马立克氏病病毒广西株G2囊膜糖蛋白gI基因的克隆和表达

根据马立克氏病病毒（MDV）强毒株GA的基因序列，设计并合成了1对引物，以强毒株G2基因组DNA为模板，通过PCR技术，扩增其囊膜糖蛋白gI基因阅读（ORF）中，除去其N－端编码水区165个碱基对（bps)以外的部分，将PCR产物按正确的阅读框架定向克隆到表性载体pGEX-6P-1中谷胱甘肽转移酶（GST）基因的下游，将重组质粒转化入大肠杆菌BL21株，在1.0mmol/LIPTG浓度和30℃的条件下诱导，gI-GST基因融合蛋白获了理想的表达，经聚丙烯酰胺凝胶电泳和Western-blotting试验，验证其表达的融合蛋白产物大小为预期的63000，将表达产物回收后免疫小鼠，所得抗血清可与MDV感染的鸡胚成纤维细胞（CEF）在免疫光试验（FA）中，呈细胞膜阳性染色，试验结果表明，在大肠杆菌中表达的G2株MDVgI基因的融合蛋白至少保留了天然蛋白的部分抗原性。     

Ⅰ型马立克氏病病毒Md11株pp24基因的真核表达

通过PCR方法扩增MDV Md11株的pp24基因,将其克隆到真核表达载体pcDNA3.1/zeo( )中.阳性克隆鉴定后,在脂质体作用下转染CEF细胞,用间接免疫荧光试验检测,结果证明阳性克隆在CEF细胞中表达了pp24蛋白.     

猪囊尾蚴T24基因的克隆与表达

采用RT—PCR方法扩增猪囊尾蚴T24免疫原基因，将扩增产物与pGEM—Teasy载体连接，重组质粒经PCR、酶切鉴定后进行测序；构建T24基因的pGEX-4T-1原核表达载体，经IPTG诱导表达后，进行SDS-PAGE、Western—blotting；用所表达的蛋白免疫小鼠，经ELISA检测血清抗体，验证其免疫原性。结果显示，所克隆的T24基因片段长716bp，含有1个678bp的开放阅读框，其编码226个氨基酸，与已报道的猪囊虫T24基因核苷酸序列同源性为100％；表达的融合蛋白大小为40ku，并能被猪囊虫阳性血清识别；免疫小鼠在免疫1周后即可检测到血清抗体，第30d达到较高水平，表明该融合蛋白具有较好的免疫原性。     

马立克氏病病毒pp38基因启动子和增强子的克隆和序列分析

参考GeneBank发表的马立克氏病病毒(MDV)国际标准强毒株GA的基因序列，设计合成一对引物，分别以RBIB，814，GD2(广东分离株)，J-1-E(北京分离株)，Md11，Md5，CV1988等不同毒株的MDV基因组DNA为模板，通过PCR扩增，获得了预期大小的PCR产物。该产物经pGEM-T-easy克隆后测序，将所得序列进行比较分析。结果发现：不同毒株间pp38基因的启动子和增强子序列间有缺失突变，序列的同源性大于95．9％，其中大多数的突变发生在MDV复制的原点附近。     

长角血蜱保护性抗原基因P27/30的克隆和原核表达

采用RT—PCR技术首次从孤雌生殖长角血蜱四川株克隆到P27／30基因,扩增序列全长670bp,包含完整的开放阅读框,编码201个氨基酸,预测蛋白相对分子质量为23．38ku。同源性分析表明孤雌生殖长角血蜱中国株与日本株P27／30基因同源性高达99．85％。经RT—PCR检测分析,该基因在孤雌生殖长角血蜱的卵、幼蜱、若蜱、饥饿成蜱和饱血成蜱这几个阶段均有表达。将该基因亚克隆后连接到pET32a（＋）原核表达载体,转化BL21（DEa）宿主菌,经IPTG诱导可成功进行表达。表达的目的蛋白大小为24ku左右,与预期大小一致;Western-blot显示兔抗长角血蜱全虫抗体能够识别该重组表达蛋白。     

