XJ-160病毒复制子型表达载体的构建

XJ-160病毒是我国首次分离的辛德毕斯病毒,全基因组测序已经完成.本文利用该病毒全基因序列首先构建了全基因组cDNA克隆质粒,在此基础上,利用基因重组技术将病毒结构基因序列替换为含有多个单酶切位点的序列,得到复制子表达载体质粒pRepxj160.为验证载体的功能,将报告基因绿色荧光蛋白(EGFP)和β-半乳糖苷酶基因(lacZ)分别插入到载体的多克隆位点,得到两个表达质粒;经体外转录获得的转录体RNA转染BHK-21细胞后14h,可检测到报告基因的表达.结果表明我们构建的XJ-160病毒复制子型表达载体具有自主复制功能,可以表达异源基因.本研究为进一步开发具有我国自主知识产权的甲病毒载体奠定了基础.     

流行性乙型脑炎病毒(JEV)全长感染性克隆的制备及恢复病毒的获得

根据JEV病毒减毒株SA14—14—2基因组序列,设计覆盖全长的4对重叠引物,以提取的活疫苗病毒RNA为模板,RT—PCR扩增出4个片段,并克隆到质粒载体中,进一步构建两个半端分子克隆,然后将全长cDNA序列克隆到一个新改造的低拷贝质粒载体pBR—kpn中,构建我国流行性乙型脑炎病毒(JEV)基因组全长cDNA克隆。经过体外转录后得到的转录子转染BHK-21细胞,重新获得JEV的恢复病毒,通过生物学特性、分子生物学水平、蛋白水平等几个方面对恢复病毒进行鉴定。结果获得了稳定的全长cDNA克隆,转录子转染BHK-21细胞后,第4天开始出现细胞病变(CPE),第6～7天时CPE为 ,经过Vero细胞进一步放大培养后,间接免疫荧光实验和RT—PCR实验均为阳性。证实了构建的JEV的全长cDNA克隆有感染性,为进一步的研究奠定了基础。     

XJ—160病毒复制子型表达载体的构建

D-160病毒是我国首次分离的辛德毕斯病毒,全基因组测序已经完成。本文利用该病毒全基因序列首先构建了全基因组cDNA克隆质粒,在此基础上,利用基因重组技术将病毒结构基因序列替换为含有多个单酶切位点的序列,得到复制子表达载体质粒pRepxjl60。为验证载体的功能,将报告基因绿色荧光蛋白(EGFP)和β-半乳糖苷酶基因(lacZ)分别插入到载体的多克隆位点,得到两个表达质粒;经体外转录获得的转录体RNA转染BHK-21细胞后14h,可检测到报告基因的表达。结果表明我们构建的XJ-160病毒复制子型表达载体具有自主复制功能,可以表达异源基因。本研究为进一步开发具有我国自主知识产权的甲病毒载体奠定了基础。     

中国首次分离的辛德毕斯病毒XJ-160病毒株感染性全基因组cDNA克隆的构建与分析

报告了中国首次分离的辛德毕斯病毒XJ-160株的感染性全基因组cDNA克隆的构建与鉴定。利用RT—PCR方法获得覆盖病毒全长基因组的cDNA片段,以低拷贝质粒pBR322作为骨架,将基因组cDNA置于SP6RNA聚合酶启动子之后,基因组3’末端带有35个连续的A,通过DNA重组技术组装成病毒基因组全长cDNA克隆。该克隆可在大肠杆菌DH5a中稳定扩增。经体外转录,RNA转录体转染BHK-21细胞,细胞发生病变,恢复病毒滴度达到10^7～10^8PFU／ml。全基因组cDNA克隆构建过程中引入的沉默突变(8453位核苷酸由C变为T)产生XbaⅠ酶切位点作为遗传标记,在子代恢复病毒的基因组中稳定存在。从细胞病变的特征、BHK-21细胞的空斑形态、病毒的抗原性、病毒在细胞中的生长动力学特征以及对乳鼠的致病性等方面比较,恢复病毒和亲本病毒XJ-160没有显著区别,提示获得了具有感染性的XJ-160病毒全长cDNA克隆。该病毒感染性全基因组cDNA克隆可以作为反向遗传学系统,为进一步研究病毒复制和致病机制,以及开发相应的载体表达系统提供分子生物学工具。     

