ＣＴＬ及对荷瘤Ｂａｌｂ／     

点突变p53肽诱导肽特异性CTL的研究

目的探索点突变ｐ５３肽诱导细胞免疫应答的可能性,为其作为肽疫苗用于肿瘤免疫治疗提供实验依据。方法人工合成小鼠点突变ｐ５３肽Ｐ１３２Ｆ（ＬＮＫＬＦＦＱＬ,１３２Ｃｙｓ→Ｐｈｅ突变）,与不完全弗氏佐剂（ＩＦＡ）或ＲＩＢＩ佐剂（ＲＡＳ）混合,皮下免疫Ｃ５７ＢＬ／６小鼠,分离脾细胞体外用肽再刺激,诱导细胞毒Ｔ淋巴细胞（ＣＴＬ）,并以５１Ｃｒ释放法检测其杀伤活性。结果Ｐ１３２Ｆ肽加上ＩＦＡ或ＲＡＳ免疫小鼠,均能诱导肽特异性ＣＤ８＋ＣＴＬ;低剂量重组白细胞介素２（ｒＩＬ－２）能增强肽免疫效果。结论所用突变的ｐ５３蛋白具有免疫原性,提示来源于突变ｐ５３的抗原肽有可能作为肽疫苗用于临床肿瘤免疫治疗     

重组结核抗原痘苗病毒Ankara株的构建及其免疫原性研究

目的 构建5种不同类型的表达结核杆菌特异抗原的重组痘苗病毒,并研究其特异免疫原性.方法 运用同源重组技术将含结核分泌抗原Ag85A和ESAT-6的基因片段插入痘苗病毒表达质粒p18中.重组质粒导入痘苗病毒Ankara(MVA)后构建重组痘苗病毒,经筛选和Western blot鉴定,得到5个种类的带有结核抗原基因的重组病毒.用构建的5种重组病毒免疫小鼠,MTT法检测免疫后小鼠脾淋巴细胞对特异结核抗原的增殖反应;ELISA检测小鼠脾淋巴细胞培养上清液中IFN-γ的含量;结核菌素纯蛋白衍化物(PPD)皮内试验以检测重组病毒引发的针对结核抗原的特异细胞免疫应答.结果 构建的5种蘑组病毒介导的细胞表达产物经Western blot鉴定确认相对分子质量与结核抗原一致.免疫小鼠两次后,5种重组病毒免疫组脾淋巴细胞体外与Ag85A-ESAT-6融合蛋白共培养后表现出明显的增殖活性(P<0.01),培养上清液中IFN-γ的浓度均较同组细胞经生理盐水刺激明显增高(P<0.05);与空痘苗病毒或生理盐水免疫后小鼠相比,5种重组MVA免疫组脾淋巴细胞与AgB5A.ESAT-6融合蛋白共培养后同样表现出明显的增殖活性(P<0.01),与Ag85A-ESAT-6融合蛋白共培养的细胞上清液中IFN-γ的浓度均升高(P<0.01).与空痘苗病毒或生理盐水免疫后小鼠相比,5种重组MVA免疫组小鼠对PPD都表现出显著的迟发型超敏反应应答(P<0.05).结论 成功构建了5种不同类型的表达结核杆菌抗原的重组痘苗病毒疫苗,其免疫小鼠后可激发针对结核杆菌抗原的特异性细胞免疫.     

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     

痘苗病毒载体与肿瘤基因治疗（下）——重组痘苗病毒载体介导的肿瘤基…

目前采用同源重组构建了多种表达外源基因的重组痘苗病毒载体，包括ＩＬ－２，ＩＬ－３，ＩＬ－４，ＩＬ－５，ＩＬ－６，ＩＦＮα，ＩＦＮγ，ＧＭ－ＣＳＦ，ＴＮＦ，ＩＬ－１０等细胞因子基因及ｐ９７，ＣＥＡ，ＭＵＣ１等肿瘤抗原基因，并应用于肿瘤基因治疗的体内外实验取得了明显的疗效。应用痘苗病毒载体进行ｉｎ ｖｉｖｏ基因治疗可望成为肿瘤基因治疗的一种新模式。     

