乙型肝炎病毒DNA聚合酶N末端蛋白基因芯片研究

目的:检测乙型肝炎病毒(HBV)DNA聚合酶N末端蛋白(TP) 的表达对肝母细胞瘤细胞HepG2基因表达谱的影响,进一步阐明TP在乙型肝炎陧性化及致肝细胞癌发生发展过程中的分子生物学机制. 方法:根据AF384372 HBVDNA病毒株序列设计、合成HBV DNA P-TP基因序列特异性的引物,以含有AF384372HBVDNA P全基因组cDNA的质粒G318A7作为模板,应用聚合酶链反应(PCR)技术扩增TP蛋白编码基因片段,以常规的分子生物学技术将获得的HBV DNA-TP 编码基因片段克隆到TA载体中进行核苷酸序列的测定, 构建真核表达载体pcDNA3.1(-)-TP.以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行DNA芯片分析. 结果:构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误.以单链可变区抗体的Western blot杂交技术证实构建的表达载体转染HepG2细胞之后有TP蛋白的表达,提取高质量的mRNA并进行逆转录成为cDNA,进行DNA 芯片技术分析.在1 152个基因表达谱的筛选中,发现有111 个基因表达水平显著上调,88个基因表达水平显著下调. 结论:应用基因表达谱芯片成功筛选了HBV DNA P-TP转染细胞后差异表达基因,为进一步阐明TP蛋白致病的分子生物学机制提供依据.     

日本血吸虫EST序列的电子延伸及结果分析

目的　建立日本血吸虫表达序列标签(SjEST)序列自动分析系统,筛选新基因、分析其表达谱。 　方法　建立本地化日本血吸虫专业数据库,整合序列同源性比较软件(BLAST)及片段整合分析软件(PHRAP),运用生物信息学策略编写程序控制EST自动延伸。建立本地化蛋白库,对延伸结果进行蛋白库同源性分析。实现对成批数据大规模自动分析 ,筛选出可能的新基因全长cDNA序列 ,并进行基因表达谱分析。　结果　延伸系统规则有效,延伸结果序列与原始序列高度同源。对552条EST进行自动分析,487条得到不同程度的延长,其中有104条EST原始序列检索无同源性 ,但经过延伸后获得高度同源性的联配序列信息。根据延伸结果尝试分析基因表达谱,筛选出27个可能新基因。　结论　建立了本地化的有效的SjEST序列自动分析系统。该系统为新基因的筛选及基因表达谱的分析提供了重要的参考信息。     

广州管圆线虫线粒体基因组比较分析

目的 目的 比较我国大陆地区广州管圆线虫线粒体基因组的多态性。 方法 方法 在种群遗传学研究基础上, 选取7条雌虫进行线粒体基因组的测定。根据广州管圆线虫线粒体基因组已知序列 （GQ398121） 设计12对引物, 进行PCR扩增, 并对目的片段进行测序和拼接。利用多种生物学软件对测定的线粒体基因组进行基因定位、 结构图绘制、 核苷酸及变异位点分析、 进化关系分析。结果 结果 获得5个不同遗传类型的线粒体基因组。这些基因组的大小相似、 结构相同, 即 13 491～13 502对碱基, 含12个蛋白编码基因, 2个核糖体基因, 22个tRNA基因, 2个较大的非编码区。上述基因紧密地排列在同一条DNA链上, 并具有相同的转录方向。通过对这些基因组的比对发现, 变异位点745个, 占整个基因组的5.5%。其中, 缺失/插入突变59个, 碱基颠换105个, 碱基转换581个。这些突变位点均匀地分布在整个线粒体基因组中。结论 结论本研究提供了丰富的广州管圆线虫线粒体基因的突变位点, 为构建种内鉴别诊断技术提供了基础。     

