鸡传染性支气管炎病毒中国分离株LX4纤突蛋白基因分子特征

应用RT-PCR方法分别扩增了中国地方分离株IBV LX4 S1和S2基因并进行了基因的克隆和序列测定.结果发现,LX4 S基因由3495个核苷酸组成,编码一条1164个氨基酸残基组成的多肽.S基因编码产物裂解后形成的S1和S2亚单位分别由539和625个氨基酸残基组成.LX4 S基因推导氨基酸切割识别位点序列为HRRRR,与A2、SD/97/02和Z株相同,而与其它国内外参考毒株不同.与国内外10株已报道的具有全S基因序列的IBV参考毒株比较,LX4与国内分离毒株SD/97/02的核苷酸和氨基酸同源性最高.与32株国内外参考毒株的S1基因进化树分析比较表明,LX4与A2、SD/97/02、Z、TJ/96/02、JX/99/01和SAIBWJ等7个国内分离株在同一亚群内.在该亚群内,LX4与A2和SD/97/02亲缘关系更近,且三者在高变区和抗原表位均具有高度的同源性,而与本亚群内其它参考毒株对应的高变区和抗原表位同源性差异较大.LX4与H120 S1基因编码氨基酸的同源性虽然高于其它国外参考毒株,但同源性仍然较低,为75%.与参考毒株比较,LX4 S2基因的点突变造成其推导的氨基酸序列有11个位点发生改变,这些突变可能影响S2与S1蛋白之间的相互作用,从而影响S蛋白与特异性抗体的结合.     

鸡传染性支气管炎中国地方分离毒株LX4mRNA1ORF1的遗传变异特征

应用RT-PCR方法扩增了冠状病毒鸡传染性支气管炎中国分离毒株LX4的mRNA1 ORF1,并将其进行了克隆、序列测定和分析.表明LX4mRNA1基因包含2个ORF,其中ORF1a由11 916个核苷酸组成,编码一条3 971个氨基酸残基组成的多肽.ORF1b由7 950个核苷酸组成,编码2 649个氨基酸残基组成的多肽.与美国Beaudette株对应的ORF1a核苷酸及推导的氨基酸序列同源性均为85%,与对应的ORF1b的同源性分别为89%和95%.二者ORF1a和ORF1b重叠区推测的"滑脱序列"(slippery sequence)核苷酸序列一致,"假结"(pseudoknot)基本结构相似.不同之处在于"假结"第2环中LX4的第7位为A,而Beaudette株在该位置为G.与Beaudette株比较,LX4 ORF1a核苷酸变异最频繁的区域集中在第2 656～3 098位碱基处,且在该区域有60个碱基的插入,导致推导的氨基酸序列插入了20个氨基酸残基.LX4 ORF1b序列缺失9个核苷酸,主要集中在第5 168～5 181位之间,导致对应的氨基酸缺失3个.但这些差异是否与病毒的生物学特性有关不明.     

中国1995～2004年鸡传染性支气管炎病毒地方分离株膜蛋白基因的遗传变异分析

应用RT-PCR方法扩增到了我国1995～2004年20株IBV现地分离株的膜蛋白(Membrane,M)基因片段.序列测定表明,20株IBV分离株M基因开放阅读框由672～681bp组成,编码由223～226个氨基酸残基组成的多肽.与我国分离株LX4相比,M基因推导氨基酸序列的变异主要发生在2～17位、221～223位,其中4～6位存在氨基酸的插入和缺失,导致IBV毒株间M蛋白糖基化位点的差异.与GenBank中34株IBV参考毒株M蛋白基因推导氨基酸序列进行比较和分析,系统进化关系显示54株IBV毒株分属于5个进化群.我国IBV分离株M基因在进化关系上较为独立,主要分布在第Ⅱ群和第Ⅳ群,其中第Ⅱ群分离株和中国台湾毒株进化关系密切.此外,参考IBV国内分离株S1基因及N基因系统发育进化树的研究结果,并与M基因进行比较,表明我国IBV也存在着基因重组现象,尤其是疫苗毒和流行毒之间的重组.     

引起免疫失败的IBV分离株的生物学特性及结构基因分析

本研究应用鸡胚尿囊腔接种的方法,从广西患传染性支气管炎的免疫失败的病鸡中分离到一株传染性支气管炎病毒(IBV)(命名GX-YES),通过间接血凝试验、动物回归试验和气管环交叉中和试验对该毒株进行鉴定和主要生物学特性的研究,同时利用RT-PCR技术扩增克隆该病毒的SI基因和N基因并测定其核苷酸序列.结果表明:该病毒株有间接血凝性;致病性较强;血清型不同于常用疫苗株H120、Ma5、4/91.同源性分析表明,SI基因核苷酸和氨基酸序列与参考株的同源性分别为63.5%～81.2%和50.7%～78.8%;N基因核苷酸和氨基酸序列与参考株的同源性分别为85.7%～87.2%和89.5%～91.7%;SI基因和N基因系统进化分析表明分离株与其它参考毒株的亲缘关系较远.SI基因和N基因分型与血清学试验结果相吻合.本研究结果提示了广西分离株GX-YL5可能是一个新的变异株,这可能是目前免疫失败的重要因素,同时也为研制适合本地使用的IBV疫苗提供了科学依据和物质基础.     

