三黄鸡a干扰素的克隆、表达及抗病毒活性初步研究

采用PCR方法从三黄鸡血液基因组中扩增鸡α干扰素全基因,并克隆和测序.序列分析表明,基因全长为582 bp,亚克隆其成熟蛋白编码基因489 bp.将该片段与表达载体pET-28a连接克隆至大肠埃希菌DH5α菌株,经测序和酶切鉴定,选取正向插入、读码框正确的阳性克隆.构建重组质粒并转化BL21(DE3),经IPTG诱导,...     

基于甲病毒复制子的犬瘟热核酸疫苗的构建及表达性研究

本试验利用T-A克隆技术,构建克隆载体pEASY-Blunt-F,BamHⅠ酶切pEASY-Blunt-F,回收F基因,将其克隆至pSCA1真核表达载体中,获得重组质粒pSCA1-F。经DNA测序、限制性内切酶分析和PCR鉴定,结果表明重组质粒pSCA1-F成功构建。通过间接免疫荧光试验和Western blotting证实F基因在转染细胞中表达。此外,细胞凋亡的检测结果表明,pSCA1-F能引起转染的细胞发生凋亡。     

Characterization of Newcastle disease viruses isolated from field cases in Japan

Seven Newcastle disease viruses isolated in Japan from 1930 to 1984 were cloned on chicken embryo fibroblasts (CEFs) and characterized biologically. All seven produced two or more types of plaques on CEFs. The plaques were classified into four types. Plaque cloning was carried out five times, and 22 cloned viruses were established. The biological characters of the cloned viruses suggested that the strains contain different clones and that their clones are different even among close cases, such as G strain and H strain.     

禽腺联病毒全基因组的克隆及感染性病毒的拯救

为了克隆禽腺联病毒(Avian adeno-associated virus,AAAV)全基因组用于构建基因转移载体研究,以鸡胚致死孤儿病毒(CELO)作为辅助病毒与AAAV共接种SPF鸡胚进行AAAV的增殖,将AAAV约4.7kb双链基因组DNA与pCR2.1载体连接,构建了含AAAV全基因组的重组质粒pAAAV并进行了测序。序列分析表明,AAAV YZ-1株的基因组为4684bp,两端具有141bp的末端倒置重复序列和Rep蛋白结合位点特征序列,与GenBank中收录的AAAV DA-1株和VR-865株的核苷酸序列同源性分别为95.0%和92.2%。将pAAAV质粒转染CELO病毒感染的鸡胚肝细胞系,获得了感染性AAAV病毒粒子,结果证明克隆的AAAV基因组中存在与病毒复制和包装相关的正确关键序列,可用于重组AAAV载体的构建。     

Properties of producer and non-producer clones of a Marek's disease turkey lymphoblastoid cell line

The MDTC-RP30 lymphoblastoid cell line established from Marek's disease (MD) tumors in turkeys consisted of a heterogeneous population of cells 10 to 25 micron in diameter. Large-cell fractions obtained from a bovine fetal serum gradient had a higher titer of cell-associated MD virus (MDV) than the small-cell fractions. Seven single-cell clones were established from MDTC-RP30 cell line: two consisted of large cells, and the other clones consisted of small cells. Infectious MDV was rescued from large-cell clones in chicken embryo fibroblast cultures but not from small-cell clones. All clones contained MDV DNA sequences when hybridized against cloned MDV DNA. All clones were positive for a Marek's-disease-tumor-associated surface antigen and surface immunoglobulins. All but two small-cell clones caused MD in susceptible chickens. The two large-cell-type clones were uniformly tetraploid, whereas one small-cell clone was diploid and the four others were a mixture of diploid and tetraploid, with an occasional triploid cell. Evidence of translocation involving the male (Z) chromosome and the chromosome #3 was seen in one clone. These results suggest that MDV transforms different subpopulations of lymphocytes.     

Reduced in vivo nonspecific resistance to Listeria monocytogenes infection during avian retrovirus-induced immunosuppression

Ten-day-old chickens infected with an avian osteopetrosis virus [MAV-2(O)] were more susceptible to challenge with Listeria monocytogenes than virus-free chickens, as demonstrated by reduced bacterial clearance from their spleens. Reduced clearance of L. monocytogenes was observed throughout a 26-day period after MAV-2(O) infection.     

