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。     

利用重组痘苗病毒表达人CD20基因

目的获得重组人 CD2 0分子并研究编码人 CD2 0的基因在痘苗病毒中的表达。方法从 p GEM- T- EASY/ CD2 0载体上酶切下编码人 CD2 0分子的 c DNA,亚克隆到 p JSA1175载体上 ,重组质粒与野生痘苗病毒共转染 TK- 143细胞。结果APAAP检测到重组病毒感染的细胞表面有 CD2 0分子表达 ,富集后病毒滴度约为 1× 10 9pfu/ ml。结论人 CD2 0基因在痘苗病毒中表达 ,为研究其功能以及研制单克隆抗体奠定了基础     

Conserved nucleotide sequence of rabies virus cDNA encoding the nucleoprotein

The cDNAs of rabies virus (the CVS strain) encoding the structural proteins (G, N, NS, and M) were cloned. Of these clones, the nucleotide sequence of the cDNA encoding the nucleoprotein was determined to compare with those of other strains of rabies virus. The comparison confirmed that the nucleotide sequences and deduced amino acid sequences are highly conserved among strains including an avirulent strain.     

表达狂犬病毒糖蛋白的非复制型重组痘苗病毒的构建 …

目的 提高表达狂犬病毒糖蛋白（ＲＧ）的重组痘苗病毒的安全性。方法 将编码中国狂犬病毒５ａＧ株糖蛋白的基因,插入痘苗病毒天坛株的ＴＫ区,获得重组病毒ＶＴＫＲＧ,通过两步同源重组,删除ＣＫ片段间与痘苗病毒毒力及宿主范围相关的基因,得到非复制型重组痘苗病毒ＶＴＫＲＧΔＣＫ。结果 经ＰＣＲ鉴定,ＣＫ间的核酸片段被成功删除,其缺失性状能稳定地遗传。     

Nucleotide sequence of the nucleoprotein gene of the RC·HL strain of rabies virus,a seed strain used for animal vaccine production in Japan

By using a phage vector (lambda ZAP II) and the mRNA extracted from IMR-32 cells infected with the RC·HL strain of rabies virus, we constructed a cDNA library from which four nucleoprotein (N)-specific cDNA clones were obtained by Southern blot hybridization. These clones contained a cDNA insert of about 1.4 kb, in which the longest open reading frame was the same length as that reported for the N cDNA of three fixed strains, CVS, PV, and SAD B19. When the nucleotide and deduced amino acid sequences were compared between the RC·HL and the three strains, homology was within the range of 91.5–91.8% and 95.1–96.0%, respectively. Of 183 nucleotides of the RC·HL N-cDNA that were not identical to that of the corresponding site of at least one of the three strains, 41 were shared with the CVS strain, whereas only three were shared with either of the other two strains. In the amino acid sequence, we found 29 residues that were not shared in common with all of the four strains, 11 of which were the substitutions with radically different amino acids that might cause conformational changes of the protein, and, in addition, five of which were located in the region close to the C terminus. The number of such amino acid substitutions between the RC·HL and CVS strains was smaller than that of the other three strains. These results are not inconsistent with the presumption that the RC·HL and CVS strains originated from the same laboratory strain of the Pasteur viruses.     

狂犬病毒CVS-N2c毒株糖蛋白基因的克隆和重组腺病毒的构建

克隆了狂犬病毒CVS-N2c毒株糖蛋白(GP)基因，并进行了GP全基因序列测定和对比分析。结果显示CVS-N2c GP基因编码区长1575bp，编码524个氨基酸，与国内外发表的狂犬病毒疫苗株3aG株、ERA株、和HEP-Flury株GP基因的核酸序列同源性分别为89．0％，89．3％，94．5％，而推导的氨基酸序列同源性分别为87．6％、88．4％、和91．2％。在此基础上，用pAdEasy载体系统构建了表达GP基因的重组腺病毒。电镜下确定重组病毒颗粒的直径约70nm，Western blot显示重组病毒表达的GP蛋白可被兔抗狂犬病毒血清识别，表达的GP蛋白分子量约为66kD，与天然的狂犬病毒GP的分子量相符。该表达CVS-N2c毒株GP基因的重组腺病毒是筛选有效狂犬病毒基因工程疫苗的基础。     

点突变p53及其抗原肽重组痘苗病毒抗肿瘤效应的比较研究

目的　比较点突变p5 3及其抗原肽重组痘苗病毒诱导的抗瘤免疫反应 ,为重组抗原疫苗用于肿瘤免疫治疗提供实验依据。方法　以人 135位Cys→Tyr点突变 p5 3为肿瘤相关抗原模型 ,观察以表达该点突变 p5 3蛋白的重组痘苗病毒rVV p5 3FL与表达包含该点突变抗原肽 p5 312 5 14 5的重组痘苗病毒rVV p5 3M 所诱导的CTL及对荷瘤Balb/c小鼠的免疫保护和治疗作用。结果　rVV p5 3FL和rVV p5 3M 均能诱导以CD8 T细胞为主的特异性CTL。用rVV p5 3FL与rVV p5 3M 免疫小鼠后 ,接种致死剂量的P815 mp5 3细胞 ,能保护部分小鼠免遭致死剂量肿瘤细胞的攻击。小鼠接种致死剂量的肿瘤细胞后 ,以rVV p5 3FL与rVV p5 3M 治疗荷瘤小鼠 ,可使小鼠平均存活时间显著延长。两种疫苗的抗瘤效应无明显区别。结论　来源于突变p5 3的抗原肽重组疫苗 ,可代替抗原大分子应用于肿瘤的免疫防治     

Expression and regulation of the vaccinia virus thymidine kinase gene in non-permissive cells

The expression and regulation of the vaccinia virus (VV) thymidine kinase (tk) gene was examined in two non-permissive cell lines, CHO and MDBK, which restrict VV development at different stages of the viral replication cycle. The VV tk gene was expressed in these two cell lines with kinetics similar to a fully permissive cell line BSC40. These results are consistent with the hypothesis that inhibition of tk mRNA translation by another viral early gene product is a normal component of the overall strategy employed to express and regulate the VV tk gene during a productive infection.     