中国2000-2004年鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析

对2000-2004年从中国9个省市分离到的13株IBV的核蛋白基因片段进行序列测定及分析的结果表明,13株IBV分离株核蛋白基因均含有一个长1230bp的ORF,但存在基因突变现象.与GenBank中的42株参考毒株核蛋白基因序列进行比较和分析,系统进化关系显示55株IBV毒株分属于9个群.第Ⅰ-Ⅲ群主要包括美国、日本、荷兰等国家以及中国的部分IBV分离株和疫苗株.其中本研究中的CK/CH/LHN/00I可能为一株分离的疫苗毒,CK/CH/LSD/03I、CK/CH/LDL/01I可能为重组毒.而中国近十年来分离的IBV毒株主要分布在第Ⅵ-Ⅷ群中,此3群内IBV毒株之间N蛋白推导氨基酸同源性为88.3%～100%,与其他各群之间同源性为62.3%～95.1%.因此,此基因型的IBV毒株可能在中国已有较长时期的存在且发生了较大程度的变异.其中第Ⅵ群中两株韩国分离株与中国IBV分离株具有较近的亲缘关系.以上结果表明,中国大多数IBV分离株在N基因进化关系上较为独立,与国外毒株相比,和韩国毒株进化关系密切.此外,中国IBV毒株基因重组现象更加普遍,尤其是疫苗毒和野毒之间的重组.     

中国2000—2004年鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析

对2000-2004年从中国9个省市分离到的13株IBV的核蛋白基因片段进行序列测定及分析的结果表明,13株IBV分离株核蛋白基因均含有一个长1230bp的ORF,但存在基因突变现象。与GenBank中的42株参考毒株核蛋白基因序列进行比较和分析,系统进化关系显示55株IBV毒株分属于9个群。第Ⅰ-Ⅲ群主要包括美国、日本、荷兰等国家以及中国的部分IBV分离株和疫苗株。其中本研究中的CK/CH/LHN/00I可能为一株分离的疫苗毒,CK/CH/LSD/03I、CK/CH/LDL/01I可能为重组毒。而中国近十年来分离的IBV毒株主要分布在第Ⅵ-Ⅷ群中,此3群内IBV毒株之间N蛋白推导氨基酸同源性为88.3%～100%,与其他各群之间同源性为62.3%～95.1%。因此,此基因型的IBV毒株可能在中国已有较长时期的存在且发生了较大程度的变异。其中第Ⅵ群中两株韩国分离株与中国IBV分离株具有较近的亲缘关系。以上结果表明,中国大多数IBV分离株在N基因进化关系上较为独立,与国外毒株相比,和韩国毒株进化关系密切。此外,中国IBV毒株基因重组现象更加普遍,尤其是疫苗毒和野毒之间的重组。     

鸡传染性支气管炎病毒变异株（793／B）核蛋白基因的克隆与序列分析

根据GenBank已经发表的传染性支气管炎病毒(IBV)N全基因组序列设计引物,对IBV793/B分离毒株N基因进行克隆与序列分析。结果表明,IBV793/B的N基因由1229bp组成,与GenBank已发表的11株IBV的N基因相比较,IBV793/B的N基因共有88处点突变,在第991位发生了一个核苷酸的缺失。N基因的核苷酸同源性为86.9%～91.4%,氨基酸同源性为75.8%～77.5%。表明IBV93/B的N基因存在着较大的变异性。     

非编码的mRNA

非编码的mRNA是近年发现的一类不含典型ORF的mRNA.目前已发现或克隆的这类基因主要有：H19基因,XIST基因,XLSIRT基因,His-1基因,bic基因,rox1和rox2基因等.它们与胚胎发育,肿瘤发生及X染色体失活密切相关.     

mRNA turnover

Nuclear RNA-binding proteins can record pre-mRNA processing events in the structure of messenger ribonucleoprotein particles (mRNPs). During initial rounds of translation, the mature mRNP structure is established and is monitored by mRNA surveillance systems. Competition for the cap structure links translation and subsequent mRNA degradation, which may also involve multiple deadenylases.     

Isolation of mRNA from specific tissues of Drosophila by mRNA tagging

To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes.     

Binary function of mRNA

Since the discovery of messenger RNA (mRNA) over half a century ago, the assumption has always been that the only function of mRNA is to make a protein. However, recent studies of prokaryotic and eukaryotic organisms unexpectedly show that some mRNAs may be functionally binary and have additional structural functions that are unrelated to their translation product. These findings imply that some of the phenotypic features of cells and organisms can also be binary, that is, they depend both on the function of a protein and the independent structural function of its mRNA. In this review, we will discuss this concept within the framework of multifunctional RNA molecules and the RNA World Hypothesis.     

Shutdown decay of mRNA

Although plasmid-borne and chromosomal toxin-antitoxin (TA) operons have been known for some time, the recent identification of mRNA as the target of at least two different classes of toxins has led to a dramatic renewal of interest in these systems as mediators of stress responses. Members of the MazF/PemK family, the so-called mRNA interferases, are ribonucleases that inhibit translation by destroying cellular mRNAs under stress conditions, while the founder member of the RelE family promotes cleavage of mRNAs through the ribosome. Detailed structures of these enzymes, often in complex with their inhibitors, have provided vital clues to their mechanisms of action. The primary role and regulation of these systems has been the subject of some controversy. One model suggests they play a beneficial role by wiping the slate clean and preventing wasteful energy consumption by the translational apparatus during adaptation to stress conditions, while another favours the idea that their main function is programmed cell death. The two models might not be mutually exclusive if a side-effect of prolonged exposure to toxic RNase activity without de novo synthesis of the inhibitor were a state of dormancy for which we do not yet understand the key to recovery. In this review, I discuss the recent developments in the rapidly expanding field of what I refer to as bacterial shutdown decay.