禽白血病病毒p27基因在原核细胞的表达及生物学特性的初步分析

将禽白血病病毒(ALV)的p27基因克隆人表达性载体pET28a,在大肠杆菌以His Tag融合蛋白的形式获得了高效表达。以表达产物免疫家兔,制备了抗ALV p27的多克隆抗体,经亲和纯化后,用此抗体建立了对ALV抗原的双抗体夹心法ELISA,并对疑似病料进行了实验室诊断。检测结果与IDEXX的禽白血病抗原检测试剂盒符合率达到98．6％,证明表达产物保留了天然p27蛋白的相关抗原性。交叉试验和群特异性试验证明,此方法具有良好的特异件,并且可以检测出A、B、J亚群的禽白血病病毒p27抗原。用此ALV抗体和所建立的ELISA方法成功地进行了禽白血病病毒抗原的实验室诊断。     

用单因子血清识别急性致瘤性ALV诱发肿瘤组织中的fps肿瘤抗原

[目的]制备鼠抗fps单因子血清,检测急性致瘤性ALV诱发肉瘤组织中的fps肿瘤抗原.[方法]以J亚群相关禽白血病/肉瘤病毒Fu-J株RNA为模板,分两段扩增v-fps肿瘤基因,利用大肠杆菌表达系统表达这两段蛋白,纯化后将两种蛋白同时免疫小鼠,获得抗血清,间接免疫荧光试验检测肉瘤组织滤过液感染的CEF和肿瘤细胞中的fps抗原,免疫组织化学方法检测肿瘤组织中的fps抗原.[结果]该单因子血清具有较好特异性,不与经典ALV-J发生交叉反应.被感染的CEF细胞、肿瘤细胞及纤维肉瘤组织切片均显示阳性.[结论]该实验制备了鼠抗fps单因子血清,建立了fps肿瘤抗原的检测方法,为进一步研究该病毒的生物学特性及其致瘤机制奠定了基础.     

禽白血病病毒J亚群内蒙株的分离与鉴定

本研究从曾见典型禽骨髓性白血病(Myeloid Leukosis,ML)病例的内蒙古某肉种鸡场随机选取的淘汰肉种鸡中,分离出一株J亚群禽白血病病毒(Avian leukosis virus Subgroup J.ALV-J)。利用PCR和间接免疫荧光反应进行鉴定,J亚群禽白血病病毒内蒙株可以被两对ALV—J特异性引物扩增(特异条带约2．2kb和545bp);且在特异性单抗的间接免疫荧光检测中呈现强阳性荧光反应。此外,对山东一例肉种鸡骨髓性白血病病例亦进行了J亚群禽白血病病毒的分离与鉴定。2．2kb PCR扩增物的测序结果表明,两株分离病毒的同源性为96．9％,与已分离的SD9901和YZ9901株ALV-J同源性达94．2％～95.4％。     

ELISA与IFA检测鸡血清ALV-A/B特异性抗体相关性比较

为了研究ELISA和IFA检测鸡血清中ALV-A/B特异性抗体的相关性,将A/B亚群禽白血病病毒接种到DF1细胞上,用ELISA检测过的鸡血清作为一抗进行IFA检测,比较ELISA检测的S/P值与IFA检测的血清效价之间的相关性。结果表明ELISA检测鸡血清中ALV-A/B抗体的S/P值与IFA检测ALV-A/B的血清效价之间存在显著正相关(r=0.974 35;P﹤0.001)。由此可推测ELISA检测鸡血清的S/P值与IFA检测的血清效价之间存在显著相关性,且呈正相关。IFA检测血清阳性率与ELISA检测结果完全一致,因此当血清样品数量少时,可用IFA检测替代ELISA检测,既节约检测成本也提高了检测的准确性。     

J亚群鸡白血病病毒AH09/2株的gp85基因的克隆与原核表达

利用PCR方法扩增出J亚群禽白血病病毒(ALV-J)AH09/2株的gp85基因全长930 bp DNA片段。经T载体克隆测序并连接到pGEX-6p-1载体上,构建了重组表达质粒pGEX-6P-1-gp85,在IPTG的诱导下进行表达。Western-blot结果分析表明,gp85融合蛋白表达产物分子量大小约61 kDa,并能与ALV-Jenv基因单抗发生特异性反应。这些结果为深入研究GP85蛋白的生物学功能及研制ALV-J检测ELISA试剂盒奠定了基础。     

