应用亲和层析法提纯 鸡马立克氏病病毒pp38基因重组产物

利用鸡马立克氏病病毒(MDV)1型特异单克隆抗体H19致敏Sepharose 4B—cNBr,从感染重组病毒BP38Ⅱ的昆虫细胞中提纯鸡马立克氏病毒pp38基因重组产物,获得良好效果。提纯的蛋白质在SDS—PAGE中表现出一条分子量约为38kDa的蛋白质条带,在免疫印迹试验中该蛋白质条带也能被单克隆抗体H19识别。利用该提纯的重组pp38免疫小鼠,所制备的小鼠抗血清在免疫荧光染色试验中不仅能与感染重组病毒的昆虫细胞Sf9反应,也能和Ⅰ型MDV型感染的鸡胚成纤维细胞反应。     

I型马立克氏病毒38kD磷蛋白与Ⅱ和Ⅲ型病毒间的交叉免疫反应

由致病性Ⅰ型马立克氏病病毒(MDV)特异性的单克隆抗体H_(19)制备亲和层析柱,从感染重组杆状病毒BP38Ⅱ的昆虫细胞系Sf9细胞中提纯MDV的pp38基因表达产物,以此免疫小鼠制备抗血清,测定其与不同型MDV毒株感染的鸡胚成纤维细胞免疫反应性,同时也用不同型MDV毒株(HVT,Z4)制备的抗血清和自然感染发病鸡血清分别与感染重组病毒的Sf9细胞进行免疫交叉反应.试验结果表明,完整的Ⅰ型MDV来源的pp38基因产物与所试Ⅱ型Z4株和Ⅲ型HVT Fc126毒株在抗原性上有显著交叉反应.这一结果表明,在Ⅱ型和Ⅲ型MDV毒株中可能存在着pp38的类似物.     

应用亲和层析法是纯鸡马立克氏病病毒PP38基因重组产物

利用鸡马立氏病病毒（ＭＤＶ）Ｉ型特异单克隆抗体Ｈ１９致敏Ｓｅｐｈａｒｏｓｅ４Ｂ－ＣＮＢｒ,从感染重组病毒ＢＰ３８Ⅱ的昆虫细胞中提纯鸡马立克氏病毒ＰＰ３８基因重组产物,获得良好效果,提纯的蛋白质的ＳＤＳ－ＰＡＧＥ中表现出一条分子量约为３８ｋＤａ的蛋白质条带,在免疫印迹试验中该蛋白质带也能被单克隆抗体Ｈ１９识别。利用该提纯的重组ＰＰ３８免疫小鼠,所制备的小鼠抗血清在免疫荧光染色试验中不仅能与感染重组病     

用杆状病毒为载体在昆虫细胞中表达马立克病病毒pp38基因

鸡马立克病病毒(MDV)38kd磷蛋白(pp38)基因中包括起始密码子和终止密码子的完整编码序列被整合进杆状病毒AcNPV的转移载体质粒pVL1392,用所得的含pp38基因的重组转移载体质粒pVLpp38I与野生型杆状病毒AcNPV的DNA共转染昆虫传代细胞系Sf9细胞后,用荧光抗体法以抗MDV单克隆抗体H_(19)筛选到能表达MDVpp38的重组杆状病毒克隆BP38 I。免疫印迹试验表明,在重组病毒BP38 I感染的Sf9细胞溶解物中,可表现一条分子量约为35—36kd的为单克隆抗体H_(19)识别的MDV特异性蛋白带。     

马立克氏病病毒38kD磷蛋白表达产物的细胞质亲和性

将pcDNA3.1/Zeo表达性载体质粒中的启动子PCMV片段用HindⅢ及BglⅡ消化去除后,在该相应位点插入GA株马立克氏病病毒(MDV)的包含上游调控序列的38kD磷蛋白(pp38)基因的完整片段,以此获得能用自身启动子表达pp38的重组表达性质粒pcDNA-pp38.转染试验表明,pp38基因的天然启动子在整合进载体质粒中后,仍能在鸡胚成纤维细胞(CEF)中启动pp38的表达.用pp38特异性单克隆抗体H19对转染的CEF做间接荧光抗体检测试验发现,在转染细胞中表达的pp38仅位于细胞浆中,基本不进入细胞核.而与此相对照的用pcDNA3.1/Zeo为载体表达的MDV的肿瘤基因meq产物则正相反,仅显现在经转染CEF的细胞核中.meq和pp38基因产物在细胞内的不同分布嗜性显然与它们不同的生物学活性相关.     

