山东省肠道病毒71型分离株SDLY11全基因组序列分析

目的了解2009年山东省临沂市分离的肠道病毒71型(EV71)分离株SDLY11的基因组序列特征,探讨病毒的神经毒力候选位点。[HTH]方法自手足口病患者的粪便标本中分离EV71,采用一步法RT PCR对病毒株基因组进行全序列扩增,运用DNAstar 和MEGA 4软件进行序列分析。[HTH]结果SDLY 11基因组全长为7 405 nt,其中5′非编码区（UTR） 742 nt,3′UTR 84nt,中间为6 579 nt的开放阅读框架（ORF）,编码2 193个氨基酸的多聚蛋白。VP1区及全基因组序列系统进化分析表明SDLY 11位于EV71病毒C4亚型的分支,同源性分析显示SDLY 11与安徽阜阳株同源性最高。序列比对结果发现,CNS累及HFMD患者病毒株在5[WTBX]′[WTBZ]UTR区出现了两处核苷酸位点的突变（T40C、C575T）,在VP2区第144位出现了氨基酸的突变（T144S）。[HTH]结论SDLY 11分离株属EV71病毒C4亚型。5′UTR区的两处突变（T40C、C575T）及VP2区的一处突变（T144S）可能与病毒的致神经毒力作用有关。     

庚型肝炎病毒多聚蛋白的翻译及基因变异研究进展

庚型肝炎病毒(HGV)是单股正链RNA病毒,其基因结构及保守序列类似于黄病毒科的成员。HGV基因组全长约9400个核苷酸(nt),与HCV相似,在5’端和3’端各有一段非翻译区(UTR),一个大开放读码框架(ORF)编码约2900个氨基酸的多聚蛋白前体,其氨基端(N端)和羧基端(C端)分别为结构蛋白(C、E1、E2)和非结构蛋白(NS2、NS3、NS4a/b、NS5a/b)。NS3区编码丝蛋白酶、解旋酶,NS5区编码RNA聚合酶。HGV与HCV全序列氨     

中国广西登革3型病毒分离株基因组序列特征分析

目的对我国广西登革3型病毒分离株桂登-1株(GD-1株)和桂登-11株(GD-11株)进行全基因组序列测定和分析,为了解其地理来源提供依据。方法根据GenBank提交的登革3型病毒基因序列设计9对引物,RT-PCR方法分段扩增GD-1和GD-11株基因序列,测序后进行拼接,得到其全基因组序列。结果两株病毒全长均为10 707nt,与国际参考株H87株核苷酸同源性为96.0%,氨基酸同源性为98.8%。GD-1株和GD-11株间仅有4个氨基酸差异,二者与H87株分别存在39和41个氨基酸差异。对3’UTR二级结构进行预测,二者与H87株存在较大差异。根据E蛋白基因序列对两株病毒进行进化分析,GD-1和GD-11均属于亚型Ⅱ,与分离自泰国的两毒株进化关系较近。结论 GD-1和GD-11均属于登革3型病毒亚型Ⅱ,二者可能是源自泰国的病原。     

人肝再生增强因子基因组DNA的克隆化与序列分析

目的克隆人肝再生增强因子(ALR)的基因组DNA,并进行序列分析。方法采用人肝再生增强因子cDNA及其编码序列,对基因的核苷酸序列数据库(GenBank)以BLAST为工具进行核苷酸序列同源性比较分析,寻找相应的ALR基因组DNA序列。结果从GenBank核苷酸序列数据库中寻找到人ALR基因组DNA全序列,由1813个核苷酸组成。人ALR基因组DNA序列由3段外显示子(exon)组成,分别为18nt,197nt和163nt。基因的编码产物由125个氨基酸残基组成,与小鼠ALR基因组DNA结构类似。基因组DNA定位于染色体的16p13.3位点上。结论克隆了人ALR基因组DNA全序列。     

2013-2022年江苏省柯萨奇病毒A6型全基因组序列特征分析

目的 研究江苏省2013-2022年分离到的柯萨奇病毒A6型(CV-A6)全基因组序列特征及对全基因组各编码区进行遗传进化分析,以期从分子流行病学特征的角度解释CV-A6取代肠道病毒A71和柯萨奇病毒A16成为江苏省引起手足口病(HFMD)主要病原的原因。方法 选取2013-2022年间江苏省地区流行的35株CV-A6毒株进行全基因组测序,采用DNASTAR、MEGA7.0和Similarity plots3.5.1等软件对获取的全基因组序列进行比对、相似性分析、系统进化分析和基因重组分析,对P1编码区和3D区主要氨基酸变异位点进行分析。结果 35株CV-A6江苏毒株的全基因组核苷酸和氨基酸序列相似性分别为87.5%～99.6%和97.0%～99.8%,与CV-A6原型株Gdula/USA/1949的全基因组核苷酸序列相似性仅为80.3%～81.0%,氨基酸序列相似性为94.7%～95.3%。基于VP1序列的系统进化树结果显示,2013-2022年江苏省有34株CV-A6分离毒株分属于D3a基因亚型,1株分属于D2基因亚型;基于3D区进化树划分不同重组模式,2013-2022年江苏省流...     