禽白血病病毒J亚群内蒙株的分离与鉴定

本研究从曾见典型禽骨髓性白血病(Myeloid Leukosis, ML)病例的内蒙古某肉种鸡场随机选取的淘汰肉种鸡中,分离出一株J亚群禽白血病病毒(Avian leukosis virus Subgroup J, ALV-J).利用PCR和间接免疫荧光反应进行鉴定,J亚群禽白血病病毒内蒙株可以被两对ALV-J特异性引物扩增(特异条带约2.2kb和545bp);且在特异性单抗的间接免疫荧光检测中呈现强阳性荧光反应.此外,对山东一例肉种鸡骨髓性白血病病例亦进行了J亚群禽白血病病毒的分离与鉴定.2.2kb PCR扩增物的测序结果表明,两株分离病毒的同源性为96.9%,与已分离的SD9901和YZ9901株ALV-J同源性达94.2%～95.4%.     

禽白血病病毒J亚群自然感染蛋鸡病毒负载、受体表达及致瘤谱关系

J亚群禽白血病病毒(Subgroup J avian leukosis virus,ALV-J)通过与宿主细胞膜上的受体chNHE1蛋白结合感染细胞并引起宿主发病,进而引起肿瘤。本研究对确诊为ALV-J感染的肿瘤携带鸡进行病理组织学观察和病毒分离,应用荧光定量PCR技术检测ALV-J自然感染蛋鸡体内ALV-J负载量与其受体chNHE1蛋白mRNA表达量,分析ALV-J负载量、受体表达量及致瘤谱之间的相关性。结果显示:ALV-J在蛋鸡及地方品种鸡体内所诱发的肿瘤呈现多样化,但是病毒负载量与肿瘤谱相关性不明显;ALV-J负载量与chNHE1蛋白mRNA表达量呈正相关;组织在发生肿瘤时chNHE1蛋白mRNA表达量升高,可见,chNHE1蛋白不仅是ALV-J感染宿主细胞的受体,而且在肿瘤形成过程中也发挥重要作用。本研究为ALV-J致瘤机制的深入研究提供了科学基础。     

猪流行性腹泻病毒流行株S蛋白高变区B细胞线性表位肽的鉴定

【背景】对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)经典毒株与流行毒株S蛋白氨基酸序列比对显示,变异位点主要集中在S蛋白的N端。【目的】鉴定PEDV流行毒株S蛋白高变区(S10A,1-496 aa)的B细胞线性表位肽,特别是流行毒株特异性B细胞表位肽。【方法】以SDS-PAGE割胶纯化大肠杆菌表达的PEDV流行株SD2014 S蛋白高变区片段(S10ASD2014),免疫新西兰大白兔制备多克隆抗体;在E. coli中谷胱甘肽巯基转移酶(Glutathione S-transferase,GST)融合表达覆盖S10ASD2014序列全长且彼此重叠8个氨基酸残基的系列16肽。以制备的抗S10ASD2014多抗为一抗,通过Western blot筛选系列16肽中的阳性反应性16肽,鉴定S10ASD2014上的B细胞线性表位肽;利用抗PEDV经典毒株(CV777)S蛋白高变区片段(S10ACV777)多抗血清鉴定S10ASD2014上的保守性B细胞线性表位肽。【结果】重组表达的S10ASD2014相对分子质量约为72 kD;纯化的S10ASD2014表位肽能被PEDV阳性血清识别。制备的抗S10ASD2014多抗可以识别PEDV DR13弱毒株。利用抗S10ASD2014血清和抗S10ACV777血清从61个GST融合表达的16肽中鉴定到28个阳性反应16肽,其中13个为2种血清共同识别16肽。对24株PEDV不同亚群代表毒株的对应区域进行同源性分析表明2个阳性16肽为保守性表位肽。【结论】确定了PEDV流行株SD2014 S蛋白高变区的B细胞线性表位肽,B细胞线性表位肽,特别是流行毒株特异性表位肽的确定有助于了解S蛋白的结构与功能,为建立以表位为基础的PEDV感染检测方法打下良好基础。     