禽网状内皮组织增生症病毒囊膜糖蛋白gp90的原核表达

根据禽网状内皮组织增生症病毒（REV）SNV株的前病毒基因组cDNA序列，设计并合成1对引物，以SNV株前病毒CDNA全基因组克隆为模板，通过PCR技术，扩增出该病毒囊膜糖蛋白（env)gp90基因部分片段。将PCR产物按正确的阅读框架定向克隆进pGEX-5X-3载体中谷胱甘肽-S-转移酶（GST）的下游，将重组质粒转化进宿主菌BL21中，在1.0mmol/L IPTG(37℃）诱导下，gp90基因部分片段以融合蛋白的形式获得了良好的表达。表达产物经聚丙烯酰胺凝胶电泳鉴定，确定其表达的融合蛋白相对分子质量为64000。将表达产物从凝胶中回收后免疫小鼠，所得的抗血清可与REV野毒株感染的鸡胚成纤维细胞（CEF）在免疫荧光试验中起反应。由此表明，体外表达的SNV株gp90-GST融合蛋白保留了天然蛋白所具有的抗原性。     

猪传染性胃肠炎病毒核衣壳(N)蛋白基因的克隆与表达

以猪传染性胃肠炎病毒亚基因组mRNA为模板根据文献设计一对引物，通过RT－PCR技术，扩增其核衣壳（N）蛋白基因的cDNA；将其按正确的阅读框架定向克隆到表达载体pProEXHTb中特异酶切位点；将重组质粒PHN转化进大肠杆菌TG1株，在浓度为1.0mM IPTG和37℃条件下诱导，PHN基因融合蛋白获得了表达；经SDS－PAGE，Western-blot试验，确定其表达的融合蛋白产物大小为预期的47kD。试验结果证明，在大肠杆菌中表达的TGEV N基因的融合蛋白产物的确具有天然蛋白的抗原性。     

流产布鲁氏菌omp25基因的克隆与原核表达

用设计的1对特异性引物对流产布鲁氏菌2型CVCC70502株总DNA进行外膜蛋白基因omp25的PCR扩增，得到了1条完整的基因，大小为642bp；测序分析证明，它与国外报道的流产布鲁氏菌omp25基因的核苷酸序列完全一致。将该基因克隆到原核表达载体pGEX-6p-1中，经酶切、PCR扩增和测序分析，表明重组表达载体构建成功。将此重组质粒转化至宿主菌BL21（DE3）中，用IPTG进行诱导。结果证实，该基因可以在大肠杆菌中表达，表达产物为分子质量约50ku的融合蛋白，与理论推测的蛋白分子质量一致；Western—blotting试验证明，表达蛋白OMP25可被流产布鲁氏菌阳性血清所识别。     

鸭疫里默氏菌OmpA与鸭IL-2融合基因的克隆与原核表达

以鸭疫里默氏菌吉林分离株JL-RA1OmpA基因(登录号HQ707077)和鸭DuIL-2成熟片段基因(登录号AY193713)为模板,分别设计带有linker的特异性引物,PCR扩增出序列大小为1182bp的OmpA-linker基因和序列大小为381bp的linkerDuIL-2成熟蛋白基因。利用SOE-PCR技术,成功构建OmpA-DuIL-2融合基因,片段大小为1551bp。将其转入大肠杆菌表达载体pET-28a中,构建pET-OmpA-DuIL-2融合表达载体,转化至大肠杆菌Rosetta(DE3)中,IPTG诱导表达,经SDS-PAGE分析得到分子量大小约为61 kDa的融合蛋白,经Western-blot分析该融合基因同时具OmpA和DuIL-2的反应原性。     

两株马立克病毒的分离鉴定及其主要致病基因分析

为了解国内鸡群马立克病毒(MDV)流行毒株的分子特征,采用细胞培养法对马立克病疑似病鸡抗凝血进行MDV分离,间接荧光法鉴定MDV血清型,PCR扩增分离病毒的meq和pp38两个主要致病基因,所得序列与MDV参考毒株的序列进行比对分析。结果显示,共分离鉴定出2株MDV I型毒株,分别命名为JS0316和JS0424。meq基因编码的氨基酸序列结果显示,2个MDV分离株缺乏弱毒疫苗株的特征(A71S、P194-),具有MDV强毒分离株的分子特征(D80Y、V115A、T139A、P176R和P217A)。同时,JS0316 Meq蛋白出现Q93R和V123A突变,pp38蛋白出现L98I和F240S突变,而JS0424 Meq蛋白出现P217T突变,pp38蛋白出现W67G和V210A突变。因此,2株MDV均具有国内流行强毒株的分子特征,同时存在新的基因突变。     

