Production and characterization of monoclonal antibodies that recognize bovine Kit receptor.

Kit receptor is a transmembrane tyrosine kinase that is the receptor for stem cell factor (SCF). The extracellular domain of bovine Kit receptor (boKit) was produced by a baculovirus expression system. Six monoclonal antibody (MAb) clones designated as bK-1 to bK-6 were obtained upon immunization of mice with the recombinant protein. Immunoprecipitation and flow cytometric analysis indicated that all of the MAbs specifically bound to boKit expressed in COS-7 cells transfected with boKit cDNA. Four of the six MAbs neutralized the biological activity of recombinant bovine SCF, whereas the other two did not. The boKit-positive and boKit-negative cell fractions were sorted from cryopreserved bovine bone marrow cells by the use of MAb bK-1. Colony formation assays indicated that the cells which were able to grow in response to bovine SCF were enriched in the boKit-positive fraction. These MAbs would be valuable in studying possible boKit-positive cell species such as bovine hematopoietic cells, and in defining the biological role of Kit receptor in cattle.     

Characterization of monoclonal antibodies directed against the canine distemper virus nucleocapsid protein

We have established four monoclonal antibodies (MAbs) against the nucleocapsid protein (NP) of canine distemper virus (CDV). A competitive binding assay has revealed that the MAbs are directed against two antigenic domains. An immunofluorescence assay using a series of deletion clones of the NP and an immunoprecipitation assay using the NP have revealed that two of the MAbs recognize the C-terminal region of the NP while the other two recognize the tertiary structure of the N-terminal domain. These MAbs reacted with all eight strains of CDV used in this study, but showed different reactivities against measles virus and rinderpest virus.     

Isolation and characterization of monoclonal antibodies against structural proteins of infectious laryngotracheitis virus

Thirteen infectious laryngotracheitis virus (ILTV)-specific monoclonal antibodies (MAbs) were isolated after immunization of mice with purified infectious laryngotracheitis virions. On the basis of their reactions in western blot analyses of ILTV-infected cells, the MAbs were assigned to five different virus proteins or protein groups. Two of the viral target proteins could be identified after transient expression of cloned ILTV genes in eucaryotic cells. The MAbs of group II detected a 60-kD protein that was shown to be the ILTV homologue of herpes simplex virus type 1 (HSV-1) glycoprotein (g)C. The MAbs of group I reacted with the positional homologue of HSV-1 gJ, which is encoded by the open reading frame (ORF) 5 gene within the unique short genome region of ILTV. The ORF 5 gene product of ILTV was previously described as a 60-kD glycoprotein (gp60), whereas multiple protein bands with apparent molecular masses of 85, 115, 160, and 200 kD were identified in the present study. Immunoelectron microscopy revealed that both gC and gJ of ILTV are localized in the envelope of virus particles, whereas the 15-kD protein detected by the MAbs of group III presumably represents a tegument component. Immunofluorescence analyses of infected cells demonstrated that the epitopes of the gC- and gJ-specific MAbs are conserved in all tested ILTV isolates originating from different parts of the world and that these MAbs are also suitable for in situ antigen detection in tissues of ILTV-infected chickens. The remaining ILTV-specific MAbs recognized viral proteins of 22 kD (group IV) and 38 kD (group V) that were not further characterized up to now.     

一株鸡源H6N1亚型禽流感病毒全基因的分子特征

2008年国家禽流感参考实验室在我国禽流感流行病学调查期间分离到1株鸡源H6N1亚型禽流感病毒(AIV)A/Chicken/ZheJiang/80/2008(H6N1)(简称为CK/ZJ/80/08),为了弄清该病毒的分子特征,我们对其8个基因片段分别进行扩增和序列测定,对每个基因进行BLAST分析,找出同源性最高的毒株。利用DNAStar中的Megalign功能进行进化分析。结果表明CK/ZJ/80/08的HA裂解位点附近的氨基酸序列为QIETR↓GLF,推测可能为一株低致病力AIV。其HA基因与日本北海道的A/duck/Hokkaido/228/2003(H6N8)和黑龙江的A/mallard/Heilongjiang/131/2006(H6N2)以及香港早期分离株A/chicken/HongKong/17/77(H6N1)等处于同一分支;NA基因在颈部没有缺失,与A/duck/Tsukuba/718/2005(H1N1)、A/goose/Guangdong/1/96(H5N1)等处于同一分支;M基因与A/duck/Hokkaido/W90/2007(H10N7)高度同源(同源性为99%);NS基因与A/duck/Denmark/65047/04(H5N2)和A/goose/Guangdong/1/96(H5N1)处于同一分支。NP、PA、PB1、PB2分别与贵州和江西分离的H5N2亚型AIV的相应基因关系密切,同源性分别为98%、97%、97%、97%。由此推测CK/ZJ/80/08可能是由H6N2、H1N1、H10N7、H5N2等多个亚型病毒重组而成。     

