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。     

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。     

特超强毒648株马立克氏病病毒的囊膜糖蛋白gE基因的克隆和序列比较

鸡马立克氏病毒特超强毒 (vv+MDV) 648株的囊膜糖蛋白 gE基因经PCR扩增并克隆入pUC1 8载体。 648株gE基因经核酸序列分析测定 ,全长为 1 494碱基。所编码的蛋白具有跨膜糖蛋白的一些特征。它含有 8个潜在的糖基化位点、N端有一段疏水区 (1～ 1 9aa)所构成的信号肽、C端有一段疏水区 (391～ 41 9aa)所构成的膜锚着序列。经 648株 (vv+MDV)和马立克氏病毒强毒 (vMDV)GA株的 gE相比较 ,在MDV血清 1型中 gE是较为保守的 ,二者仅有 2个核苷酸的差异 (第 51 2位、第 1 472位 ) ,并导致了有二个相应的氨基酸的改变 (第1 71位Leu/Pro、第 491位Arg/Lys)。     

小鼠白细胞介素-33基因的克隆及序列分析

目的:克隆小鼠IL-33基因全长编码区cDNA,并对其进行序列分析。方法:从BALB/c小鼠的脊髓组织中提取总RNA,逆转录为cDNA,用热启动PCR技术,扩增小鼠IL-33基因全长编码区cDNA,经双酶切后,克隆入pcDNA3.1( )载体中,构建真核表达载体pcDNA3.1-mIL-33,然后进行酶切鉴定与序列分析。结果:小鼠IL-33基因的PCR产物和重组载体经凝胶电泳和酶切鉴定、测序分析证实,其序列与GenBank中数据一致。小鼠IL-33基因的全长编码序列为801 bp,编码266个氨基酸。结论:小鼠IL-33基因成功的克隆并构建了其真核表达载体,为进一步进行IL-33的表达与功能研究奠定了基础。     

黑曲霉脯氨酸蛋白内肽酶基因的克隆与序列分析

以黑曲霉(A.niger)TCCC41013的总RNA作为模板,通过RT-PCR扩增出含自身信号肽的脯氨酸蛋白内肽酶基因,将其插入pUCm-T载体上,经PCR和酶切鉴定后进行测序并分析.所克隆的PEP基因全长为1 581bp,编码526个氨基酸残基,成熟肽为504个氨基酸残基.与GeneBank中已报道的A.niger CBS513.88的PEP序列同源性最高,达到99.75%.脯氨酸蛋白内肽酶是一种新型的丝氨酸蛋白酶,黑曲霉PEP与已克隆的其他菌种的PEP同源性不高.     

大乳头水螅RACE cDNA文库的构建

目的:为筛选和克隆大乳头水螅发育调控相关基因的全长cDNA,构建大乳头水螅RACE cDNA文库.方法:提取大乳头水螅总RNA后从其中分离mRNA,运用SMART技术构建RACE cDNA文库.为鉴定所构建文库的质量,根据GenBank中大乳头水螅actin基因cDNA序列设计5'RACE和3'RACE的引物及用于扩增actin基因编码区全长序列的引物.结果:琼脂糖凝胶电泳结果表明,RACE cDNA文库中全长cDNA的长度集中在500-2 000bp之间.5'RACE、3'RACE PCR及扩增actin基因编码区全长序列时均以本文构建的大乳头水螅RACE cDNA文库为模板,这3个PCR反应均能扩增出产物,产物大小与目标片段预计大小相似.PCR产物分别经T/A克隆及测序后证明为大乳头水螅actin基因cDNA的相应序列.结论:RACE cDNA文库的成功构建为通过RACE方法获得大乳头水螅功能基因cDNA全长序列奠定了基础.     

一株具有急性致瘤特性的马立克氏病病毒的分离与鉴定

应用鸭胚成纤维细胞(DEF)从免疫过CVI988/Rispens疫苗株患马立克氏病(MD)的三黄鸡中分离到一株马立克氏病病毒(MDV)(命名为YL040920株)。从该分离株蚀斑克隆获得的9个克隆在蚀斑形成时间及其形态大小上均无明显差别,表明它较为单一;应用聚合酶链式反应(PCR)技术扩增并测定了毒株的致瘤相关基因meq的核苷酸序列,并与其他MDV-1参考毒株的序列进行比较分析,发现其序列具有MDV-1强毒株的特征;用基于抗MDV-1 MEQ蛋白的单克隆抗体3G12E6的免疫荧光试验(FA)对毒株的DEF培养物进行检测,发现有特异性的荧光定位于细胞核内;应用毒株感染霞烟鸡,最早在接种后(PI)21d即可诱发明显的内脏器官,在各器官肿瘤中以心脏、肝脏和皮肤的肿瘤发生率最高;用禽肿瘤病三重PCR鉴别诊断技术对毒株的DEF培养物以及感染鸡的内脏器官组织进行检测,均能扩增到MDV-1强毒株的特异性带,而网状内皮增生症病毒(REV)和禽白血病病毒(ALV)的检测则均为阴性。研究的结果表明,分离株YL040920为单一的MDV-1强毒株,无REV、ALV以及疫苗株CVI988/Rispens的混杂,并具有以心脏、肝脏和皮肤肿瘤为主的急性致瘤特性。     

