EB病毒胸苷激酶基因的定向克隆

采用异硫氰酸胍（ＧｕＳＣＮ）和硅藻从Ｂ９５－８细胞中快速抽摸板ＤＮＡ。根据ＥＢ病毒（ＥＢＶ）Ｂ９５－８株ＤＮＡ全序列及编码ＥＢＶ胸苷激酶（ＴＫ）的开放读框ＢＸＬＦ１的结构,设计合成一对引物,并在引物的５′一端分别引入ＥｃｏＲＩ和ＰｓｔＩ切点,用ＰＣＲ技术扩增出一含完整的ＥＢＶＴＫ基因的１．８４３ＫｂＤＮＡ片段,ＮｃｏＩ酶切分析鉴定,ＥｃｏＲＩ／ＰｓｔＩ双酶切ＰＣＲ产物和载体,使目的基因定向克隆至选     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

狂犬病毒aG株糖蛋白在pET原核系统中的表达及纯化

在狂犬病的临床诊断和基础研究中都迫切需要大量纯度高且价廉的狂犬病毒糖蛋白抗原。本文应用带有Ｈｉｓ６尾的ｐＥＴ原核表达系统对狂犬病毒（ＲＶ）ａＧ株的糖蛋白（ＧＰ）进行表达和纯化。构建的融合表达载体ｐＥＴ－ａＧ１和ｐＥＴ－ａＧ２（－５７ｂｐ）分别含有ＲＶａＧ株ＧＰ基因的全序列及删除了为ＧＰ信号肽编码的５８个碱基的序列。用ＳＤＳ 聚丙烯酰胺凝胶电泳、免疫印迹、间接ＥＬＩＳＡ检测都证明表达产物为ＲＶＧＰ,且位于菌体中的包涵体内。经固定化金属螯合层析（ＩＭＡＣ）提纯,ｐＥＴ－ａＧ１表达产物有较高的特异性和纯度,可用作测定ＲＶＧＰ抗体的免疫诊断试剂。     

黄地老虎颗粒体病毒DNA片段的克隆与颗粒体蛋白基因的定位

用ｐＵＣ１９质粒作载体,克隆了黄地老虎颗粒体病毒（Ａｇｒｏｌｉｓｓｅｇｅｔｕｍｇｒａｎｕｌｏｓｉｓｖｉｒｕｓ,简称ＡｓＧＶ）ＤＮＡＰｓｔＩ－Ｄ．Ｅ．Ｆ．Ｇ．Ｈ．Ｊ．Ｋ．等７个片段。以［￣（３２）Ｐ］－ｄＣＴＰ标记的油桐尺蠖核型多角体病毒（Ｂｕｚｕｒａｓｕｐｐｒｅｓｓａｒｉａｎｕｃｌｃａｒｐｏｌｙｈｅｄｒｏｓｉｓｖｉｒｕｓ简称ＢｓＮＰＶ）多角体蛋白基因为探针,在３７℃条件下对ＡｓＧＶ）颗粒体蛋白基因进行了定位,将其分别定位于ＢｓｌⅡ－Ｓ或ＴＰｓＴＩ－Ａ或Ｂ和ＥｃｉＲＩ－Ａ片段上。     