两株不同毒力EV71全基因组序列测定及分析

目的了解济南市2008年EV71的流行和变异情况,寻求潜在的EV71毒力决定位点。方法采用分段扩增的RT-PCR方法对EV71JN200803和JN200804株的基因组全长进行扩增、测序。对两株EV71的全基因组核苷酸、氨基酸序列进行比较,对非编码区进行RNA二级结构的预测和分析,并对VP1区和全基因组进行遗传进化分析。结果 EV71JN200803和JN200804株的基因组全长分别为7 405nt和7 404nt,编码区没有核苷酸的插入和缺失,全基因组序列的组成和结构均符合肠道病毒71型的特征。JN200803和JN200804全基因组有106个核苷酸突变,编码区98个,非编码区8个,编码区多数属于同义突变,仅造成16个氨基酸突变。遗传进化分析显示,EV71JN200803和JN200804株与2008年国内其他流行株同属C4亚型的C4a进化分支,与Fuyang/17.08/1株进化关系最近。结论 2008年济南市流行的EV71属于C4a亚型,济南分离株的毒力决定位点不但具有非单一性,而且具有较为独特的济南区域特点。     

柯萨奇病毒A组4型分离株全基因序列分析

目的分析1株柯萨奇病毒A组4型(coxsackievirus A4,CV-A4)毒株的全基因组序列和系统进化特征。方法使用细胞培养法对肠道病毒阳性标本中的病毒进行分离。提取病毒RNA,通过RT-PCR法分段扩增全基因组,经测序、比对和组装后获得CV-A4分离株的全基因组序列。利用MEGA7.0软件分别基于全长VP1序列和全基因组序列构建系统发育树;使用Geneious Prime 2020.1.2软件对全基因组及编码区各区段的核苷酸和氨基酸序列同源性进行分析,并对编码区氨基酸变异情况进行比较分析。结果从人类横纹肌肉瘤(human rhabdomyosarcoma, RD)细胞上分离到1株CV-A4分离株,命名为R09220/YN/CHN/2009。基于全长VP1序列的系统进化分析显示,该分离株属于C2基因亚型。在全基因组核苷酸序列上与其同源性最高的是CV-A4毒株CVA4/SZ/CHN/09(核苷酸为95.4%,氨基酸为98.7%)。与C2基因亚型内16株CV-A4代表株的全序列核苷酸相似性为88.4%～95.4%,氨基酸序列相似性为97.5%～98.7%。通过比较C2基因亚型内的23株CV-A4分离株的全基因组氨基酸序列,R09220/YN/CHN/2009共存在23个氨基酸变异位点,其中10个为其独有的变异位点。结论 R09220/YN/CHN/2009为肠道病毒CV-A4,属于C2基因亚型,该亚型在中国大陆流行的CV-A4病毒中占据优势地位。     

一株埃可病毒16型510/YN/CHN/2010的全基因组分析北大核心CSCD

目的分析一株埃可病毒16型(Echovirus 16,E16)云南分离株510/YN/CHN/2010全基因组序列及其遗传特性。方法设计针对E16的引物,提取病毒RNA,RT-PCR扩增病毒目的基因并测序,利用BioEdit 7.2.3、MEGA 7.0和Simplot 3.5.1软件分析其全基因组。结果E16510/YN/CHN/2010全基因组核苷酸序列全长7432 nt,是编码含2196个氨基酸的多聚蛋白。其全VP1和全基因组与原型株Harrington的核苷酸和氨基酸同源性分别为80.68%、49.33%和81.26%、97.26%。与其他埃可病毒原型株的全基因组核苷酸和氨基酸同源性分别为75.72%~88.44%和83.01%~87.65%。系统进化分析将E16分为3个基因型,分离株510/YN/CHN/2010和其他中国分离株均属于B基因型。重组分析显示,510/YN/CHN/2010株可能在非结构编码区的P2段重组。结论510/YN/CHN/2010分离株为E16 B基因型,可能为重组株。     