表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析

鸡传染性法氏囊病（IBD）是一种严重危害养禽业的高度致死性和免疫抑制性传染病。为研制IBD重组火鸡疱疹病毒（HVT）活载体疫苗,本研究构建了表达鸡传染性法氏囊病病毒（IBDV）保护性抗原VP2基因的重组HVT并对其体外生物学特性进行了分析。通过RT-PCR扩增IBDV超强毒株VP2基因并克隆入pCI载体,获得重组真核表达质粒pCI-VP2。用限制性内切酶将携带CMV启动子的VP2基因表达框架切下,连接于入门质粒pENTR,构建获得重组入门质粒pENTR-VP2。将pENTR-VP2与HVT重组黏粒H3-Kan/ccdB进行LR重组反应,构建重组表达黏粒H3-VP2。用H3-VP2与其他4个相互重叠并覆盖HVT全基因组的黏粒共同转染鸡胚成纤维细胞（CEF）,拯救获得重组病毒rHVT-VP2。将重组病毒在CEF中连续传至20代后用PCR、间接免疫荧光试验和免疫印迹试验进行检测,并绘制重组病毒体外生长曲线,分析其体外复制特性。结果表明,重组病毒rHVT-VP2能够稳定表达VP2蛋白,rHVT-VP2在CEF中的复制能力与亲本病毒无明显差异。重组病毒rHVT-VP2免疫鸡后能够诱导产生IBDV中和抗体,并对IBDV强毒株攻击引起的死亡提供90%免疫保护。重组病毒rHVT-VP2的构建为研制IBD重组HVT活载体疫苗奠定了基础,对IBD的防控具有重要意义。     

Diagnostic complementary DNA probes for genome segments 2 and 3 of epizootic hemorrhagic disease virus serotype 1

Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced. Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated. The polyadenylated RNA was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired. The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method. Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis. The cDNA clones of RNA segment 3 of EHDV-1 cross hybridized with the corresponding segment of EHDV serotype 2 by results of cDNA/RNA blot hybridization, but not with RNA of bluetongue virus serotypes isolated in the United States. After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.     

表达H5N1亚型禽流感病毒HA和NA基因的重组鸭瘟病毒的构建

利用PCR技术扩增出鸭瘟病毒(DPV)生长非必需的TK基因(约1.1kb),将其克隆入pGEM-Teasy载体获得载体pGTK。根据已知的绿色荧光蛋白载体pEGFP-C1的序列设计了一对引物,PCR扩增出pEGFP-C1上含CMV启动子、EGFP及其多克隆位点的完整的基因表达盒插入pGTK的TK上,获得质粒pGTK-EGFP。根据Genbank已发表的H5N1亚型禽流感病毒的血凝素(HA)和神经氨酸酶(NA)基因序列,设计了两对引物,分别从pT-HA和pT-NA两个质粒上扩增出HA和NA基因,克隆到pGTK-EGFP的表达盒的多克隆位点Kpn2I与SmaI之间,构建含EGFP及HA和NA基因的转移质粒载体pGTK-EGFP-HA-NA。将这质粒载体与DPV34F2疫苗毒共转染鸡胚成纤维细胞(CEF),通过荧光方法筛选,获得了表达HA和NA基因的重组DPV(rDPV-HA-NA)。     

凋亡素基因的克隆

为了探索应用凋亡素杀死肿瘤细胞的可能性,用ＰＣＲ方法扩增了鸡贫血病毒（ｃｈｉｃｋｅｎａｎｅｍｉａｖｉｒｕｓ,ＣＡＶ）的ＶＰ３基因（凋亡素）片段,然后将该片段重组于ｐＵＣ１９载体,并进行了限制性内切酶鉴定与序列分析,证实该片段与鸡贫血病毒Ｃｕｘ－１株的ＶＰ３ＤＮＡ序列一致,该ＤＮＡ全长３６３ｂｐ,编码１２１个氨基酸。     

Development of genomic probes to Sarcocystis cruzi (Apicomplexa).

A genomic library of Sarcocystis cruzi sporozoite DNA was constructed in bacteriophage lambda gt10. Recombinant phages containing insert DNA were selected by growth on Escherichia coli strain C600 hflA150. Of 14 clones examined, 11 contained DNA inserts ranging in size from approximately 1.45 kilobase (kb) to 6.18 kb. Insert DNA from four of these clones specifically hybridized to 32P-labelled S. cruzi merozoite DNA. One of these insert DNA, clone SL41, was selected and labelled with 32P. This probe did not hybridize with the other ten DNA inserts nor with bovine cellular DNA, but it hybridized with sporozoite, merozoite and bradyzoite DNA preparations. The SL41 probe could detect merozoite DNA in as little as 17 ng total DNA. Genomic probes detecting developmental stages of Sarcocystis spp. could provide an improved means is diagnosis of acute bovine sarcocystosis.     