我国地方品种鸡分离到的一个禽白血病病毒新亚群的鉴定

为探明我国地方品种鸡群禽白血病病毒(Avian leukosis virus,ALV)的特点,通过接种DF-1细胞及细胞培养上清液p27抗原的检测,从芦花鸡中分离得到三株外源性ALV禽白血病病毒,分别是JS11C1、JS11C2和JS11C3,并对其进行亚群鉴定分析。用PCR方法扩增env基因测序,并与已知鸡源各亚群ALV的囊膜蛋白(gp85)作氨基酸同源性比较。这三株ALV的env基因的gp85大小为1 005bp,编码335个氨基酸;env基因的gp37大小为609bp,编码203个氨基酸。三个毒株之间gp85的同源性为91.9%～97.0%。与A、B、C、D和E五个经典亚群在GenBank中已发表的18个毒株的gp85的同源性仅在77.7%～84.6%间,显著低于鸡群中常见的A、B、E各亚群内的同源性范围(分别为88.2%～98.5%,91.6%～98.8%和97.9%～99.4%),而与J亚群参考株的同源性更是只有34.2%～36.5%。上述结果表明,芦花鸡分离到的三株病毒可能是不同于鸡源ALV已知6个亚群的一个新亚群,按国际上对ALV亚群分类的习惯,初步将其定名为K亚群。     

禽白血病病毒J亚群囊膜蛋白env基因的克隆和表达

禽白血病病毒J亚群（ALV－J）是90年代鉴定出的ALV的新亚群,其囊膜蛋白env基因序列别与ALV A－E亚群的有相当大的差别。为ALV－J env基因及春表达产物的特点,用PCR方法扩增出ADOL－4817毒株的env基因,并克隆进TA载体,经电泳鉴定大小为1.7kb。将克隆出的env基因与杆状病毒pBlue-Bac4表达质粒DNA连接,构建成转移性载体pBac4817env,通过与Bac-N-Blue杆状病毒DNA共转染,区得了重组病毒rBac4817env-2。该重组杆状病毒感染Sf9细胞,能高效表达env基因产物,免疫荧光分析结果证明,单克隆抗体G2或多价兔抗env gp37血清能识别Sf9细胞,能高效表达env基因表达的特异性抗原;Western blotting分析结果表明,表达的重组基因产物的分子量大小约为90kD-94kD。用这些重组基因产物免疫鸡可以诱导鸡导鸡产生出高滴度的抗ALV－J特异性抗体。这一结果提示,这种杆状病毒表达的重组基因产物有助于ALV－J env基因生物学特性的深入研究。     

二株B亚群禽白血病病毒全基因组序列及其在细胞上的复制性比较

本研究比较了从山东地方品系鸡群分离到的二株B亚型禽白血病病毒(ALV)SDAU09E3和SDAU09C2的全基因组序列及它们在细胞培养上的复制动态。这二株ALV-B的同源性为95.4%,与GenBank中3株B亚群参考株之间的同源性也均在91.0%～94.9%间,而与其它亚群参考株的同源性均低于87.9%。与亚群无关的gag、pol基因和LTR的核苷酸序列比较表明,这二株ALV-Bgp85基因的gag和pol基因与所有比较的参考株的同源性均在93%以上。LTR与其他外源性ALV参考株的LTR间的同源性在72.6%～88.3%范围内,但与E亚群内源性ALV的LTR的同源性只有51.5%。然而,这二个ALV-B的LTR的同源性也只有74.8%,远低于其他基因组部分的同源性,特别是它们的LTR的U3区同源性只有68.8%,二者在二个CAAT分布上也显著不同。对这二株ALV-B在DF-1细胞上的复制动态比较表明,它们在细胞培养上清液中的TCID50值非常类似,但SDAU09E3株核衣壳蛋白p27抗原的含量显著高于SDAU09C2株。这表明,同一亚群的不同毒株在复制过程中,所表达的p27抗原量与所形成的具有传染性的病毒量间没有平行关系。这一差异与LTR-U3区的相关性则有待应用感染性克隆技术来做进一步深入研究。     