表达绿色荧光蛋白的重组鸡传染性支气管炎病毒的构建

为探讨鸡传染性支气管炎病毒(IBV)作为载体表达外源基因的可行性,本研究根据IBV H120疫苗株的全基因组序列设计引物,采用RT-PCR方法分10个片段对其基因组进行扩增,并克隆至pMD19-T载体中;同时构建IBV基因组5a基因编码区被增强型绿色荧光蛋白(EGFP)基因替换的重组质粒。采用体外拼接策略,将BsaI酶切处理的10个基因片段顺序连接,构建5a基因编码区被EGFP基因替换的基因组全长cDNA,其5’端具有完整的T7 RNA聚合酶启动子核心序列,3’端具有polyA尾巴结构。然后通过T7 RNA聚合酶体外转录系统合成病毒基因组RNA,脂质体转染BHK-21细胞进行病毒拯救。结果表明成功的从基因组全长cDNA拯救出重组病毒H120-5a/EGFP株,其在鸡胚中能有效的复制和传代,并表达绿色荧光蛋白;5a基因的缺失并不影响病毒对鸡胚的致病性。本研究为进一步开展IBV的分子致病机理、载体疫苗等研究奠定了基础。     

口蹄疫病毒多基因重组质粒的体外表达及衣壳组装

PCR扩增获得包含口蹄疫病毒P1、2A、3C、3D及部分2B编码区的目的基因片段P12X3C3D,将P12X3C3D经AflⅡ和XbaⅠ双酶切后,定向克隆于真核表达质粒载体pcDNA3.1( );另将PCR扩增获得的P12X3C3D直接与真核表达质粒载体pTARGET^TM连接,进行筛选、鉴定及DNA序列分析,分别获得重组质粒pcDNA3．1／P12X3C3D、pTARGET／P12X3C3D。将重组质粒分别转染BHK-21细胞,通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中表达的口蹄疫病毒抗原,用磷钨酸负染,以电子显微镜观察转染重组质粒的细胞中组装的口蹄疫病毒空衣壳。结果表明,口蹄疫病毒基因组片段正确克隆到真核表达质粒载体上,重组质粒pcDNA3．1／P12X3C3D、pTARGET／P12X3C3D均可在BHK-21细胞中表达FMDV目的蛋白。其中重组质粒pTARGET／P12X3C3D表达的口蹄疫病毒抗原蛋白,能够在细胞内正确组装成病毒空衣壳。     

猪水泡病病毒HK′1／70株基因组全长cDNA的构建及其序列测定

从实验感染猪水泡病病毒(SVDV)的乳鼠组织中提取RNA,利用长距离的RACE技术,扩增出覆盖SVDVHK′1/70株全基因组的2个忠实性的cDNA重叠片段(3′PCR片段和5′PCR片段),分别克隆进pGEM-T Easy载体。利用AatⅡ和BssHⅡ酶切含5′PCR片段的重组质粒,回收目的片段,定向克隆于含3′PCR片段的重组质粒,构建出了SVDV HK′1/70株全长cDNA重组质粒,然后进行序列测定。结果表明,HK′1/70株基因组全基因组序列长7 401 nt(poly A除外),其中5′NCR长743 nt,该毒株蛋白编码区的核苷酸序列为6 558 nt,编码一个长2 185个氨基酸的聚合蛋白,3′NCR长102 nt,其后是至少含有74个A碱基的poly A尾。在HK′1/70株全长cDNA序列的5′端引入了T7启动子序列,在poly A3′端引入了Psp1406Ⅰ识别序列。通过序列同源性和进化树分析,结果表明,HK′1/70属于第Ⅱ抗原遗传群,SVDV与CB5的遗传关系最近,且位于CB5遗传进化树的分支上。HK′1/70株全长cDNA的序列测定及构建,为拯救SVDV和在分子水平进一步深入研究SVDV打下坚实的基础。     