传染性法氏囊病病毒中国强毒株A节段cDNA基因的克隆和序列分析

以来自哈尔滨传染性法氏囊病病毒（ＩＢＤＶ） 强毒株（Ｈａｒｂｉｎ 毒株,Ｈ） 的基因组ＲＮＡ为模板,用反转录聚合酶链反应（ＲＴ－ ＰＣＲ） 的方法得到了其Ａ 节段的全长ｃＤＮＡ 片段,分５＇端（１ ６５９ｂｐ） 和３＇端（１ ４４４ｂｐ） 上下两段分别克隆到ｐＧＥＭＢ○Ｒ － Ｔ 载体上,测定了其核苷酸顺序,在长为３ １０１ ｂｐ 中含有两个阅读框ＯＲＦＡ１ 和ＯＲＦＡ２ ,分别编码１ ０１２ 个氨基酸的前体蛋白（ＶＰ２ － ４ －３） 和１４５ 个氨基酸的ＶＰ５,ＯＲＦＡ１ 和ＯＲＦＡ２ 有部分的重叠。将核苷酸序列及推测出的氨基酸序列与已报道的ＩＢＤＶ 血清Ⅰ型和Ⅱ型毒株的相应序列进行了比较,结果表明：Ｈ 毒株与其它血清Ⅰ型毒株之间,在核苷酸水平上存在２５ｂｐ － ２６７ｂｐ 的差异;在氨基酸水平上存在１７ ～４０ 个氨基酸的差异。在ＶＰ２ － ４ － ３ 内比较显示,Ｈ 毒株与Ｐ２ 、Ｃｕ－ １ 之间氨基酸的差异最小为１ ．７％ ,Ｈ 毒株与ＵＫ６６１ 之间氨基酸的差异最大为３ ．９ ％ 。变异主要发生在ＶＰ２ 的可变区（２０６ － ３５０ 位氨基酸） ,在Ｈ 毒株所特有的１２ 个氨基酸当中,该区就占５ 个,代表１ ．７６ ％ 的变异。ＶＰ４、ＶＰ３ 和ＶＰ５区各有     

利用脊髓灰质炎病毒5‘NCR和RNA聚合酶基因可提高外源基因表达

将含脊髓灰质炎病毒（ＰＶ）ＲＮＡ聚合酶的不同长度基因片段克隆到载体ｐＳＧ５质粒上,分别构建了４个表达ＲＮＡ聚合酶的质粒。体外转录实验证明,ｐＳＧ５－ＰＯＬ１．９９和ｐＳＧ５－ＰＯＬ２．０３质粒转染细胞的提取物促进了特异的ＲＮＡ转录,表明两质闰可表达ＲＮＡ聚合酶。将ＰＶ的５’ＮＣＲ序列插在载体ｐＧＲＥＥＮ ＬＡＮＴＥＲＮ－１的ＣＭＶ启动子下游,构建了ｐＧＲＥＥＮ ＬＡＮＴＥＲＮ－１－５’ＮＣＲ质粒;     

萝卜抗真菌蛋白1（Rs—AFP1）在大肠杆菌中的融合表达及其对大丽轮 …

利用聚合酶链式反应（ＰＣＲ）获得了萝卜（Ｒａｑｈａｎｕｓ ｓａｔｉｖｕｓ Ｌ．）抗真菌蛋白１（Ｒｓ－ＡＦＰ１）基因编码区核苷酸序列。将整个阅读框架片段和云除了Ｎ－端信号肽序列的片段分别装入原核表达载体ｐＥＴ－３２ｇ（＋）中,在大肠杆菌中表达,发现带有信号 的Ｒｓ－ＡＦＰ１不能在大肠杆菌中表达,而当这一序列去除后,表达出约２７ｋＤ的Ｒｓ－ＡＦＰ１的融合蛋白。用凝血酶处理融合蛋白以云除Ｎ－端Ｈｉｓ．ｔ     

草鱼出血病病毒多肽的基因定位

用聚丙烯酰胺凝胶电泳分离纯化的草鱼出血病病毒ＧＣＨＶ８７３的基因组ｄｓ－ＲＮＡ的１１个片段,分别在麦胚无细胞翻译体系中进行翻译。其翻译产物经ＳＤＳ－ＰＡＧＥ系统分析。结果表明,基因组片段１、２、３、４、５、和１０分别编码病毒核心衣壳的结构多肽ＶＰ１、ＶＰ２、ＶＰ３、ＶＰ４、ＶＰ５和ＶＰ１０,片段６和７分别编码病毒外层衣壳的结构多肽ＶＰ６和ＶＰ７。片段８和９分别编码５２ｋＤ和４１ｋＤ的多肽,片段１１编码两种多肽,分子量分别为２９ｋＤ和１９．５ｋＤ,它们与病毒结构多肽无明显对应关系。病毒基因组与多肽大体是一一对应的关系。     