应用基因表达谱芯片技术筛选乙型肝炎病毒HBsAg基因转染细胞差异表达基因

目的应用基因表达谱芯片技术研究乙型肝炎表面抗原(HBsAg)主蛋白的表达对肝母细胞瘤系HepG2基因表达谱的影响.方法设计并合成HBsAg主蛋白基因序列特异性的引物,以含有HBV全基因组的质粒G376 A7作为模板,应用聚合酶链反应(PCR)技术扩增HBsAg蛋白编码基因片段,以常规的分子生物学技术构建表达栽体pcDNA3.1(-)-HBsAg.以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行DNA芯片分析并比较.结果在1152个基因表达谱的筛选中,发现有30个基因表达水平显著上调,29个基因表达水平显著下调.结论应用基因表达谱芯片成功筛选了HBsAg转染细胞后差异表达的基因,为深入研究HBsAg可能的生物学功能提供依据.     

胆固醇损伤内皮细胞差异表达基因的生物信息学分析

目的利用差异表达基因克隆方法（抑制消减杂交）获得大量的动脉粥样硬化相关候选基因和表达序列标签后，探讨如何进行后续基因表达及功能的研究。方法利用Internet网络上的数据库及生物学分析软件对胆固醇损伤内皮细胞后获得的差异表达基因进行核酸序列和蛋白质序列分析，探索差异表达基因克隆后的研究方法和思路。结果通过电子延伸得到一个684bp的全长cDNA序列；通过核酸序列分析，该序列定位在线粒体基因组的7587位～8270位，含有一个完整的开放阅读框，编码与氧化磷酸化相关的9个亚基。对其中一个亚基细胞色素氧化酶Ⅱ（COX2）分析得知，细胞色素氧化酶Ⅱ基因编码一段25．6kDa的弱酸性的信号锚蛋白，细胞色素氧化酶Ⅱ蛋白的三维结构是一个典型的椅式结构，它含有一段疏水区域，一个跨膜结构域和一个胞质结构域。运用蛋白的进化分析，得知细胞色素氧化酶Ⅱ蛋白胞质结构域的氨基酸序列在进化过程中高度保守。结论生物信息学技术是一种高效的获取疾病相关基因信息的方法，利用生物信息学方法对细胞色素氧化酶Ⅱ基因进行分析，获得了基因及其编码蛋白的相关信息，该蛋白参与电子传递，可能与细胞的氧化应激有关。     

基因表达谱芯片技术筛选XTP4基因转染细胞差异表达基因

目的 应用基因芯片技术,检测乙型肝炎病毒(HBV)X蛋白(HBxAg)反式激活基因XTP4的表达对肝母细胞瘤细胞系HepG2基因表达谱的影响,进一步阐明XTP4蛋白可能的分子生物学功能。方法 设计并合成XTP4基因序列特异性的引物,应用聚合酶链反应(PCR)技术扩增XTP4基因片段,以常规的分子生物学技术将获得的XTP4编码基因片段克隆到TA载体中进行核苷酸序列的测定,构建真核表达载体pcDNA3.1(-)-XTP4。以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空表达载体pcDNA3.1(-)的HepG2细胞进行cDNA芯片分析。结果 构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误。提取高质量的mRNA,逆转录为cDNA,进行cDNA芯片分析。在1152个基因表达谱的筛选中,发现有21个基因表达水平显著上调,38个基因表达水平显著下调。结论 应用基因表达谱芯片成功筛选了XTP4转染细胞后差异表达基因,为进一步阐明XTP4蛋白可能的生物学功能提供依据。     