Isolation of monoclonal antibodies that inhibit the binding of infectious bursal disease virus to LSCC-BK3 cells

Three hybridoma cell lines producing monoclonal antibodies (MAbs) against LSCC-BK3 cells which are susceptible to infectious bursal disease virus (IBDV) infection were produced and characterized. The MAbs, designated T7, Q11 and Q13, inhibited the attachment of IBDV to LSCC-BK3 cells. Furthermore, these MAbs bound to LSCC-BK3 but not to nonpermissive cells in flow cytometry. MAb T7 detected a 110-kDa membrane protein of LSCC-BK3 cells, whereas Q11 and Q13 reacted with membrane proteins of molecular weights 58-, 85-, 90- and 110-kDa. These observations imply that the 110-kDa protein recognized by all the MAbs is associated with IBDV binding. The MAbs established in this study are useful for studying the interaction between IBDV and its target cell.     

Monoclonal antibodies to Quebec strain (Q17) of bovine rotavirus.

Fourteen monoclonal antibodies (MAbs) against "Quebec" strain (Q17) of bovine rotavirus were isolated and characterized. Four were specific for viral protein Vp7 and ten were specific for viral protein Vp6. Five different isotypes were represented by this group of antibodies. All of the anti-Vp6 and none of the anti-Vp7 antibodies were sensitive to the effects of periodate on their antigen. The antibodies could be separated into three groups based on their relative resistance to the dissociation of their antigen-antibody complex by thiocyanate. The MAbs cross-reacted with the proteins of porcine and human rotaviruses both by immunoprecipitation and immunoblot analyses. These techniques revealed the differences in Mw of the viral proteins from different serotypes.     

Class Ⅰ新城疫病毒核衣壳蛋白在体外细胞感染过程中表达定位的检测

为分析核衣壳蛋白(NP)在新城疫病毒(NOV)感染真核细胞过程中细胞内定位的情况,本研究以NDV Class I强毒株9a5b作为免疫原制备4株NP特异性单克隆抗体(MAb),并将病毒株9a5b按5 MOI感染量接种DF1单层细胞,当接种后1 h～3 h时,NP有极少量表达并逐量增加,主要呈点状分布于细胞浆内;当接种后4 h～20 h时,NP聚集于细胞浆中,聚集数量与感染时间成正比;感染24 h后,细胞核碎裂,NP散在分布于细胞浆中.表明NP作为立早期蛋白,始终在胞浆中进行复制;在病毒感染初期,NP介导核糖核蛋白复合物发挥转录和翻译功能.本研究为深入开展NP与病毒mRNA转录、复制和包装的相互关系机制的研究奠定了基础.     

Development and Characterization of Monoclonal Antibodies against the Snakehead Rhabdovirus

Abstract

Monoclonal antibodies (MAbs) directed against the snakehead rhabdovirus (SHRV) were produced. These MAbs were characterized by immunofluorescence and neutralization tests, and by their ability to immunoprecipitate viral proteins. Of 15 MAbs developed, 9 were isotyped as IgG1 and 6 were IgG2a. Eight of the MAbs recognized the viral glycoprotein in an immuneprecipitation assay. Three of these, designated E1-9A, P10C, and O10F, had neutralizing activity. By immunofluorescence, 12 MAbs showed good binding activity in SHRV-infected epithelioma papulosum cyprini cells. In an indirect fluorescence assay, the MAbs gave varied staining patterns depending upon the viral structural proteins recognized.     

Production and characterisation of monoclonal antibodies specific for chicken interleukin-2.