大鼠BDNF基因真核表达载体的构建

从23日龄大鼠脑组织中抽提总RNA,应用RT-PCR技术扩增脑源性神经营养因子(BDNF)基因片段,将此片段克隆入T载体,酶切反应鉴定。然后双酶切BDNF-T载体和空质粒pEGFP(N1),将所获目的片段和线性空载体用T4 DNA连接酶连接,构建真核表达载体pEGFP(N1)-BDNF,并进行酶切反应鉴定及DNA测序。序列测定的结果与GenBank比较,所克隆的BDNF基因从起始密码子ATG到终止密码子TAG全长共750 bp序列完全相同。结果表明成功构建真核表达载体pEGFP(N1)-BDNF,为进一步研究BDNF基因表达,神经系统疾病治疗和生物制药奠定基础。     

Forensic investigation of a 1986 outbreak of osteopetrosis in commercial brown layers reveals a novel avian leukosis virus-related genome

Avian leukosis virus (ALV) is known to cause several neoplastic conditions in chickens, such as B-cell lymphomas, myelocytomas, erythroblastosis, and other types of neoplasia including osteopetrosis. We describe herein the identification of unique ALV-related proviral DNA sequences in an archived chicken bone affected with osteopetrosis. The osteopetrotic bone was obtained from an affected 46-week-old brown layer during an outbreak of osteopetrosis in Costa Rica in 1986. Analysis of proviral DNA in the 23-year-old osteopetrotic bone revealed unique exogenous ALV-related sequences that were named CR-1986 (Costa Rica, 1986). The 5' and 3' long terminal repeats (LTR) in the proviral DNA were identical to each other. The U3 regions in the LTRs were most similar to equivalent sequences in ALV-J, while U5 was identical to known endogenous ALV-E sequences. The predicted CR-1986 envelope protein was most similar to the envelope of myeloblastosis associated virus type 1 (MAV-1), although the percentage of amino acid sequence similarity to MAV-1 was low (90.4%). The variable and hypervariable regions of gp85 displayed several mutations compared to representative strains of ALV. The gp37 (transmembrane or TM) envelope protein showed three leucine to serine mutations that may represent important changes in the conformation of this protein, a finding that is currently being investigated. Several recombination events may have contributed to the emergence of CR-1986 because each analyzed segment was similar to a different ALV. CR-1986 may represent a unique ALV based on distinctive characteristics of its predicted envelope protein in comparison to previously reported ALVs.     

Recombinant DNA probes for Mycoplasma synoviae

A genomic library was prepared from Mycoplasma synoviae (MS) strain WVU 1853 cloned in plasmid vector pUC8 and transformed in Escherichia coli host JM83. In dot blot assays, four transformed E. coli clones hybridized with 32P-labeled chromosomal DNA of MS but not with 32P-labeled chromosomal DNA of M. gallisepticum (MG) strain S6. In Southern hybridization, each of the CsCl-purified recombinant plasmid clones was shown to contain two MS DNA fragments between 1.0 to 2.3 kbp in length. 32P-Labeled probes prepared from each of the four recombinant plasmids hybridized in dot blot assays with MS strain WVU 1853 and nine MS field isolates but not with MG strains S6, K810, F2F10, four MG field isolates, and 15 other species of avian mycoplasmas.     

绵羊Myostatin基因真核表达载体的构建及在其原代成纤维细胞中的瞬时表达

Myostatin(MSTN)基因是胚胎期肌肉形成和出生后骨骼肌生长的主要调控因子之一,通过抑制肌细胞的扩增和分化而调控肌肉的生长和发育。为了进一步揭示MSTN在绵羊成纤维细胞中的生物学功能,采用RT-PCR从绵羊肌肉组织中扩增MSTN基因,将其cDNA终止密码子TGA删除,采用定向克隆技术连接到带有水母绿色荧光蛋白(AcGFP)报告基因的真核表达载体pAcGFP-N1中,构建融合蛋白重组质粒,经XhoⅠ/SacⅡ双酶切、测序鉴定后,用脂质体介导质粒转染绵羊原代成纤维细胞,观测荧光表达及用RT-PCR和Western blotting方法检测基因转录、蛋白质表达情况。结果表明,成功克隆绵羊MSTN基因,通过PCR方法在MSTN阅读框两端引入了XhoⅠ和SacⅡ克隆位点,成功构建pAcGFP-MSTN融合蛋白真核表达载体,重组质粒转染绵羊成纤维细胞24 h后在荧光显微镜下观察到绿色荧光,通过RT-PCR扩增出1138 bp的转录产物,并用Western blotting检测到78 ku目的蛋白的表达。本试验为研究MSTN基因在成纤维细胞和脂肪分化调控中的具体机制奠定基础。     