应用基因表达谱芯片技术筛选XTP6基因转染细胞差异表达基因

目的应用基因芯片技术,检测乙型肝炎病毒(HBV)X蛋白(HBxAg)反式激活基因XTP6的表达对肝母细胞瘤系HepG2基因表达谱的影响,进一步阐明XTP6蛋白可能的分子生物学功能.方法设计并合成XTP6基因序列特异性的引物,应用聚合酶链反应(PCR)技术扩增XTP6基因片段,以常规的分子生物学技术将获得的XTP6编码基因片段克隆到TA载体中进行核苷酸序列测定,构建真核表达载体pcDNA3.1(-)-XTP6.以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空表达载体pcDNA3.1(-)的HepG2细胞进行cDNA芯片分析.结果构建的表达载体经过限制性内切酶分析的DNA序列测定,证实准确无误.提取高质量的mRNA,逆转录为cDNA,进行cDNA芯片分析.在1 152个基因表达谱的筛选中,发现21个基因表达水平显著上调,18个基因表达水平显著下调.结论应用基因表达谱芯片成功筛选了XTP6转染细胞后差异表达基因,为进一步阐明XTP6蛋白可能的生物学功能提供依据.     

应用基因表达谱芯片技术筛选HCV p7蛋白反式调节基因

目的应用基因芯片技术对pcDNA3.1(-)和pcDNA3.1(-)-p7分别转染的HepG2细胞的基因表达谱进行分析,筛选能被HCV p7蛋白反式调节的靶基因,研究p7蛋白的生物学功能。方法以含有HCV全基因组的pBRTM质粒作为模板,应用聚合酶链反应(PCR)扩增p7蛋白编码基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1(-)-p7。以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行DNA芯片分析并比较。结果构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误,提取高质量的总mRNA并逆转录成cDNA,进行DNA芯片技术分析。在1152个基因表达谱的筛选中,有1个基因表达水平显著上调,22个基因表达水平显著下调。结论 HCV p7蛋白是一种反式调节因子,p7基因的表达对于肝细胞基因表达谱有显著影响。     

丙型肝炎

丙型肝炎病毒不同片段抗体蛋白芯片的制备及评价；HCV非结构蛋白3反式激活基因6转染肝癌细胞的基因表达谱芯片分析；血清HCVRNA检测在慢性丙型肝炎诊断中的意义；丙型肝炎病毒结构蛋白E1在昆虫细胞中的表达及定位；应用抑制性消减杂交技术克隆筛选丙型肝炎病毒F蛋白结合蛋白2反式激活基因；丙型肝炎病毒C和E1区的DNA改组。[编者按]     

C2株蓝氏贾第鞭毛虫SUMO特异性蛋白酶基因的克隆、生物信息学分析及其催化活性区的原核表达

目的 克隆C2株蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)的SUMO-Specific Protease(SENP)基因,并对其序列进行生物信息学分析,原核表达贾第虫SENP的催化活性区。方法 提取C2株贾第虫基因组DNA,以基因组DNA为模板获得SENP编码区全长片段,连入克隆载体pGM-T,测序后进行生物信息学分析;根据分析结果克隆SENP的催化活性区,构建其原核表达载体pET-28a(+)-SENPc,在E.coli Rosetta(DE3)中诱导表达,SDS-PAGE及Western blot观察表达结果。结果 成功克隆了C2株贾第虫SENP编码区全长序列,生物信息学分析显示C2株贾第虫SENP蛋白序列与WB株相同,二级结构以无规则卷曲为主,其催化活性区位于126-497aa,被一段插入序列分割成两个部分;构建了SENP催化活性区原核表达载体并在大肠杆菌中高效表达,在相对分子量约43 kD的位置出现目的 蛋白条带,与理论值相符。结论 成功克隆了贾第虫SENP基因并原核表达了其催化活性区,为贾第虫SENP蛋白功能的研究提供了基础。     

Isolation and characterization of rat skeletal muscle and cytoplasmic actin genes.