Using genetic immunisation of mice, we produced antibodies against chicken interleukin-2 (ChIL-2), the first produced against a non-mammalian interleukin. After a final injection with a recombinant ChIL-2 protein, two stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of the two MAbs to recombinant ChIL-2 produced by Escherichia coli and COS-7 cells was demonstrated in an indirect ELISA, Western blotting and dot blots. Both of them were able to neutralise the biological activity of the ChIL-2, but neither allowed the detection of ChIL-2 by flow cytometry.     

鸡传染性支气管炎病毒分离株核衣壳基因克隆、序列分析和真核表达

根据GenBank公开序列自行设计一对引物，采用RT—PCR扩增出鸡传染性支气管炎病毒（Infectious bronchitis virus，IBV）W和C9001分离株完整的核衣壳（N）基因，并将其克隆至pMD18-T载体进行核苷酸序列测定和分析。结果表明，扩增的2个IBV分离株核衣壳基因片段长度均为1230bp，编码409个氨基酸，彼此间核苷酸和氨基酸同源性分别为88．0％和89．5％，与GenBank中有代表性的参考毒株相应基因核苷酸和氨基酸序列比较显示，w株核苷酸序列与GenBank中的广东分离株（AY646283）同源性最高，为94．1％，氨基酸序列同源性为94．6％；与国内部分毒株核苷酸序列同源性在86．1％～88．0％之间，氨基酸序列同源性在88．0％～90．7％之间；C9001株与国内部分毒株核苷酸序列同源性在86．4％～99．8％之间，氨基酸序列同源性在88．0％～99．8％之间。从核衣壳基因编码的氨基酸序列的系统进化树可见，W株与C9001株处于不同的进化分枝，亲缘关系较远。同时将核衣壳基因构建于真核表达质粒pVAX1中，用脂质体法将重组质粒转染入COS-7细胞中，间接免疫荧光检测出核衣壳蛋白的体外表达。研究结果为进一步研究IBV核衣壳蛋白的结构与功能以及基因工程疫苗的研制奠定了基础。     

The complete nucleotide sequences of L3 and S7 segments of Ibaraki virus encoding for the major inner capsid proteins, VP3 and VP7

The complete nucleotide sequences of the genes encoding two of the major inner capsid proteins of Ibaraki virus (IBAV), belonging to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) were determined. The L3 RNA segment is 2768 nucleotides in length which encodes VP3 polypeptides of 899 amino acid residues (M.W. 103 kDa). The S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and encodes 349 amino acids (M.W. 38 kDa). These RNA segments had the characteristic consensus motifs of Orbivirus RNA segments in termini, namely 5'-GUUAAA... and ...ACUUAC-3'. The comparison of the IBAV L3 and S7 sequences with those of other two EHDV-2 isolates revealed the higher homologies of 93% and 92% against EHDV-2 Australia isolate (EHDV-2AUS) and lower homologies of 80% and 81% against EHDV-2 North America isolate, respectively. The phylogenetic analysis based on L3 and S7 genes also indicated close relationships between IBAV and EHDV-2AUS. KEY WORDS: dsRNA gene, lbaraki virus, inner capsid, VP3, VP7.     

抗蓝舌病病毒VP6和VP7蛋白单克隆抗体的制备及鉴定

为制备针对蓝舌病病毒(BTV)的单克隆抗体(MAb),本研究利用血清型1型BTV(BTV1)免疫BALB/c鼠,将其脾淋巴细胞与SP2/0进行融合,并用BTV1包被ELISA板,通过间接ELISA方法筛选出3株稳定分泌抗BTV1的MAb的杂交瘤细胞株(2B10、3D4和4H8)。利用表达BTV1主要蛋白的真核表达重组质粒转染BHK-21后,对所制备的杂交瘤细胞株上清进行间接免疫荧光(IFA)以及western blot鉴定,结果显示:2B10和4H8与VP7蛋白反应,而3D4与VP6蛋白反应。同时,IFA鉴定结果进一步表明,3株MAb与24个血清型的BTV均可以发生反应。本研究制备的MAb为建立BTV免疫学检测方法和相关病毒蛋白的功能研究奠定了基础。     