马立克氏病病毒６４８株囊膜糖蛋白gI基因的克隆和表达的研究

根据马立克氏病病毒（ＭＤＶ）强毒株ＧＡ的基因序列，设计和合成一对引物，以特超强毒６４８株基因组ＤＮＡ为模板，通过ＰＣＲ技术，扩增其囊膜糖蛋白gI基因阅读框（ＯＲＦ）中，除去其Ｎ－端编码疏水区的１６５个碱基对（bp)以外的蓁部分；将ＰＣＲ产物按正确的阅读框架定向克隆到表性载体pGEX-6P-1中谷胱甘肽转移酶（ＧＳＴ）基因的下游，将重组质粒转化进大肠杆菌ＢＬ２１株，在１．０mMIPTG浓度和３０℃的条件下诱导，gI-GST基因融合蛋白获得了理想的表达；经聚丙烯酰胺凝脉电泳，West-ern-blot试验，通信班下其表达的融合蛋白产物大小为预期的６３ＫＤ。将表达产物回收后免疫小鼠，所得抗血清可与ＭＤＶ感染的鸡胚成纤维细胞（ＣＥＦ）在免疫荧光试验（ＦＡ）中呈细胞膜阳性染色。试验结果表明，在大肠杆菌中表达的６４８株ＭＤＶgI基因的融合蛋白产物保留了天然蛋白的某些抗原性。     

The detection of the meq gene in chicken infected with Marek's disease virus serotype 1

In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.     

Identification and characterization of Marek's disease virus serotype 1 (MDV1) ICP22 gene product: MDV1 ICP22 transactivates the MDV1 ICP27 promoter synergistically with MDV1 ICP4

A previous report [Virus Genes 6 (1992) 365-378] has shown that the US1 gene of Marek's disease virus serotype 1 (MDV1) encodes a homologue of herpes simplex virus type 1 infected cell protein No. 22 (ICP22). In the present study, we expressed and identified a product of the MDV1 US1 gene in chicken embryo fibroblasts (CEFs) with the aid of a recombinant baculovirus expressing a Flag epitope-tagged MDV1 US1 gene, under control of the SRalpha promoter (composed of the enhancer region of the simian virus 40 early promoter and the R region of the human T-cell leukaemia virus type 1 long terminal repeat). In CEF infected with the recombinant baculovirus, MDV1 ICP22 was specifically and efficiently expressed in the presence of n-butyric acid. The apparent M(r) of the expressed protein was 30,000. Reporter gene assays revealed that MDV1 ICP22 by itself transactivated an MDV1 ICP27 promoter/reporter construct weakly but specifically, and furthermore, worked synergistically with MDV1 ICP4 to efficiently up-regulate the MDV1 ICP27 promoter. MDV1 ICP22 may be a regulatory protein that stimulates viral promoters in co-operation with other viral regulatory proteins such as MDV1 ICP4.     

表达鸡新城疫病毒F蛋白的重组马立克氏病毒的构建及其鉴定

为构建表达鸡新城疫病毒(NDV)融合蛋白(F)的重组马立克氏病毒(MDV),本研究采用RT-PCR方法从NDV强毒株F48E9基因组中扩增出病毒的融合蛋白F基因,构建由CMV启动子和BGH polyA组成的2.7 kb F基因表达盒。将其插入带有黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因(gpt)和MDV US2同源臂的中间转移载体pUAB-gpt中获得重组MDV转移载体pUAB-gpt-wF。将该转移载体与MDV-814疫苗株感染的鸡胚成纤维细胞(CEF)总DNA共转染CEF,经同源重组及gpt选择系统筛选,获得带有F基因表达盒的重组MDV(rMDV814-wF)。其体外增殖与亲本病毒没有差异。经间接免疫荧光试验、PCR、Southern-blot及western blot等试验证明,重组病毒在CEF中传至13代以上仍稳定表达NDV的F蛋白。该重组病毒的构建为MDV活载体疫苗的筛选及应用奠定了基础。     

Comparative analysis of Marek's disease virus (MDV) glycoprotein-, lytic antigen pp38- and transformation antigen Meq-encoding genes: association of meq mutations with MDVs of high virulence