马立克氏病病毒pp38／pp24聚合体结构及其对pp38基因上游双向启动子活性的影响

前期以氯霉素乙酰转移酶（CAT）活性为指标的研究证明了,马立克氏病病毒（Marek’s disease virus,MDV）的pp38和pp24的同时表达可显著增强基因组中pp38基因与1．8kb mRNA转录子之间双向启动子的转录活性．本研究又以增强型绿色荧光蛋白（EGFP）表达水平作为pp38基因上游双向启动转录活性的标志,更直观地证明了只有当同一细胞内同时表达pp38和pp24时,该启动子活性才有完整的启动活性．为了证明这两个蛋白能否相结合,分别以单独表达pp38或pp24的重组质粒pcDNA-pp38或pcDNA-pp24及能同时表达这两个基因的重组质粒pBud-pp38-pp24质粒转染鸡胚成纤维细胞（CEF）,用pp38特异的单克隆抗体H19对转染细胞的裂解标记物进行免疫沉淀实验．结果表明,H19可沉淀pp38,但pp24只是在pp38同时存在时才被H19沉淀,而在pcDNA-pp24单独转染的处理样品中不能显示pp24的条带．这证明了,pp24是通过与pp38的结合而被共沉淀下来的,显示pp24和pp38在天然状态下可以形成异二聚体或多聚体．上述两个独立的实验结果表明,pp38和pp24是以聚合体的形式结合于该双向启动子发挥作用的．     

马立克氏病病毒pp38基因上游的一个双向启动子研究

马立克氏病病毒（MDV）pp38基因上游是病毒基因组DNA复制原点。在其两侧均含有启动子TATAbox、CAATbox等特征性的保守基元,推测是一个天然的双向启动子。为了在体外验证其双向启动活性,本研究以MDVpp38为报告基因,并将其ORF插入到pUC18中,构建了pUCpp38质粒。将包含该启动子完整区域的789bp序列分别以正反两个方向克隆进pUCpp38质粒中pp38报告基因的上游,获得的重组质粒pProfpp38和pProrpp38。将所获得的重组质粒分别转染鸡胚成纤维细胞（CEF）,通过间接免疫荧光试验检测pp38基因的表达以验证该启动子的双向启动活性。结果表明,马立克氏病病毒复制原点区的启动子无论以何种方向插入pUCpp38质粒中,在转染细胞24h内能检测到pp38基因的表达,48h后能获得高效和持续的表达。逐渐缩小该启动子的范围,最终在320bp时,仍能检测到两个方向较强的启动活性。     

接种不同毒力的马立克氏病病毒后鸡病毒血症的动态比较

1日龄非免疫鸡分别接种马立克氏病病毒（MDV）I型强毒GA株、I型MDV疫苗毒CVI988株和III型火鸡疱疹病毒（HVT）疫苗株后第4日起,定期采血并用抗MDV囊膜糖蛋白B（gB）单克隆抗体介导的间接免疫荧光试验检测MDV在外周血液单核细胞（PBMC     

马立克氏病血清Ⅰ型致瘤株病毒感染的早期检测

马立克氏病（MD）是养禽业最重要的疫病之一,一直缺少有效的早期诊断方法。根据血清Ⅰ型马立克氏病毒（MDVⅠ）meq基因的核酸序列设计了一对寡苷酸核引物,分别对MDVR1致瘤株（京－1株）、非致瘤株（MD11／75C株、CV1988株）、MDV2（SB－1株）、HVT（Fv-126株）的核酸进行扩增。结果表明：京－1株扩增到约1.15kb核酸片段,MD11／75C株和CVI988株的扩增产物都与Digoxigenin标记的meq基因探针杂交,说明都是特异性的扩增产物,对MSB1细胞DNA及MDV感染鸡的血液及肝、肾肿瘤等DNA扩增都得到1.15kb条带,将京－1株和CV1988株感染的细胞DNA混合再扩增,同时得到1.15kb和1.0kb的核酸条带,所以根据拉增产物大小可以区别致瘤株京－1株及非致瘤株CV1988株,这表示可从CV1988株病毒免疫鸡体内检测到MDV强毒,适于早期确诊强毒感染。     