家蚕表皮蛋白基因的生物信息学分析

家蚕的表皮蛋白(cuticular protein)是家蚕重要的结构蛋白,与几丁质一同作为家蚕表皮的重要组成部分。运用生物信息学的方法对家蚕表皮蛋白进行了广泛分析。通过保守的基序检索家蚕基因组,得到163条候选的表皮蛋白序列,包括RR、Tweedle、CPF&CPFL等家族,并且发现具有确定的保守基序的表皮蛋白基因在染色体上都是以串联集群形式分布。氨基酸组成分析表明,这些表皮蛋白富含甘氨酸、丙氨酸、亮氨酸、组氨酸等。运用Sig-nalP server预测这部分蛋白质有85%都具有信号肽。进化分析显示,负选择是家蚕表皮蛋白基因进化的主要驱动力。采用TFSEARCH等在线服务软件分析发现,在70%含有RR基序的家蚕表皮蛋白基因的核心启动子区具有通用的TATAAA序列。     

Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains six structural proteins the roles of which are not completely understood. In a preceding study, immunization with the dutch isolate I10 of PRRSV had led to the development of MAbs against four structural proteins [Wieczorek-Krohmer, M., 1994. Herstellung und Charakterisierung von monoklonalen Antik?rpern gegen das Virus des Porzinen Reproduktiven und Respiratorischen Syndroms (PRRSV). Inaugural-Dissertation, Ludwig-Maximilians-Universit?t, München] here finally identified by reaction with individual plasmid-expressed PRRSV proteins as products of ORFs 3 (GP3), 4 (GP4), 5 (GP5) and 7 (N). Surprisingly, the MAbs against GP5 revealed the presence of two antigenically distinct virus populations in the isolate I10, the population PRRSV-'PPV', isolated from plaques and the PRRSV-'EPV', gained by end point dilution. MAbs against GP3, GP4 and N reacted with both I10 populations as well as with natural PRRSV isolates. However, the anti-GP5 MAbs exclusively recognized PRRSV-'PPV'. In this study immunization of mice with both separated I10 populations confirmed that solely PRRSV-'PPV' possesses the property to induce an immune response ultimately leading to the establishment of MAbs against GP5. Whereas the 15 anti-GP5 MAbs (derived from four independent fusions) reacted exclusively with PRRSV-'PPV' of the isolate I10, anti-GP4 MAbs detected their target antigen on various isolates of European origin and were able to neutralize them. As indicated by competition assays and selection of neutralization-resistant virus mutants, all GP5 MAbs are directed against a single antigenic site on the ORF 5 protein. Both groups of neutralizing antibodies bound to the surface of purified virions demonstrating that the recognized epitopes represent surface structures of the virion envelope. However, anti-GP5 MAbs mediated the binding of more gold granules than anti-GP4 MAbs. Comparison of the neutralizing effect of anti-GP4 and anti-GP5 MAbs revealed the anti-GP5 MAbs as the more efficient antibodies. For the complete neutralization of about 100 ID50 of PRRSV-'PPV' anti-GP5 culture supernatant was effective up to a dilution of 1:1280 whereas the most effective anti-GP4 antibodies exhibited a comparable effect only up to 1:64. These results indicate that PRRSV GP5 in principle is a major target for neutralizing antibodies, as is found for other arteriviruses, but that in nature 'ORF 5 escape mutants' may develop as easily as in vitro.     

Improvement of a rapid diagnosis kit to detect either influenza A or B virus infections

To improve the sensitivity of a kit, ESPLINE INFLUENZA A&B for rapid diagnosis of influenza by detecting influenza A or B virus specific nucleoproteins (NP), the ESPLINE INFLUENZA A&B-N was developed by using newly established monoclonal antibodies (MAbs) to the respective NP molecule. MAbs FVA2-11 and FrB1-03 recognize the epitope on the amino acid region 59-130aa of the NP molecule of influenza A virus, and that on the region 72-191aa of the NP of influenza B virus, respectively. The new kit detected influenza A and B virus antigens with a detection limit of 10(2.0)-10 (2.7) pfu/test, which is 4-1000 times higher than that of the original kit. Importantly, this kit detected each of influenza A viruses of the known hemagglutinin (HA) subtypes (H1-H15) including the H5N1 viruses recently isolated from human and avian sources in Asia. The kit also detected all of the 15 representative influenza B virus strains tested. The ESPLINE INFLUENZA A&B-N is thus a rapid and highly sensitive and specific kit for the diagnosis of either influenza A or B virus infections.     