Southern blots of rat genomic DNA indicate the existence of at least 12 EcoRI DNA fragments containing actin gene sequences. By using specific probes and stringent conditions of hybridization, it was found that only one of these fragments contains sequences of the skeletal muscle alpha-actin gene. Recombinant bacteriophages originating from eight different actin genes were isolated from rat genomic DNA libraries. One of them, Act 15, contains the skeletal muscle actin gene. Another clone, Act I, contains a gene coding for a cytoplasmic actin, identified tentatively as the beta-actin gene. Both genes have a large intron very close to the 5' end of their transcribed region, followed by several small introns. DNA sequence analysis and comparison with the available data on actin genes in other organisms indicated an interesting relationship between the positions of introns and the evolutionary relatedness. Several intron sites are conserved from at least the echinoderms to the vertebrates; others appear to be present in some actin genes and not in others.     

全基因组芯片结合通路分析筛选心力衰竭患者心肌细胞差异基因

目的 利用人全基因组芯片分析心力衰竭(心衰)患者与正常人心肌细胞差异表达基因,结合已知基因功能分类着重对细胞信号通路进行分析.方法 应用华联公司人全基因组芯片,检测4例心衰患者及4例脑死亡的正常人心肌细胞的基因表达谱,并以实时荧光定量PCR技术对基因芯片检测结果 进行验证.将心衰差异表达量≥1.2倍或≤-1.2倍的基因经BioCarta通路和KEGG通路进行信号通路分析.结果 4例心衰心肌标本与正常对照比较,表达量变化1.2倍以上的基因有2806条;变化2倍以上的有399条基因.经BioCarta通路分析,心衰时涉及11条通路蛋白.经KEGG通路分析,涉及16条通路.结论 心衰时心肌细胞巾有大量的基因呈现差异表达,并激活了多条促进细胞凋亡通路及涉及转录调控的通路.运用基因芯片和生物学通路相结合的方法 ,分析基因表达谱能准确而且有的放矢地研究与心衰病理生理的相关基因.     

Cytochrome c gene-related sequences in mammalian genomes.

We use a rat cytochrome c gene that we previously isolated and determined the sequence of to estimate the number of related sequences present in the rat genome. Approximately 25 different EcoRI restriction endonuclease fragments from total rat DNA hybridize to the gene of known structure. Four of these correspond to homologous sequences present in four different lambda Charon 4A-rat cytochrome c recombinants previously isolated. Intact or nearly intact genes appear to reside on almost all of the genomic fragments, because they hybridize strongly to gene subfragments representing both 5' and 3' portions of the coding sequence as well as to 3' noncoding DNA that is found specifically associated with the coding region. A subgroup of about six of the fragments also shares homology within the 73 nucleotides immediately preceding the AUG codon. An intron-specific probe reveals only the EcoRI fragment from which it was derived and one other genomic fragment. On the basis of the temperature of complete dissociation of the coding region probe in 0.75 M NaCl/0.075 M Na3 citrate/50% (vol/vol) formamide, the 25 fragments are separable into three stringency classes of 40-50 degrees C, 50-55 degrees C, and 55-60 degrees C. The latter, high-stringency group of about seven fragments includes those cloned in the recombinant phage isolates, whose regions homologous to cytochrome c are shown to differ from the purified gene of known sequence by an amount equivalent to about 2% mismatched bases. Families of cytochrome c gene-related sequences are also found in the genomes of several other mammals, including humans.     

Mapping of the genes encoding the HLA-DR alpha chain and the HLA-related antigens to a chromosome 6 deletion by using genomic blotting.