系统感染TMV的番茄叶胞外β-1,3-葡聚糖酶

系统感染ＴＭＶ （ｔｏｂａｃｃｏ ｍ ｏｓａｉｃ ｖｉｒｕｓ）的番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．）叶胞外蛋白提取液经冰冻干燥浓缩、－ ２０℃丙酮沉淀、ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析和Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶层析纯化,获得ＰＡＧＥ均一的β－１,３－葡聚糖酶．ＳＤＳ－ＰＡＧＥ证明,它包含分子量为３６ ｋＤ 和２７ ｋＤ的两个同工酶．以昆布多糖为底物,酶的最适ｐＨ 在４．８—５．２之间,在ｐＨ ４—８稳定;酶的最适温度在３０—４０℃之间,在４０℃保温１ｈ 后酶活性不变;Ｋｍ 值为９．２ ｍ ｇ／ｍ Ｌ．在系统感染ＴＭＶ 的番茄叶胞外蛋白提取液中,有分子量为２２ ｋＤ、２７ ｋＤ和３６ ｋＤ的３个β－１,３－葡聚糖酶同工酶     

甜菜坏死黄脉病毒内蒙分离物RNA3序列分析及编码25kD蛋白基因在大肠杆菌中的表达

以甜菜坏死黄脉病毒（ＢＮＹＶＶ）内蒙分离物的总ＲＮＡ为模板,通过反转录－ＰＣＲ扩增获得ＢＮＹＶＶＲＮＡ３全长ｃＤＮＡ。将其克隆到ｐＧＥＭ－７Ｚｆ（＋）上,得到重组质粒ｐＧＢＹ５６。序列分析结果表明,内蒙分离物ＲＮＡ３基因组全长为１７７５ｎｔ,其中包含３个开放阅读框架,分别编码２５ｋＤ蛋白、４．６ｋＤ蛋白和一种由５９个氨基酸组成的Ｎ蛋白。与法国Ｆ２分离物、德国Ｇ１分离物和日本Ｓ分离物相比,其核苷酸序列的同源性分别为９６．４％、９６．８％和９７．３％。将２５ｋＤ蛋白编码基因克隆到ｐＪＷ２上,构建了该基因的原核表达载体。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析结果表明,２５ｋＤ蛋白基因在Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）中经温度（４２℃）诱导后,可特异地表达２５ｋＤ蛋白     

腺病毒4型E3区核苷酸序列测定及其基因结构与功能分析

本文将Ａｄ４基因组相当于７３．３－８９．２基因图谱单位的ＤＮＡ片段进行了序列测定及基因结构分析,它包括了Ａｄ４Ｅ３区全基因及该区两侧的部分序列。序列分析表明,Ａｄ４ Ｅ３区从ＴＡＴＡＡ ｂｏｘ起至该区基因结束共４７７８ｂｐ,编码１１个大于６ｋＤ的开放读码框架。对Ａｄ４ Ｅ３区ＯＲＦ分析结果表明,Ａｄ４ Ｅ３区编码的１９．３ｋ,１５ｋ,１０．４ｋ蛋白,分别与Ａｄ２ Ｅ３区的ｇｐ１９ｋ,１４．ｋ和１０     

Genotype and allele frequencies of polymorphic cytochromes P450 CYP1A2 and CYP2E1 in Mexicans

CYP1A2 and CYP2E1 are two of the main cytochrome P450 isoforms involved in the metabolism of commonly used drugs and xenobiotic compounds considered to be responsible for or possible participants in the development of several human diseases. Individual susceptibility to developing these pathologies relies, among other factors, on genetic polymorphism which depends on ethnic differences, as the frequency of mutant genotypes varies in different human populations. Thus the aim of this study was to investigate the frequency of CYP1A2 5'-flanking region and CYP2E1 Rsa I/Pst I polymorphisms in Mexicans by PCR-RFLP methods. The DNA of 159 subjects was analysed and mutant allele frequencies of 30% for CYP2E1 Rsa I/Pst I sites and 43% for CYP1A2 5'-flanking region were found. These frequencies are higher than those previously reported for other human populations.     