表达虎源H_5N_1亚型禽流感病毒NP蛋白重组犬2型腺病毒的构建及鉴定

将A/Tiger/Harbin/132/2007(H5N1)的NP基因克隆入pVAX1载体中,然后将含有NP基因的表达盒(CMV+NP+PolyA)克隆入pVAXE3的SspⅠ酶切缺失处,获得含有NP基因表达盒的穿梭载体pVAXΔE3-NP。用SalⅠ+NruⅠ分别对pVAXΔE3-NP和pPoly-2-CAV-2进行双酶切,将含有NP基因表达盒的片段定向克隆入pPoly-2-CAV-2,获得在E3区缺失处插入NP基因表达盒的重组质粒pCAV-2-NP。释放CAV-2-NP重组基因组,脂质体介导转染DK细胞,获得了能使DK细胞产生典型腺病毒样细胞病变的CAV-2-NP重组病毒。从形态学、基因组水平、NP基因的转录、NP蛋白的表达以及重组病毒的生长特征等方面进行鉴定。结果表明,CAV-2-NP具有典型的CAV-2形态特征,在繁殖过程中没有对H5N1NP表达盒片段进行缺失或重排,并且能够转录H5N1NP基因的mRNA,ELISA和免疫荧光分析表明重组表达产物可被流感病毒NP基因单克隆抗体1G6G4所识别。     

猫importin基因生物信息分析及其在犬细小病毒感染F81细胞表达的动态变化

试验旨在系统研究猫源不同亚型importin（importin α1、importin α3、importin α4、importin α5、importin α6、importin α7、importin α8和importin β）基因序列和蛋白基本特性,探讨其在犬细小病毒（canine parvovirus,CPV）感染F81细胞表达的动态变化。本研究利用RT-PCR方法扩增F81细胞中improtin基因全长序列,应用生物学软件预测其氨基酸序列及编码蛋白的结构及功能;进一步利用实时荧光定量RT-PCR技术检测importin基因在CPV感染F81细胞后24、48和72 h表达水平。结果显示,不同亚型importin α基因全长约在1 500 bp,而importin β基因全长为2 600 bp;预测importin亚型蛋白序列发现,其等电点均在4.60左右,且发现importin α1、importin α5、importin α6、importin α7和importin β为不稳定蛋白;分析发现这些importin蛋白均无信号肽、无跨膜区、无细胞定位;采用Predictprotein软件预测蛋白二级结构,结果显示importin亚型蛋白序列主要以α-螺旋和无规则卷曲形式存在。实时荧光定量RT-PCR结果显示,CPV感染F81细胞后随着感染时间的延长importin α1（P<0.01）和importin β表达量逐渐降低,而importin α3、importin α4、importin α5、importin α6（P<0.01）、importin α7（P<0.01）和importin α8（P<0.01）表达量逐渐提高,其中importin α8在病毒感染细胞后表达量最高。本研究结果为进一步分析CPV进入细胞核的转运机制及开发治疗药物相关研究奠定基础。     

Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and gastro and extra-intestinal infections in humans. Monoclonal antibodies (MAbs) were produced against outer membrane proteins (OMPs) of E. tarda ET-7, isolated from diseased snakehead (Ophiocephalus punctatus). Two stable hybridoma clones, designated as 3F10 and 2C3 MAbs were found to be potentially specific for E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These MAbs recognized major immunogenic OMP band at 44kDa in Western blotting. Both MAbs belonged to the IgG1 isotype and recognized different epitopes of OMP as seen by competitive ELISA. These MAbs strongly reacted with all 17 isolates of E. tarda used in our study by indirect ELISA and Western blotting. Interestingly, no reaction was observed with the reference strain of E. tarda (MTCC 2400). The sensitivity of 3F10 MAb to detect whole cells of E. tarda was up to a level of 1x10(4)CFU/ml in indirect ELISA. No cross-reactivity of MAbs were seen with Escherichia coli, Salmonella arizonae, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae, Flavobacterium ferrugineum and Mycobacterium tuberculosis. These MAbs could be used for specific detection of E. tarda infection in fish by immunoassays.