葡萄球菌A蛋白基因的克隆及其IgG受体区新型表达载体的构建

旨在克隆SPA基因并将该基因的IgG结合区亚克隆至毕赤酵母表达载体中。以金黄色葡萄球菌CowanI菌株基因组为模板,对葡萄球菌A蛋白(SPA)基因全长序列进行PCR扩增,再将PCR产物克隆入pMD18-T质粒,将DNA测序所得的结果用Blastn软件进行在线同源比对,经鉴定为SPA基因序列后,在线对其进行功能区域(IgG-Fc受体区)的预测,将该区域亚克隆入表达载体pPICZaA。结果显示,从CowanI菌株中成功地扩增到SPA基因,与NCBI中公布的序列同源性高达97%,同时构建了IgG-Fc受体区新型的表达载体     

人脑内-含有ACP样结构域新基因的发现

为寻找脑内新基因,以正常成人全脑cDNA为模板,采用锚定PCR方法进行扩增,将经琼脂糖DNA电泳 鉴定获得的一约1200bp大小的特异性条带回收,并克隆入Teasy载体.用310 Genetic Analyzer进行自动测序. 所得序列进行生物信息学分析BLAST相似性分析结果证明所得序列为新序列,读框分析表明,该序列中存在 一完整编码区,编码含357个氨基酸的蛋白质.ProDom软件分析发现其含有酰基携带蛋白(ACP)样结构域. 随后,经3'RACE法克隆到该基因的全长cDNA,其全长为2024bp,染色体定位在14q11.2,含有16个外显子, 15个内含子,该基因已登录到GenBank.经设计编码区引物,从Teasy载体扩增出编码区后再克隆入pGEX-4T1 表达载体,经异丙基硫代-D-乳糖苷(IPTG)化学诱导表达.其编码区克隆人pGEX-4T1表达载体后,转入 JM109宿主菌,经IPTG诱导已得到表达.点杂交及RNA印迹表明,该基因在正常成人脑内广泛高表达.     

The RNA subunit of telomerase is encoded by Marek's disease virus

Marek's disease virus (MDV) is a herpesvirus of chickens that induces T lymphomas and tumors within 4 to 5 weeks of infection. Although the ability of MDV to induce tumors was demonstrated many years ago and although a number of viral oncogenic proteins have been identified, the mechanism by which the MDV is implicated in tumorigenesis is still unknown. We report the identification of a virus-encoded RNA telomerase subunit (vTR) within the genome of MDV. This gene is found in the genomic DNA of the oncogenic MDV strains, whereas it is not carried by the nononcogenic MDV strains. The vTR sequence exhibits 88% sequence identity with the chicken gene (cTR). Our functional analysis suggests that this telomerase RNA can reconstitute telomerase activity in a heterologous system (the knockout murine TR(-/-) cell line) by interacting with the telomerase protein component encoded by the host cell. We have also demonstrated that the vTR promoter region is efficient whatever the species of cell line considered and that vTR is expressed in vivo in peripheral blood leukocytes from chickens infected with the oncogenic MDV-RB1B and the vaccine MDV-Rispens strains. The functionality of the vTR gene and the potential implication of vTR in the oncogenesis induced by MDV is discussed.     

Deoxyribonucleic Acid of Marek''s Disease Virus in Virus-Induced Tumors

DNA was extracted from [(3)H]thymidine-labeled Marek's disease virus (MDV) and purified by two cycles of CsCl gradient centrifugation in a fixed-angle rotor. The DNA was transcribed in vitro into (32)P-labeled complementary RNA (cRNA). MDV cRNA did not hybridize with DNA from chicken embryo fibroblast cultures or from chicken spleen, but hybridized efficiently with DNA from MDV particles or MDV-infected cell cultures. Five Marek's disease tumors from different chickens and different organs (ovary, liver, testis) were all found to contain MDV DNA sequences. The relative amount of MDV DNA varied from tumor to tumor and was between 3 and 15 virus genome equivalents per cell. The content of virus DNA per cell in spleens from tumor-bearing chickens was much lower than in tumors from the same animals. MDV-infected cell cultures contained a large proportion (28-59%) of virus antigen-positive cells, as measured by immunofluorescence, but tumor cells were negative in this respect (<0.02% positive cells). These data indicate that MDV is present in a provirus form in tumor cells.     