转录病毒载体介导的稳定表达口蹄疫病毒3D基因的BHK21细胞系的建立

[目的]研究口蹄疫病毒RNA聚合酶在BHK-21细胞中的稳定表达状况,为研究RNA聚合酶生物学活性及其基囚工程疫苗研制提供科学依据.[方法]从重组质粒pMD18-T-3D扩增口蹄疫病毒3D基因,通过分子克隆技术构建莺组逆转录病毒表达载体pBPSTR1-3D.用pBPSTR1-3D和pVSV-G双质粒瞬时转染GP2-293包装细胞,收获重组逆转录病毒,然后感染BHK-21细胞,嘌呤霉素持续筛选12 d后获得阳性克隆,并用有限稀释法挑选单个阳性细胞克隆.[结果]应用PCR、RT-PCR技术可从体外反复传代的阳性细胞中扩增到3D基因,证实目的外源基因能转录并被稳定整合进宿主细胞基因组中.经SDS-PAGE、Western blot、间接免疫荧光检测到在不同代次的阳性细胞中有目的蛋白表达.[结论]本试验利用逆转录病毒载体介导的基因转移技术,将外源基因插入到靶细胞的基因组中,构建了稳定表达口蹄疫病毒RNA聚合酶的包装细胞系,为研究3D基因表达及其蛋白定位提供了方便,也为下一步研究RNA聚合酶生物学功能和疫苗研制提供了科学依据.     

同源重组法制备口蹄疫病毒多基因重组腺病毒

通过细菌内同源重组的方法成功构建了含有O型口蹄疫病毒P1-2A和3C蛋白酶基因和3D基因的重组腺病毒表达载体.首先将P1-2A、3C和3D基因亚克隆连接到穿梭质粒pShuttle-CMV上,再将重组穿梭质粒用PmeI线性化后电转化携带有腺病毒骨架载体pAdeasy-1的大肠杆菌BJ5183感受态菌,经细菌内同源重组产生pAdcmv-p12x3c和pAdcmv-p12x3cd重组腺病毒质粒,经序列测定证实目的基因已正确的插入到腺病毒骨架载体中.重组腺病毒质粒经PacI线性化后转染HEK293细胞,转染1w内细胞出现典型病变.取转染细胞裂解液上清连续传代至第4代时,细胞于24~48h内即病变完全,收取接毒后24h细胞进行PCR和RT-PCR检测,表明目的基因已整合到腺病毒基因组内,且在mRNA水平上有表达.取第4、6、8和10代病毒,用蛋白酶K处理后可扩增出目的基因,证明此重组病毒可稳定存在.本研究为FMDV腺病毒活载体疫苗的研究奠定了基础.     

Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1).

The gene (HSD-1) coding a human sperm membrane protein (hSMP-1) was isolated from a human testis cDNA expression library using antibodies found in the serum of an infertile woman. HSD-1 was localized to a single locus on chromosome 9 and assigned to band 9p12-p13 by fluorescent in situ hybridization (FISH) mapping and DAPI (4,6-diamidino-2-phenylindole) banding, using rat/human somatic cell hybrids and metaphase chromosomes of human lymphocytes. In rescreening a testis lambdagt10 cDNA expression library, the full-length cDNA (HSD-1) and several truncated cDNAs with heterologous regions were isolated from positive clones. The heterology consisted of deletion, insertion and alteration of the 5'-end. These heterologous truncated fragments may be produced by alternative splicing of mRNAs. Two recombinant prokaryotic expression vectors were constructed with one of the heterologous fragment (clone #26) with and without the alternative 5'-end. Escherichia coli transfected with the construct containing the alternative 5'-end failed to produce the recombinant product, whereas those transfected with the vector lacking the 5'-end produced hSMP-1. DNASIS analysis of the structure of #26 mRNA suggests that the 5'-end has a stable secondary configuration that may maintain the mRNA in an inactivated state, whereby hindering its translation and preventing the expression of the gene.     

Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice

The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8⁺ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8⁺ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8⁺ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.     

重组黄热病毒E蛋白结构域Ⅲ作为亚单位疫苗的抗原性和免疫原性

目的：表达纯化黄热病毒（YFV）囊膜蛋白（E蛋白）结构域Ⅲ,研究其作为亚单位疫苗预防YFV、日本脑炎病毒（JEV）感染的可能。方法：扩增YFVE蛋白结构域Ⅲ（YFDⅢ）的cDNA片段333bp,将其连接到原核表达载体pET-32a（＋）中,构建原核表达载体pET-YFDⅢ,转化感受态大肠杆菌Rosetta（DE3）,IPTG诱导表达重组YFDⅢ;用纯化的YFDⅢ免疫新西兰兔和BALB/c鼠,检测相关抗体滴度。结果：在大肠杆菌中可溶性表达了YFDⅢ融合蛋白,表达量约占菌体蛋白的50%;Western印迹及ELISA分析表明,纯化的YFDⅢ具有良好的抗原性和免疫原性;利用纯化的YFDⅢ免疫新西兰兔,获得了高达1∶4×105滴度的抗YFV抗体和1∶2×104滴度的抗JEV抗体;利用纯化的YFDⅢ免疫BALB/c鼠,获得了1∶7×104滴度的抗YFV抗体和1∶2×103滴度的抗JEV抗体。结论：重组YFDⅢ有较好的免疫原性,具有开发成亚单位疫苗的潜能。     

Identification of a nuclear pheromone-sensitive protein kinase not identical to p34CDC28 in Saccharomyces cerevisiae

Abstract A fragment containing the full length cDNA from Aspergillus oryzae α-amylase has been amplified by PCR using specific synthetic oligonucleotides. The amplified cDNA was designed to favour its expression in yeast by modifying its upstream untranslated region. It was subcloned in the expression vector pYEXα1, placed under the control of the yeast CYC1-GAL10 promoter and used to transform Saccharomyces cerevisiae . Cells were then able to express and secrete active α-amylase to the medium in a regulated fashion. The recombinant enzyme had similar electrophoretic mobility and catalytic properties to the original A. oryzae α-amylase.     

单纯疱疹病毒2型糖蛋白D成熟肽基因毕赤酵母表达载体的构建及序列分析

构建单纯疱疹病毒2型包膜糖蛋白D成熟肽基因毕赤酵母表达载体,并对序列进行分析,为进行高抗原性的真核表达重组gD蛋白奠定基础。采用PCR扩增HSV2-gD成熟肽基因,将该段基因克隆于pGEM-T克隆载体,转化鉴定后,与巴斯德毕赤酵母表达载体(pPIC9K)酶切连接,转化大肠杆菌DH5α,筛选测序确定构建了pPIC9K?gD的真核表达载体,对克隆的序列进行分析,预测表达产物的理化特性及抗原性。结果显示,获得的重组的酵母表达载体pPIC9K-gD,测序结果证实为HSV2-gD成熟肽基因,序列分析其高度保守,预测蛋白分子量40.63kD,等电点pI为7.15,包含完整成熟肽分值达1.7的多个抗原决定簇。成功构建了HSV2-gD成熟肽基因的毕赤酵母表达载体。     

重组人白细胞介素11在毕氏酵母中的表达

将人白细胞介素11基因选用酵母偏爱密码子人工合成全基因,克隆到酵母分泌型表达载体pGENYk中,酶切线性化后原生质体转化导入酵母细胞进行整合,G418筛选得到多拷贝转化子,甲醇诱导表达,纯化制备产物,经过SDS－PAGE、Western印迹及体内外生物学活性等分析表明,产物活性与E.coli融合表达的Neumega一致。     