We have used genomic blotting with DNA from a human cell line that has a small deletion on chromosome 6 (6.3.6) and from its parent cell line (T5-1) to map DNA fragments complementary to cloned DNA sequences encoding the HLA-B7 antigen (class I) and the alpha chain of the HLA-DR antigen (class II). The 6.3.6 variant fails to express the HLA-A, -B, -C, and -DR and MB specificities associated with one of the parental T5-1 haplotypes and has a visible deletion in the short arm of one chromosome 6 (1). The gene locus assignment was based on the expectation that, if the chromosomal location of the DNA sequences used as a hybridization probe were within the deletion, then the relative amount or size (or both) of genomic restriction fragments that hybridize to the probe in T5-1 and in 6.3.6 DNAs should differ predictably. By comparing the genomic blot patterns from T5-1 and 6.3.6 DNAs, we have shown directly that the loss of haplotype expression was due to deletion of the structural genes and have mapped the structural gene for the HLA-DR alpha chain to the chromosomal location (6p2105-6p23) defined by the 6.3.6 deletion. A cDNA clone encoding the alpha chain of the HLA-DR antigen hybridized to two genomic fragments, 4.2 and 3.8 kilobases long, generated by Bgl II digestion of T5-1 DNA. The 4.2-kilobase fragment was absent from DNA derived from the 6.3.6 deletion variant. Thus, this fragment could be assigned to the parental chromosome 6 with the A1, B8, DR3 haplotype, and the 3.8-kilobase fragment, to the chromosome 6 with the A2, B27, DR1 haplotype. In addition, comparison of the T5-1 and 6.3.6 genomic blot patterns obtained with the HLA-B7 probe revealed dosage differences for all of the class I genomic fragments generated by BamHI digestion, suggesting that all of the class I loci map to the region 6p2105-6p23.     

A 78-kilobase region of mouse chromosome 3 contains salivary and pancreatic amylase genes and a pseudogene.

Genetic studies have demonstrated that salivary and pancreatic amylase genes are closely linked in human and mouse. To analyze the arrangement of genes within the amylase cluster, a library of YBR mouse genomic DNA was cloned in the cosmid vector pJB8. Clones containing amylase genes were identified by hybridization with amylase cDNA probes. Salivary and pancreatic amylase genes were isolated on separate cosmid clones, but no overlapping clones were evident from the initial screening. A strategy for the rapid isolation of terminal noncoding fragments from the cosmid clones was developed. By using these terminal fragments for chromosome "walking," a map of 78 kilobases of the amylase gene region was constructed. The salivary and pancreatic amylase genes are present within this region in the same 5'-to-3' orientation, separated by 22 kilobases of genomic DNA. A truncated amylase pseudogene is located 10 kilobases downstream from the pancreatic amylase gene.     

病毒性心肌病小鼠心肌线粒体基因表达变化

目的观察实验性病毒性心肌病小鼠心肌线粒体基因表达变化情况,探讨线粒体基因表达变化在病毒性心肌病中的发病作用。方法用柯萨奇病毒B3m反复增量感染Balb/c小鼠,建立心肌病动物模型,用包容35 852条基因的cDNA微矩阵芯片分析心脏组织特异的mRNA表达,筛选出明显差异表达的线粒体相关基因。采用原位末端脱氧核糖核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL)观察心肌细胞凋亡情况。结果共有31个心肌病小鼠心肌线粒体能量代谢相关基因、心肌细胞线粒体凋亡及相关调节基因明显差异表达。心肌病小鼠心肌细胞凋亡的数目明显高于对照组(t=8.85,P<0.01)。结论心肌线粒体功能受损伴有心肌细胞凋亡发生是病毒性心肌病的重要发病机制。     

Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning

To accelerate gene isolation from plants by positional cloning, vector systems suitable for both chromosome walking and genetic complementation are highly desirable. Therefore, we developed a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, that can accept and maintain large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. Furthermore, it has the cis sequences required for Agrobacterium-mediated gene transfer into plants. We cloned large genomic DNA fragments of Arabidopsis thaliana into the vector and showed that most of the DNA fragments were maintained stably. Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into Arabidopsis with high efficiency and shown to be inherited faithfully among the progeny. Furthermore, we demonstrated the practical utility of this vector system for positional cloning in Arabidopsis. A TAC contig was constructed in the region of the SGR1 locus, and individual clones with ca. 80-kb inserts were tested for their ability to complement the gravitropic defects of a homozygous mutant line. Successful complementation enabled the physical location of SGR1 to be delimited with high precision and confidence.