传染性支气管炎病毒江苏分离株核蛋白基因序列测定与同源性分析

将本室鸡传染性支气管炎病毒（IBV）江苏省地方分离肾型毒株JS／95／03接种鸡胚,分离、纯化病毒,提取单股RNA做为反转录－聚合酶链反应（RT－PCR）的扩增模板。用Genbank公开序列多重比较后设计一对引物,使用单管RT－PCR方法,物异扩增IBV核蛋白（N）基因5'端854bp的片段,扩增产物纯化后测,序列分析表明,IBV N基因也存在较大变异,此毒株与呼吸型疫苗株M41序列同源性最高。     

表达IBDV VP2融合蛋白的重组MDV的构建及其免疫特性

将增强型绿色荧光蛋白基因(eGFP)与鸡传染性法氏囊病病毒(IBDV)的VP2基因融合,插入马立克氏病毒(MDV)CVI988/Rispens的非必需区US10片段中,成功构建表达VP2融合蛋白的MDVCVI988转移载体pUC18-US10-VP2。将转移载体质粒与CVI988/Rispens疫苗毒共转染鸡胚成纤维细胞(CEF),筛选获得表达VP2融合蛋白的重组MDV(rMDV)。聚合酶链式反应(PCR)和间接免疫荧光实验(IFA)证明,rMDV传至第31代仍能稳定表达VP2融合蛋白。用rMDV免疫SPF鸡,进行IBDV攻毒保护试验,1日龄SPF鸡分别用1000PFU、2000PFU、5000PFU的rMDV进行免疫,33日龄用100LD50的IBDVJS超强毒进行攻毒,鸡的免疫保护率分别为50%、60%、80%。值得注意的是,5000PFU的rMDV一次免疫1日龄SPF鸡,其法氏囊组织病理损伤等级与IBD中等毒力活疫苗常规二次免疫相当(2·0/1·5),其保护效果无显著差异(p>0·05),而与非重组病毒免疫组相比较,保护效果差异显著(P<0·01),这表明构建的表达IBDVVP2融合蛋白的rMDV可以有效地为SPF鸡提供免疫保护作用。     

有限延伸法检测端粒酶活性

人端粒酶是一种核蛋白体,通过其内含的RNA模板与端粒末端配对把重复端粒片段添加在端粒3＇末端|因此,端粒酶活性与细胞凋亡、衰老、永生化有密切关系,是癌症临床预测诊断的一个生物标签．现有的端粒酶活性检测方法,存在灵敏度低和不易定量等问题．本研究采用错配有限延伸法检测端粒酶活性：在人端粒酶延伸人工合成的游离端粒酶底物时,只加入dATP和dGTP,端粒酶只能把底物延伸4个脱氧核糖核苷酸AGGG．然后加入dNTP,让端粒酶延伸的产物和一条长的引物配对从而延伸出PCR模板|再加入引物进行热启动PCR．PCR后进行非变性PAGE (polyacrylamide gel electrophoresis),得到希望的唯一1条目标带．同时,用不同的端粒酶浓度梯度进行优化,发现有限延伸法检测端粒酶活性的下限达到250个HeLa细胞．     

The simple chicken major histocompatibility complex: life and death in the face of pathogens and vaccines

In contrast to the major histocompatibility complex (MHC) of well-studied mammals such as humans and mice, the particular haplotype of the B-F/B-L region of the chicken B locus determines life and death in response to certain infectious pathogens as well as to certain vaccines. We found that the B-F/B-L region is much smaller and simpler than the typical mammalian MHC, with an important difference being the expression of a single class I gene at a high level of RNA and protein. The peptide-binding specificity of this dominantly expressed class I molecule in different haplotypes correlates with resistance to tumours caused by Rous sarcoma virus, while the cell-surface expression level correlates with susceptibility to tumours caused by Marek's disease virus. A similar story is developing with class II beta genes and response to killed viral vaccines. This apparently suicidal strategy of single dominantly expressed class I and class II molecules may be due to coevolution between genes within the compact chicken MHC.