鹅细小病毒VP3基因真核表达载体的构建及其在Vero细胞中表达

根据已发表的鹅细小病毒(GPV)B株核苷酸序列,设计并合成了1对引物,PCR扩增GPV吉林分离株主要结构蛋白VP3基因,获得大小为1605bp的核苷酸片段。将该片段纯化后克隆入pMD18-T载体,转化感受态大肠杆菌JM109,双酶切鉴定,筛选阳性重组质粒并测序。经过酶切和连接反应,将VP3基因克隆入真核表达载体pVAX1,转化感受态大肠杆菌DH5α,筛选阳性克隆,提取质粒,进行了PCR和酶切鉴定。通过脂质体法将pVAX1-VP3转染Vero细胞,RT-PCR和间接免疫荧光法检测。结果显示,VP3基因克隆成功,与GPVB株核苷酸序列同源性为96.2%;PCR和酶切鉴定结果证实,成功构建了含VP3基因的GPV真核表达载体pVAX1-VP3。提取转染该质粒的Vero细胞RNA,RT-PCR扩增,在1000～2000bp可见一明显DNA条带;间接荧光抗体染色转染细胞,在细胞表面可见特异荧光。     

梅花鹿鹿茸表皮生长因子基因原核表达载体的构建及鉴定

通过T-A克隆技术,成功地构建了克隆载体pMD-18T-EGF,用EcoRⅠ和HindⅢ双酶切克隆质粒pMD-18T-EGF后,回收EGF基因,并将此基因定向克隆至相同双酶切回收后的pET32a原核表达载体中,获得重组质粒pET32a-EGF。经限制性内切酶分析和PCR鉴定,结果表明:成功地构建了原核重组质粒。     

牦牛病毒性腹泻病毒E1基因的克隆及生物信息学分析

参考GenBank中发表的BVDV毒株的基因组序列设计2对引物,利用套式RT-PCR方法首次成功克隆牦牛体内分离鉴定出的牛病毒性腹泻病毒E1基因,并扩增出预期的585 bp目的片段。将扩增产物克隆至pMD18-T Vector,经质粒PCR鉴定及酶切鉴定获得阳性重组质粒并进行测序。测序结果经BLAST同源性比较分析,克隆得到的E1基因与Osloss株同源性最高,但核苷酸同源性仅为73.3%,推导氨基酸同源性仅为82.6%,表明牦牛病毒性腹泻病毒存在较大的基因突变。这可能是该病毒为适应牦牛这种特有生物体和牦牛所生活的高原生态环境的结果,或该病毒也可能具有独立的遗传衍化来源。     

Cloning of Mycoplasma synoviae genes encoding specific antigens and their use as species-specific DNA probes.

A genomic library of Mycoplasma synoviae (MS) was generated by using bacteriophage lambda gt11 as a cloning and expression vector. Identification of recombinant clones highly specific to MS was achieved by screening the library for expression of MS proteins with polyclonal antiserum that had been preadsorbed with 6 heterologous avian mycoplasma species antigens. Expression of the recombinant clones in Escherichia coli followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total cell lysates and immunoblot yielded a predominant reactive fusion protein of 165 kD. Two clones (MS2/28 and MS2/12) that yielded inserts of different size were selected. The 2 MS DNA inserts were subcloned in a plasmid vector, labeled with digoxigenin, and used as probes for the specific recognition of several MS strains. A high degree of conservation was demonstrated for the MS2/12 and MS2/28 genes in tested MS strains. In addition, neither DNA fragment recognized any other avian mycoplasma species (M. gallisepticum, M. meleagridis, M. gallinarum, M. iners, M. anatis, and M. iowae), thus indicating their high specificity to MS. The sensitivity of the slot blot hybridization method using digoxigenin-labeled MS2/12 and MS2/28 probes for direct detection of MS from broth cultures of field isolates was 10(5) colony-forming units/ml. These results demonstrate the effectiveness of adsorbed antisera for the isolation of species-specific mycoplasma DNA and the potential for its use as probes for the specific and direct detection of MS from broth cultures of field isolates.     

A型肉毒毒素基因的克隆及序列分析

目的:克隆肉毒梭菌(Clostridium botulinum)A型肉毒毒素(BoNTa)编码基因。方法:提取肉毒梭菌国际标准株(62A)基因组DNA,根据肉毒梭菌BoNTa基因(GenBank登录号M30196)序列设计引物,采用LA-PCR方法,扩增出目的基因片段,与pMD18-T载体连接,通过酶切鉴定、测序分析克隆到的A型肉毒毒素基因序列。结果:该基因片段与Genbank中的BoNTa基因序列(GenBank登录号M30196)一致性为100%,预测氨基酸序列一致性为100%。结论:成功克隆肉毒梭菌的A型肉毒毒素基因序列,为肉毒梭菌的快速检测,以及进一步用基因工程方法生产A型肉毒毒素奠定了基础。