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.     

Sequence duplication and internal deletion in the integrated human papillomavirus type 16 genome cloned from a cervical carcinoma.

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.     

对虾白斑综合症病毒重组cDNA克隆的构建与分析

取经人工注射感染了对虾白班综合症病毒40-45h的凡纳对虾鳃组织,分离mRNA,以mRNA为模板合成双链cDNA,并克隆于PUC质粒的Not I/Sal I位点,构建了1000余株对虾感染后期鳃细胞的重组cDNA克隆,重组质粒经PCR鉴定插入片段,DNA斑点杂交分析目的片段,测定了20株对虾白斑综合症病毒的重组cDNA克隆的末端DNA序列,并对其进行了包含存在的开放阅读框架,启动区上游序列、编码产物的特性等分析。结果显示,PCR产物在0.3-1.6kb之间;大于1kb的克隆中有31.8%的克隆为白斑综合症病毒的重组cDNA克隆。已测序的不包含同源序列的13株克隆中含有14个开放阅读框,其中11个上游可检出启动子基序,4个可检出启动子调制元件,ORF转译产物的特性基序分析显示,有2个ORFs可检出锌指基序,3个ORFs可检出亮氮酸拉链基序,2个ORFs可检出NTP结合基序,未检定核定位信号基序。     

Analysis of the replication region of a mycobacterial plasmid, pMSC262.

We determined the nucleotide sequence of a DNA fragment which contains the replication region of pMSC262, a Mycobacterium scrofulaceum plasmid used to construct the Mycobacterium-Escherichia coli shuttle vector. The complete sequence of the fragment contained 2,504 bp with an overall G+C content of 69.8%. By deletion analysis, we found that the minimum length required for plasmid replication in M. bovis BCG was about 1.6 kb. Within this region, several open reading frames (ORFs) and a putative replication origin (ori) were identified by computer analysis. One of the ORFs, ORF2, which encodes a putative 28.9-kDa basic protein with characteristics of DNA-binding proteins, appeared to be involved in replication of the plasmid in BCG. By separation of ORF2 and the putative ori region, it was revealed that the relative locations of ORF2 and the putative ori region are likely important for replication in BCG. No DNA or amino acid homologies were found between this replication region and that of pAL5000, another mycobacterial plasmid used for vector plasmid construction. In addition, we found that this replicon did not lead to replication in E. coli and was compatible in BCG with pAL5000-derived vector plasmid pYUB75 (R. G. Barletta, D. D. Kim, S. B. Snapper, B. R. Bloom, and W. R. Jacobs, J., J. Gen. Microbiol. 138:23-30, 1992).     

胸腺素α_1 基因的克隆表达及其生物活性

 胸腺素α1(thymosinalpha 1 ,Tα1)作为一种免疫增强剂 ,临床用途广泛 .为大量制备Tα1,按大肠杆菌惯用密码子合成Tα1基因 ,克隆于质粒pUC1 9的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后 ,串联为 4串体 (Tα1④ ) ,经再次测序确认后克隆入pThioHisA的EcoRⅠ和PstⅠ位点 .转化大肠杆菌T0P1 0 ,酶切鉴定正确后 ,经 1mmol LIPTG诱导 4h ,获得硫氧还蛋白与Tα1④的融合表达 ,用离子交换层析纯化融合蛋白 .溴化氰裂解融合蛋白 ,释放出Tα1单体 ,经离子交换色谱纯化出Tα1.采用3 H TdR参入法进行生物活性测定 ,证实融合蛋白和Tα1均具有刺激小鼠脾淋巴细胞分裂增殖的能力 .     

Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.