鸡马立克氏病病毒814株细菌人工染色体的构建

为构建全基因组鸡马立克氏病病毒814株感染性细菌人工染色体(bacterial artificial chromosome, BAC), 首先通过构建表达Eco-gpt(xanthine-guanine phosphoribosyl transferase, XGPRT, gpt)的哺乳动物细胞基因转移遗传选择标记(1.3 kb)和带有细菌人工染色体的基本功能基因序列的鸡马立克氏病病毒重组病毒转移载体pUAB-gpt-BAC11, 将重组病毒转移载体与鸡马立克氏病病毒细胞总DNA共转染鸡胚成纤维细胞, 在选择培养基中经过8轮加压筛选, 获得并纯化重组病毒; 将重组病毒细胞总DNA电转化大肠杆菌, 筛选共获得38个BAC分子克隆化病毒, 提取BAC-DNA转染鸡胚成纤维细胞以拯救重组病毒。结果表明, MDV-BAC2 DNA再次启动病毒感染, 拯救了重组鸡马立克氏病病毒。成功构建了鸡马立克氏病病毒814株基因组全长感染性细菌人工染色体, 为方便利用现代RED/ET基因重组系统对病毒进行反向遗传操作提供了技术平台; 同时为研究鸡马立克氏病病毒的基因功能和开发新型马立克氏病疫苗奠定了基础。     

表达强毒pp38决定簇的马立克病病毒疫苗毒CVI988点突变株的构建和特性

用鸡马立克病病毒（ＭＤＶ）强毒ＧＡ株的３８ｋＤ磷蛋白（ｐｐ３８）基因克隆ＤＮＡ转染Ｉ型弱毒疫苗ＣＡＩ９８８／Ｒｉｓｐｅｎｓ株ＭＤＶ感染的鸡胚成纤维细胞,再用能识别Ｉ型强毒ｐｐ３８的单克隆抗体Ｈ１９做免疫荧光试验,筛选到能在ｐｐ３８基因上表达强毒株特异性抗原决定簇的定向点突变弱毒株ＣＶＩ／ｒｐｐ３８。用＾３５Ｓ－蛋氨酸标记的细胞裂解物做免疫沉淀反应表明,单抗Ｈ１９不能识别天然ＣＶＩ９８８株ＭＤＶ中的     

Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties.

The gene encoding the gIV glycoprotein of bovine herpesvirus 1 has been inserted into the genome of Autographa californica baculovirus in lieu of the coding region of the A. californica baculovirus polyhedrin gene. Recombinant protein was identified by its reactivity with gIV-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in Spodoptera frugiperda (SF9) cells. The recombinant glycoprotein had an apparent molecular mass of 63 kDa, indicating that it was incompletely glycosylated. However, it was transported to and expressed on the cell surface of infected SF9 cells. Furthermore, reactivity with polyclonal and monoclonal antibodies specific for gIV suggested that most epitopes were functionally unaltered on the recombinant gIV. Immunization of cattle with recombinant gIV in crude, partially purified, or pure form resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gIV. However, the neutralizing antibody titers were lower than those elicited by an equivalent amount of affinity-purified authentic gIV, which appeared to be mainly due to reduced recognition of one of the neutralizing antigenic domains of gIV, designated domain I. The potential use of this recombinant gIV glycoprotein as a vaccine to bovine herpesvirus 1 infection in cattle is discussed.     

Expression of the Marek's disease virus (MDV) homolog of glycoprotein B of herpes simplex virus by a recombinant baculovirus and its identification as the B antigen (gp100, gp60, gp49) of MDV.

A gene encoding a homolog of glycoprotein B of herpes simplex virus (gB homolog) has been identified on the Marek's disease virus (MDV) genome (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989); however, the molecular and immunological characteristics of the gene product(s) are still not clear. In the present study, the gB homolog of MDV was expressed in insect cells by a recombinant baculovirus, and it was characterized to determine its molecular and antigenic properties. The expressed recombinant protein had three molecular sizes (88 to 110, 58, and 49 kDa) and was recognized by antisera from chickens inoculated with each of the three serotypes of MDV. By immunofluorescence analysis, it was shown that the protein was expressed in the cytoplasm and on the surface of the recombinant baculovirus-infected cells. The gB homolog of MDV was processed similarly to pseudorabies virus and varicella-zoster virus with respect to cleavage and the intramolecular disulfide bond between the cleaved products. Interestingly, the expressed protein reacted with monoclonal antibody M51, specific to the B antigen (gp100, gp60, gp49) of MDV, although the locations of the gene encoding the B antigen and of the gene encoding the gB homolog were reported to be different. Moreover, competitive experiments revealed that anti-gB homolog serum and monoclonal antibody M51 recognized the same molecules. From these results, the gB homolog and the B antigen of MDV seem to be the same glycoprotein.     