致蛋鸡血管瘤J亚群禽白血病病毒cDNA全序列分析

【目的】了解近年来我国商品蛋鸡群中以血管瘤为主要表型的J亚群禽白血病病毒(Avian Leukosis Virus subgroup J,ALV-J)的分子生物学特性,为控制ALV-J在鸡群中流行提供基础资料。【方法】采用PCR扩增和序列分析技术,对分离自血管瘤或者血管瘤与髓样细胞瘤(Myeloid Leukosis,ML)并存的3株蛋鸡ALV-J毒株前病毒DNA的全序列及3株商品蛋鸡血管瘤型分离毒和1株商品蛋鸡ML型分离毒的致瘤关键性序列进行研究。【结果】来自血管瘤或者血管瘤与ML并存的商品蛋鸡分离毒株与来自肉鸡分离毒株的全序列差异明显,在遗传进化树上分属两个大的分支;研究发现商品蛋鸡血管瘤及ML混合病例分离毒JS09GY3与JS09GY6株的引物结合位点(Primer Binding Site,PBS)-Leader中出现极为罕见的连续19bp的插入突变,其与劳斯相关病毒1(Rous Associated Virustype1,RAV-1)、劳斯相关病毒2(Rous Associated Virustype2,RAV-2)及劳斯肉瘤病毒施密特-鲁宾二氏[Rous sarcoma virus(strain Schmidt-RuppinB),RSV-SRB]毒株序列相同;通过对U3区调控元件的分析,发现血管瘤商品蛋鸡病例分离毒NHH与JS09GY5的U3区各发生1处连续序列缺失,出现了极为独特的c-Est-1、TCF11及C/EBP结合位点,这些调控元件可能与病毒的致肿瘤特性相关;所测的5株血管瘤商品蛋鸡分离毒均保留完整E元件,而所有肉鸡分离毒的E元件均发生了几乎相同的大部分序列缺失;首次发现血管瘤商品蛋鸡分离毒JS09GY3的E元件中有11bp的连续插入序列。【结论】商品蛋鸡血管瘤型ALV-J与肉鸡分离毒在全序列上差异明显,U3、DR1和E元件等区域有一部分特殊的突变与毒株的宿主类型和肿瘤表型有一定关系,其功能尚需进一步研究。而血管瘤型、髓细胞瘤型ALV-J可能是ALV-J与其它反转录病毒的重组毒。     

芦花鸡中B亚群禽白血病病毒的分离与鉴定

通过接种DF-1细胞(C/E)系,从山东某地方品系芦花鸡的鸡群中分离到一株外源性白血病病毒(ALV)SDAU09C2。与GenBank中已发表的不同亚群鸡ALV参考株的囊膜蛋白gp85的氨基酸序列比较,表明该分离株与B亚群ALV(ALV-B)2个参考株的gp85的氨基酸同源性最高,均为92.5%;与A、C、D、E亚群ALV的gp85的氨基酸同源性仅在73.2%~87.9%之间;而与J亚群gp85的氨基酸同源性更低至30.3%~32.4%。这是我国地方品系鸡群中第一次分离和鉴定ALV-B及其gp85基因的报道。     

Isolation and identification of a subgroup A avian leukosis virus from imported meat-type grand-parent chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%–90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for ALV of subgroup A isolated from imported breeders.     

The follicle promotes the evolution of variants of avian leukosis virus subgroup J in vertical transmission

Avian leukosis virus (ALV) is one of the main causative agent of tumor development, which brings enormous economic losses to the poultry industry worldwide. ALV can be transmitted horizontally and vertically, and the latter often give rise to more adverse pathogenicity. However, the propagation and evolution of ALV underlying vertical transmission remain not-well understood. Herein, an animal model for the evolution of variants of ALV subgroup J (ALV-J) in the vertical transmission was built and different organs from infected hens and plasma from their ALV-positive progenies were collected, and then three segments in the hypervariable regions of ALV (gp85-A, gp85-B, LTR-C) were amplified and sequenced using conventional Sanger sequencing and MiSeq high-throughput sequencing, respectively. The results showed that the genomic diversity of ALV-J occurred in different organs from ALV-J infected hen, and that the dominant variants in different organs of parental hens, especially in follicle, changed significantly compared with original inoculum strain. Notably, the dominant variants in progenies exhibited higher homologies with variants in parental hens’ follicle (88.9%–98.9%) than other organs (85.6%–91.1%), and most consistent mutations in the variants were observed between the progenies and parental hen’s follicle. Furthermore, HyPhy analysis indicated that the global selection pressure value (ω) in the follicle is significantly higher than those in other organs. In summary, an animal model for vertical transmission was built and our findings revealed the evolution of variants of ALV in the process of vertical transmission, moreover, the variants were most likely to be taken to the next generation via follicle, which may be related to the higher selection pressure follicle underwent.     