狂犬病毒糖蛋白及核蛋白在非复制型痘苗病毒天坛株中的共同表达

目的　提高重组痘苗病毒狂犬疫苗的有效性和安全性。方法　利用TK区表达狂犬病毒糖蛋白 (RG)的重组痘苗病毒作为亲本株 ,通过两步同源重组 ,删除痘苗病毒基因组CK片段间与毒力和宿主范围相关的核酸片段 ,同时将狂犬病毒核蛋白 (RN)基因插入C K片段之间 ,获得了含有RG基因和RN基因的非复制型重组痘苗病毒VTKRGΔCKLacZRN。结果　经PCR鉴定 ,RN基因已插入CK区 ;Westernblot结果显示 ,该株病毒能同时表达RG和RN ,相对分子质量 (Mr)分别为 6 5× 10 3 和 5 0 5× 10 3。VTKRGΔCKLacZRN在人源细胞如TK 143细胞中不能正常繁殖 ,在鸡胚成纤维细胞 (CEF)中能正常复制。与非复制型重组病毒VTKRGΔCK相比 ,VTKRGΔCKLacZRN免疫小鼠后能更快地诱生较高滴度的中和抗体 ,效果相当于复制型重组病毒VTKRG免疫组 ;它们均能保护小鼠针对致死剂量狂犬病毒国际标准攻击毒株 (CVS)的攻击。结论　VTKRGΔCKLacZRN具有良好的免疫效果和安全性     

人CD19基因在痘苗系统中的表达

目的 在痘苗系统中表达人ＣＤ１９基因,为研究ＣＤ１９分子的豚研制抗ＣＥ１９的单克隆抗体（ｍＡｂ）打下基础。方法 用ＰＣＲ技术扩增人ＣＤ１９全长基因,并 组人克隆载体ｐＧＥＭ－Ｔ,进行序列测定。将ＣＤ１９全长基因克隆人痘苗表达载体ｐＪＳＡ１１７５中,用脂 本介导的方法,与野生 人转染ＴＫ－１４３细胞。运用蓝斑筛选获避人ＣＤ１９全长基因的重组痘苗病毒,并用此重组病毒感染ＴＫ－１４３细胞,以ＡＰＡＡＰ法     

中国呼吸道合胞病毒地方株G蛋白在重组痘苗病毒中的表达

目的　构建表达中国呼吸道合胞病毒 (RSV)地方株G蛋白基因的重组痘苗病毒 ,以用于RSV感染防治的研究。方法　用基因克隆技术将中国RSV地方株G蛋白基因插入到痘苗病毒载体中 ,与痘苗病毒共感染获得重组病毒。用免疫印迹、ELISA、蚀斑减少试验等方法检测表达产物的免疫原性及生物活性。结果　RSV地方株G蛋白在痘苗病毒中获得较好的表达 ,表达产物的糖基化程度较高 ,且该重组病毒免疫家兔可诱发特异性抗体产生。蚀斑减少试验证明 ,用该表达产物制备的抗血清具有中和病毒的活性。结论　重组病毒表达的中国RSV地方株G蛋白具有较好的抗原性、免疫原性等生物活性     

Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry

We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser₆₅ to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.     

Complete genome sequence of a raccoon rabies virus isolate

The entire genome of a mid-Atlantic raccoon strain rabies virus (RRV) isolated in Canada was sequenced; this is the second North American wildlife rabies virus isolate to be fully characterized. The overall organization and length of the genome was similar to that of other lyssaviruses. The nucleotide sequence identity of the raccoon strain ranged between 32.7% and 85.0% when compared to other lyssaviruses, while the deduced amino acid sequence identity ranged between 22.9% and 94.2% with the nucleoprotein and polymerase being the most conserved. Notable features of RRV include the phosphoprotein's four amino acid extension compared to most other rabies viruses, and a nucleotide substitution immediately prior to the normal start codon that results in an additional methionine at the beginning of the L protein. This is the first report of the RRV L gene sequence and its 2128 amino acid product. Rates of non-synonymous and synonymous nucleotide changes within the lyssavirus L gene identified the conserved blocks II, III and IV as being most constrained. Analysis of L gene codon substitution patterns favoured models that supported positive selection, but only one site, corresponding to Leu62 of the RRV L protein, was identified as being under weak positive selection.     

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。     

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA^r) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.     

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.     

Functional organization of variola major and vaccinia virus genomes

Comparison of the genomic organization of variola and vaccinia viruses has been carried out. Molecular factors of virulence of these viruses is the focus of this review. Possible roles of the genes of soluble cytokine receptors, complement control proteins, factors of virus replication, and dissemination in vivo for variola virus pathogenesis are discussed. The existence of buffer genes in the vaccinia virus genome is proposed.     

Inhibition of vaccinia virus thymidine kinase by the distal products of its own metabolic pathway

Vaccinia virus thymidine kinase activity is inhibited by low concentrations (10 microM) of dTDP or dTTP, but not by dTMP. This inhibition is specified for the thymidine nucleotides as dATP, dGTP, and dCTP have no effect. The viral enzyme phosphorylates thymidine to dTMP with typical first-order kinetics. However, evidence was obtained to indicate that the observed dTTP inhibition was noncompetitive in nature. This suggests that thymidine and dTTP interact with different sites in the native viral enzyme.