应用痘苗病毒体表达猴轮状病毒VP4抗原基因

把编码猴轮状病毒Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ｐＪＳＡ１１７５－Ｖｐ４。应用磷酸钙沉淀技术将ｐＪＳＡ１１７５－Ｖｐ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ 存在下筛选蓝色蚀斑。     

乙型脑炎重组痘苗病毒J3免疫性的研究

用含有日本脑炎病毒（ＪＥＶ）结构蛋白ＰｒＭ、Ｅ和非结构蛋白ＮＳ１、ＮＳ２Ａ基因的重组痘苗病毒Ｊ３免疫家兔，能较快地诱生抗ＪＥＶ抗体，其滴度可随免疫次数的增加和免疫时间的延长而增高。此抗血清可被动保护不同毒株ＪＥＶ的致死性攻击，该保护作用与其中抗体和血凝抑制抗体有关。     

重组杆状病毒表达的EIAV Env蛋白与含有env基因重组痘苗病毒的联合免疫

目的：构建新型的马传染性贫血病毒(EIAV)的候选疫苗：方法：利用BAC—To—BAC杆状病毒表达系统，将中国马传贫驴白细胞弱毒疫苗(EIAV DLV)及其亲本株(EIAV LN)env基因导入到杆状病毒基因组中。转染昆虫细胞后，得到的重组病毒用SDS—PAGE和Western blot检测表达产物。以本实验室构建的含有EIAV env基因的重组痘苗病毒，单独或与重组杆状病毒表达的EIAV Env蛋白联合免疫小鼠。结果：构建的重组杆状病毒能正确表达全长Env蛋白。与单独免疫组相比，联合免疫组免疫应答显著增强，其中中和抗体的滴度提高5～9倍。结论：含有EIAV env基因的重组痘苗病毒与Env蛋白抗原联合免疫，能够诱导高滴度的中和抗体。     

Comparative sequence analysis of the M gene among rabies virus strains and its expression by recombinant vaccinia virus

Nucleotide sequences and the deduced amino acid sequences of the gene encoding the matrix (M) protein of the Nishigahara and the CVS strains of rabies virus have been determined. The M gene is 609 nucleotides long and is capable of coding for a peptide composed of 202 amino acids. Sequence comparison of these M genes with those of other stains [Pasteur (PV), ERA, Avol] revealed that there is 89.7–91.5% homology at the nucleotide level, and 90.1–92.1% homology at amino acid level, between almost all combinations of these strains. However, in the combinations of the PV and ERA strains, and the virulent CVS and the avirulent CVS-derived Avol strains, much higher homology was observed both at the nucleotide and amino acid levels. The predicted secondary structure and hydropathy profiles also exhibited similar features. Recombinant vaccinia virus containing the M gene was constructed. Sodium dodecyl sulfate (NaDodSO₄)-polyacrylamide gel electrophoresis of the precipitates obtained by immune reaction of the recombinant virus-infected cell lysate with a monoclonal antibody against the M protein revealed that electrophoretic mobility of the expressed protein is indistinguishable from that of the authentic M protein from rabies virions.The nucleotide sequence data of the M genes of the CVS and Nishigahara (RCEH) strains reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers D90450 and D90451.     

Cytotoxic T lymphocyte responses to wild-type and mutant mouse p53 peptides

Cytotoxic T lymphocytes (CTL) recognize peptides presented at the cell surface in association with major histocompatibility complex (MHC) class I molecules. The finding that peptides binding to MHC class I molecules share common amino acid motifs renders feasible the selection of antigenic peptides by simply scanning protein sequences, and thus, provides the possibility of inducing CTL to pre-defined specificities. Tumor cells possess antigens known to generate MHC class I-restricted CD8⁺ CTL responses. Thus, these antigens represent good targets to induce tumor-specific immunity. Among these antigens, the p53 tumor suppressor gene product is an attractive candidate for cancer immunotherapy. Mutations in the p53 gene have been found to be very frequently associated with a malignant transformation and often lead to p53 protein overexpression. Thus, we investigated the possibility of inducing CTL to wild-type or mutant p53 peptides in a BALB/c (H-2^d) mouse model. Peptides possessing the H2-K^d binding motif were selected and tested for binding to the H-2K^d molecules in vitro. Synthetic peptides p53_122–130 wild-type or “mutant” (Lys → Glu substitution at position 129) were shown to be the best binder peptides and were tested for their immunogenicity in mice. H-2K^d-restricted p53-specific CD8⁺ CTL were generated following immunization of mice with either wild-type (wt) p53_122–130 or mutant (mut) p53_122–130 (E129) peptides. Only low-affinity CTL can be obtained by immunization with the wt sequence. In contrast, CTL elicited with the mut peptide recognized the mut sequence at a 10–100-fold lower concentration. This indicates that CTL elicited with the mut peptide recognized the mut sequence very efficiently, whereas the wt sequence is poorly recognized, if at all. Taken together, these results thus suggest that p53-specific tumor immunotherapy may be successful only if the mutated protein is taken into consideration.     