Marek's disease (MD) is a highly contagious lymphoproliferative and demyelinating disorder of chickens. MD is caused by Marek's disease virus (MDV), a cell-associated, acute-transforming alphaherpesvirus. For three decades, losses to the poultry industry due to MD have been greatly limited through the use of live vaccines. MDV vaccine strains are comprised of antigenically related, apathogenic MDVs originally isolated from chickens (MDV-2), turkeys (herpesvirus of turkeys, HVT) or attenuated-oncogenic strains of MDV-1 (CVI-988). Since the inception of high-density poultry production and MD vaccination, there have been two discernible increases in the virulence of MDV field strains. Our objectives were to determine if common mutations in the major glycoprotein genes, a major lytic antigen phosphoprotein 38 (pp38) or a major latency/transformation antigen Meq (Marek's EcoRI-Q-encoded protein) were associated with enhanced MDV virulence. To address this, we cloned and sequenced the major surface glycoprotein genes (gB, gC, gD, gE, gH, gI, and gL) of five MDV strains that were representative of the virulent (v), very virulent (vv) and very virulent plus (vv+) pathotypes of MDV. We found no consistent mutations in these genes that correlated strictly with virulence level. The glycoprotein genes most similar among MDV-1, MDV-2 and HVT (gB and gC, approximately 81 and 75%, respectively) were among the most conserved across pathotype. We found mutations mapping to the putative signal cleavage site in the gL genes in four out of eleven vv+MDVs, but this mutation was also identified in one vvMDV (643P) indicating that it did not correlate with enhanced virulence. In further analysis of an additional 12 MDV strains, we found no gross polymorphism in any of the glycoprotein genes. Likewise, by PCR and RFLP analysis, we found no polymorphism at the locus encoding the pp38 gene, an early lytic-phase gene associated with MDV replication. In contrast, we found distinct mutations in the latency and transformation-associated Marek's EcoRI-Q-encoded protein, Meq. In examination of the DNA and deduced amino acid sequence of meq genes from 26 MDV strains (9 m/vMDV, 5 vvMDV and 12 vv+MDVs), we found distinct polymorphism and point mutations that appeared to correlate with virulence. Although a complex trait like MDV virulence is likely to be multigenic, these data describe the first sets of mutations that appear to correlate with MDV virulence. Our conclusion is that since Meq is expressed primarily in the latent/transforming phase of MDV infection, and is not encoded by MDV-2 or HVT vaccine viruses, the evolution of MDV virulence may be due to selection on MDV-host cell interactions during latency and may not be mediated by the immune selection against virus lytic antigens such as the surface glycoproteins.     

应用双重实时荧光定量PCR方法检测鸡马立克氏病血清1型病毒

本研究建立了鸡马立克氏病血清1型病毒（MDV1）绝对定量检测方法。研究中选择MDV1特有的Meq基因的一段保守序列作为检测对象，将其克隆到质粒载体中，作为阳性标准品；同时将管家基因．鸡卵铁转蛋白（Ovo）特异性基因片段克隆到质粒载体上作为内参照的标准品。经荧光定量PCR（FQ-PCR）法扩增获得MDV1的FQ-PCR两条标准曲线，建立了MDV1双重FQ-PCR检测方法。应用该方法绝对定量检测了实验攻毒鸡及吉林省某地发病鸡只的羽髓、淋巴细胞等组织样本中单位细胞病毒拷贝数，并与琼脂扩散（AGP）、常规PCR等检测方法进行比较。结果表明，不论实验攻毒鸡还是自然发病鸡，羽髓中病毒富含量均高于其它组织，每百万宿主细胞内病毒含量为10^7～10^8拷贝；FQ-PCR检测MD发病鸡只的阳性率高于AGP，达100％；该方法的灵敏度比常规PCR检测高10～100倍，在单位细胞内可灵敏地检测到2．78个拷贝的病毒。该方法可以在不同的样品中有效的绝对定量检测MDVl。     

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.     

马立克氏病病毒囊膜糖蛋白B基因原核表达产物单抗的制备

用大肠杆菌BL21表达了马立克氏病病毒（MDV）强毒GA株的囊膜糖蛋白B（gB)基因，通过SDS－聚丙烯酰胺凝胶龟泳（SDS－PAGE）分离表达蛋白条带，切下并碾碎后作为免疫原制备单克隆抗体。通过酶联免疫吸附试验（ELISA）和间接免疫荧光试验（IFA），得到1株GA株阳性的单克隆抗体杂交瘤细胞株7C8。该单抗腹水的ELISA效价为1：2^12，IFA效价为1：800，与MDV不同致病型毒株CVI988、GA、RB1B、MD11、648A株感染的鸡胚成纤维细胞（CEF）均呈IFA阳性。1：100稀释时与GA感染的CEF在斑点酶联免疫吸附试验（dot-ELISA)中呈阳性。