同源重组法制备口蹄疫病毒多基因重组腺病毒

通过细菌内同源重组的方法成功构建了含有O型口蹄疫病毒P1—2A和3C蛋白酶基因和3D基因的重组腺病毒表达载体。首先将P1—2A、3C和3D基因亚克隆连接到穿梭质粒pShuttle—CMV上,再将重组穿梭质粒用PmeI线性化后电转化携带有腺病毒骨架载体pAdeasy—1的大肠杆菌BJ5183感受态菌,经细菌内同源重组产生pAdcmv—p12x3c和pAdcmy—p12x3cd重组腺病毒质粒,经序列测定证实目的基因已正确的插入到腺病毒骨架载体中。重组腺病毒质粒经PacI线性化后转染HEK293细胞,转染1w内细胞出现典型病变。取转染细胞裂解液上清连续传代至第4代时,细胞于24～48h内即病变完全,收取接毒后24h细胞进行。PCR和RT—PCR检测,表明目的基因已整合到腺病毒基因组内,且在mRNA水平上有表达。取第4、6、8和10代病毒,用蛋白酶K处理后可扩增出目的基因,证明此重组病毒可稳定存在。本研究为FMDV腺病毒活载体疫苗的研究奠定了基础。     

α-葡萄糖苷酶基因序列分析及真核表达载体构建

目的：Aspergillus niger（CU-1）菌株α-葡萄糖苷酶基因（agdA）克隆并对其序列进行分析,构建该基因的真核表达载体。方法：设计合成的一对特异性引物,采用PCR以总DNA为模板,扩增得到DNA片段（D1）;采用RT-PCR方法扩增得到DNA片段（D2）。将DNA片段D1和D2转入大肠杆菌中并进行了序列测定,序列进行BLAST比对分析。将α-葡萄糖苷酶基因的cDNA片段与表达载体pGAPZαA连接,构建重组表达载体。结果：基因agdA大小为3 127bp,含有3个外显子和4个内含子。该基因的cDNA序列大小为2 958bp,包含完整的编码框,编码985个氨基酸。agdA基因序列与已发表的α-葡萄糖苷基因序列同源性达99%,其中有6个位置的碱基发生了变化。已成功构建重组表达载体pGAPZαA-agdA。结论：Aspergillus niger（CU-1）菌株α-葡萄糖苷酶基因序列分析及重组表达载体pGAPZαA-agdA的构建为在毕赤酵母中表达Aspergillus niger（CU-1）菌株的α-葡萄糖苷酶奠定基础。     

Mammalian and viral DNA sequences which interfere with the maintenance of a centromeric vector in yeast.

We constructed a recombinant plasmid by inserting into the pRS314 yeast centromeric plasmid vector the mouse DNA sequence responsible for the maintenance in transgenic mice of plasmid p12B1 (1). Such constructs could constitute convenient shuttle vectors between yeast and mouse cells. However, the recombinant molecule could not be established as a stable plasmid in Saccharomyces cerevisiae. A region with a limited similarity to the yeast centromere (CEN element) is present in this mouse sequence as well as in two other sequences subsequently identified in a data bank search using the CEN consensus. One of them is localized in Bovine Papillomavirus Type 1 DNA, and the other one in the human beta-globin locus. Once inserted in pRS314, these two sequences showed the same inhibitory effect on plasmid maintenance as the p12B1 mouse DNA fragment. This effect appears to depend on the simultaneous presence in the construct of one of the "CEN-like regions" and of an authentic CEN element. Non-centromeric yeast plasmids carrying one of the three sequences could replicate autonomously, and were even stabilized to a significant extent. These results identify in the genomes of higher eukaryotes and their viruses a family of sequences which cannot be simply cloned in centromeric yeast vectors.     

A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in Saccharomyces Cerevisiae

A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.