Purification and characterization of the DNA-binding activity of the Epstein-Barr virus DNA polymerase accessory protein BMRF1 gene products, as expressed in insect cells by using the baculovirus system.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) BMRF1 gene product, the EBV DNA polymerase accessory protein, under the control of the polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced two phosphoproteins of 52 and 50 kDa and one unphosphorylated protein of 48 kDa, recognized by anti-BMRF1 protein-specific monoclonal antibody. The major protein bands were 50 and 48 kDa. The expressed BMRF1 gene products were purified to near homogeneity from the nuclear extract of the recombinant baculovirus-infected insect cells by double-stranded DNA-cellulose column chromatography followed by heparin-agarose column chromatography. The purified BMRF1 gene products exhibited higher binding affinity for double-stranded DNA than for single-stranded DNA without ATP hydrolysis. The protein-DNA interaction did not necessarily require a primer terminus. The present system will open the way for the biochemical characterization of the EBV DNA polymerase accessory protein.     

Topoisomerase activity is associated with purified SV40 T antigen.

Purified SV40 T antigen has been assayed for topoisomerase activity. The ability to relax negatively-supercoiled SV40 DNA was found in preparations of T antigen purified either from human 293 cells infected with Ad5-SVR111 virus or from insect Sf9 cells infected with recombinant baculovirus 941T. The T antigen-associated relaxing activity was stimulated by MgCl2 and was not dependent on ATP, suggesting that it is not due to cellular topoisomerase II. The topoisomerase activity was immunoprecipitated by a monoclonal antibody specific for T antigen, but not by a control monoclonal antibody. In addition, immunoblotting of purified T antigen from human 293 cells with antihuman topoisomerase I and anti-human topoisomerase II antibodies failed to detect cellular topoisomerases I or II. Sedimentation analysis of purified T antigen revealed that the topoisomerase activity co-sedimented with the hexameric form of T antigen at 23S. The topoisomerase activity is, therefore, either inherent to T antigen or due to a cellular topoisomerase I tightly bound to, and co-purifying with, T antigen.     

Production of native creatine kinase B in insect cells using a baculovirus expression vector

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.     

Identification of a unique Marek's disease virus gene which encodes a 38-kilodalton phosphoprotein and is expressed in both lytically infected cells and latently infected lymphoblastoid tumor cells.

The identification of unique Marek's disease (MD) virus (MDV) antigens expressed not only in lytically infected cells but also in latently infected MD lymphoblastoid tumor cell lines is important in understanding the molecular mechanisms of latency and transformation by MDV, an oncogenic lymphotropic herpesvirus of chickens. Through cDNA and nucleotide sequence analysis, an open reading frame (designated the pp38 ORF) which encodes a predicted polypeptide of 290 amino acids was identified in BamHI-H. Demonstration that the pp38 ORF spans the junction of the MDV long unique and long internal repeat regions (MDV has an alphaherpesvirus genome structure) precludes the presence of the gene encoding the B-antigen complex (gp100, gp60, and gp49) in the same region of BamHI-H, where it was originally thought to exist. Duplication of the complete pp38 ORF was not observed in BamHI-D, but part of it (encoding 45 amino acids) was found in the long terminal repeat region of the fragment. By use of trpE-pp38 fusion proteins, antisera against pp38 were prepared. By immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a predominant virus-specific 38,000-dalton polypeptide (designated pp38) and a minor 24,000-dalton polypeptide (designated p24) were found. No precursor-product relationship was found between pp38 and p24 by pulse-chase analysis, and only pp38 was detected by Western blot (immunoblot) analysis with antiserum to pp38. pp38 was found to be phosphorylated and present in oncogenic serotype 1-but not nononcogenic serotype 3-infected cells. Expression of the gene encoding pp38 was relatively insensitive to phosphonoacetic acid inhibition, suggesting that pp38 may belong to one of the early classes of herpesvirus proteins. pp38 was also detected in the latently infected MSB-1 lymphoblastoid tumor cell line. The detection of antibody against pp38 in immune chicken sera indicates that pp38 is an immunogen in birds with MD. Most of the properties described here for a protein detected by methods based on finding the ORF first are identical to those of a 38-kDa phosphoprotein reported by others, suggesting that they are the same. Collectively, the data reported here provide (i) more definitive information on the complete ORF of another MDV gene and the protein that it encodes, (ii) clarification of the gene content within a specific region of the MDV genome, and (iii) the molecular means to conduct further studies to determine whether pp38 plays a role in MDV latency and transformation.