乳腺癌p53和p27蛋白的表达及意义

目的　探讨p5 3蛋白和p2 7蛋白在乳腺癌中的表达及意义。方法　应用免疫组化S -P法检测 6 7例乳腺癌中p5 3蛋白和p2 7蛋白的表达情况。结果　 6 7例乳腺癌中p5 3蛋白和p2 7蛋白的阳性表达率分别为 5 8 3%和 5 9 7% ,p5 3蛋白的表达与乳腺腋窝淋巴结转移有相关性 (P <0 0 1) ,p2 7蛋白的表达也与乳腺腋窝淋巴结转移有关 (P <0 0 5 ) ,乳腺癌p2 7和p5 3蛋白表达之间无明显相关。结论　p5 3蛋白的过表达和p2 7蛋白的失表达均与乳腺癌发生发展相关 ,二者可作为判断乳腺癌预后的有用指标     

Intracellular processing and immunogenicity of the envelope proteins of human T-cell leukemia virus type I that are expressed from recombinant vaccinia viruses

Two types of recombinant vaccinia viruses (VVs) expressing the env gene of the human T-cell leukemia virus type I (HTLV-I) were reported previously. One recombinant VV, WR-proenv1, synthesized the authentic env protein. In the other recombinant VV, WR-env17, the env gene was inserted within the signal sequence of the VV hemagglutinin (HA) gene, so that the reading frame for the env gene was in phase with that for the HA gene. Comparative studies were performed on the mode of expression and processing of the env proteins in relation to their immunogenicity. In WR-env17-infected cells, translation was initiated exclusively from the initiation methionine of the HA to produce nascently the chimeric env protein, including the altered HA signal peptide. Both this altered HA signal peptide and the internalized env signal peptide functioned as insertion signals for the endoplasmic reticulum. Although about half of the nascent chimeric protein was cleaved at the carboxyl terminus of the internalized env signal peptide to produce the authentic env protein, the other half was cleaved at the carboxyl terminus of the altered HA signal peptide alone to synthesize the chimeric protein. These events led to a less efficient transport of the env protein produced by WR-env17 from the rough endoplasmic reticulum to the Golgi apparatus than that of the authentic env protein synthesized by WR-proenv1. The efficiency of the processing and transport of the env protein affected the immunogenicity of these two recombinant VVs.     

接种CEA重组痘苗病毒对小鼠CEA^＋肿瘤抑制作用的机制探讨

目的 探讨CEA重组痘苗病毒（rV-CEA)治疗CEA^ 肿瘤的机制。方法 以我室构建的rV_CEA,腹腔接种供体C57/BL小鼠,取其脾细胞及腹腔巨噬细胞（Mφ）,分别过继转移给荷CEA^ -HePa肝癌细胞的C57/BL小鼠,检测该公共体小鼠脾细胞、腹腔Mφ及相应受体的脾细胞体外杀瘤细胞的效应,结果 接种rV-CEA的供体小鼠的脾细胞及腹腔Mφ过继免疫给受体小鼠,具有明显抑制受体CEA阳性肿瘤生长的作用,体外实验表明,该供体脾细胞及接种了供体Mφ的受体脾细胞对同一靶细胞的杀伤活性明显增强,但供体Mφ体外的细胞毒活性无明显增加,结论 rX-CEA对CEA^ 肿瘤的抑制作用,可能主要通过CEA特异性免疫反应激活T细胞而实现。Mφ作为抗原提呈细胞可通过激活T细胞而杀伤肿瘤细胞,具体机制值得进一步研究。