Expression of Endogenous Retrovirus ev/J gp85 Gene and Analysis of Its Immunoreactivity in Comparison with Exogenous Viral Protein

The envelope gene gp85 of ev/J,a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian ieukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC<,10200> strain).     

禽白血病病毒J亚群env基因的克隆和序列分析

应用多聚酶链反应（PCR）的方法扩增出ADOL-4817毒株的囊膜蛋白env基因,并克隆进大肠杆菌。经核酸序列分析证明,env基因的大小为1746 bp,其中gp85和gp37由1554 bp组成,可翻译成517个氨基酸,分子量为57.7 D。根据糖基化位点N-X-S/T的特点,发现ADOL-4817的env蛋白有15个潜在的糖基化位点。同源性分析证明,ADOL-4817的env基因与其它ALV-J的env基因序列同源性为88.8%～92.4%,而与外源性ALVs的相应序列的同源性仅为40.5%～51.4%,然而,与内源性的EAV-HP毒株的类env基因的同源性高达91.2%;另外,ADOL-4817毒株的gp37在C末端多了13个氨基酸。这些结果提示,ALV-J的env基因存在广泛的变异性,env基因可能来源于内源性和外源性ALVs的重组。     

Two functionally distinct forms of a retroviral receptor explain the nonreciprocal receptor interference among subgroups B, D, and E avian leukosis viruses

Subgroups B, D, and E avian leukosis viruses (ALV-B, -D, and -E) share the same chicken receptor, TVB(S1), a tumor necrosis factor receptor (TNFR)-related protein. These viruses, however, exhibit nonreciprocal receptor interference (NRI): cells preinfected with ALV-B or ALV-D are resistant to superinfection by viruses of all three subgroups, whereas those pre-infected by ALV-E are resistant only to superinfection by other subgroup E viruses. In this study, we investigated the basis of this phenomenon by characterizing the interaction of TVB(S1) with ALV-B Env or ALV-E Env. Sequential immunoprecipitation analysis using surface envelope immunoglobulin fusion proteins revealed the existence of two separate types of TVB(S1) that are encoded by the same cDNA clone. One form, designated the type 1 receptor, is specific for ALV-B and ALV-E. The other form, the type 2 receptor, is specific for ALV-B. We show that a protein consisting of only the first and second extracellular cysteine-rich domains of TVB(S1) is capable of forming both receptor types. However, the third extracellular cysteine-rich domain is required for efficient formation of the type 1 receptor. We also demonstrate that heterogeneous N-linked glycosylation cannot explain the difference in activities of the two receptor types. The existence of two types of TVB(S1) explains the NRI pattern between ALV-B and -E: subgroup B viruses establish receptor interference with both receptor types, whereas subgroup E viruses interfere only with the type 1 receptor, leaving the type 2 receptor available to mediate subsequent rounds of ALV-B entry. The formation of a TVB receptor type that is specific for cytopathic ALV may also have important implications for understanding how some subgroups of ALV cause cell death.     

禽白血病病毒J亚群env基因产物的抗原性分析

用PCR扩增方法将ALV Jenv基因不同片段进行了克隆 ,并构建了env基因片段GST融合蛋白载体。用Westernblot实验证明 ,大肠杆菌表达的不同env基因片段的GST融合蛋白能与相应的单克隆抗体产生特异性反应性 ,单克隆抗体JE9和G2识别的抗原位点位于gp85的氨基酸 6 5～ 1 5 5区域 ,而I45识别的抗原表位位于env基因的另一区域 (1 5 6～ 2 3 3位氨基酸 )。ALV J氨基酸多肽而非糖基化位点决定ALV J的亚群特异性