福建省肠道病毒71型分离株（2010FJLY008）全基因组核苷酸序列分析

目的了解肠道病毒71型在福建省的遗传背景,追踪EV71流行过程中可能发生的变异。方法对2010年福建1例手足口病患儿标本中分离到的1株肠道病毒71型分离株（2010FJLY008）进行全基因组核苷酸序列测定,并对基因组序列进行同源性比较及遗传进化分析。结果2010FJLY008株全长7 403bp,核苷酸及编码氨基酸序列同源性比较,均与08年阜阳流行株Fuyang17.08 2同源性最高,整个基因组核苷酸同源性为98.2%,氨基酸同源性为99.5%;全长基因组核苷酸序列遗传进化分析及VP1区基因遗传进化分析,与Fuyang17.08 2株进化关系较为接近,同属于C4a基因亚型。结论2010年福建省EV71型病毒流行株（2010FJLY008）在病毒基因组结构、基因组核苷酸同源性比较、编码氨基酸同源性比较、全长基因组核苷酸序列遗传进化分析及VP1区基因遗传进化分析等方面,均与2008年阜阳流行株（Fuyang17.08 2）关系密切,未产生明显的抗原漂移及变异。     

丙型肝炎5''''非编码区末端快速基因扩增方法的建立及应用

目的 建立快速获得丙型肝炎（HCV）基因组5’非编码区（5’uTR）真末端序列的分子生物学方法。方法 逆转录后利用末端聚合酶（TOT）进行加尾反应，再利用套式聚合酶链反应（PCR）扩增出目的末端基因的cDNA片段，A-T克隆，用限制性内切酶片段长度多态性分析（RVLP）与PCR鉴定重组子，全自动序列分析仪测定插入子序列。结果 cDNA末端快速扩增技术（RACE）获得5株HCV5’UTR克隆，包括3株全长克隆和2株缺失克隆。2株缺失克隆，一条在5’末端缺失53个碱基，另一条缺失144个碱基。结论 RACE技术快速、有效、实用，可有效获得丙型肝炎病毒基因组的5’非编码区末端序列。     

幽门螺杆菌中国株cagA全长基因克隆及其分子特征分析

目的克隆幽门螺杆菌中国株MEL-Hp 27cagA全长基因,并进行分子特征分析。方法参考Hp26695基因组序列,分别针对cagA基因编码区保守序列和位于cagA基因上、下游的基因序列设计两对PCR引物,交叉分段扩增包括cagA基因编码区、5′、3′非编码区在内的全长基因序列,并对cagA基因调控序列、编码序列及源自中国的CagA分子EPIYA基序分布特点进行分析。结果Hp27cagA基因编码区长3510bp,编码1169个氨基酸。5′端非编码区长649bp,-10区、-35区分别位于起始密码子ATG上游89bp和154bp处,3′端非编码区长476bp。Hp27cagA基因编码区核苷酸序列与东亚菌株同源性为96%,与西方菌株的同源性仅为86%左右。Hp27CagA分子存在典型的EPIYA基序,属于ABD型,与国际参考株NCTC11637的AB-CCC型不同。比较源自中国的HpCagA序列的EPIYA基序分布特点,未发现中国Hp分离株之间存在CagAEPIYA基序与疾病结局(胃癌、慢性胃炎和十二指肠溃疡)的聚类关系。结论成功克隆幽门螺杆菌全长ca-gA基因;cagA基因编码区及非编码区序列存在东西方差异;未发现中国Hp分离株之间存在CagA EPIYA基序与疾病结局的聚类关系。     

Cloning and sequence analysis of cDNA for human argininosuccinate lyase.

Using antibodies specific for argininosuccinate lyase (EC 4.3.2.1), we isolated two cDNA clones by screening a human liver cDNA library constructed in the lambda gt11 expression vector. The identity of these isolates was confirmed by in vitro translation of plasmid-selected mRNA. One of these isolates was used to rescreen the cDNA library and a 1565-base-pair (bp) clone was identified. The entire nucleotide sequence of this clone was determined. An open reading frame was identified which encoded a protein of 463 amino acids with a predicted molecular weight of 51,663. The clone included 115 bp of 5' untranslated sequence and 46 bp of 3' untranslated sequence. A canonical poly(A) addition site was present in the 3' end, 16 bp from the beginning of the poly(A) tract. Comparison of the deduced amino acid sequence of the human enzyme with that of the yeast enzyme revealed a 56% homology, when conservative amino acid changes were taken into consideration. The yeast protein is also 463 amino acids long, with a molecular weight of 51,944. By use of a genomic DNA panel from human-Chinese hamster somatic cell hybrids, the human gene was mapped to chromosome 7. Another hybridizing region, corresponding to a portion of the 5' end of the cDNA, was found on chromosome 22.     

Identification of 5′ capped structure and 3′ terminal sequence of hepatitis E virus isolated from Morocco

AIM: To examine 5′ and 3′ terminal sequences of hepatitis E virus (HEV) isolated from Morocco, to confirm 5′ methylated cap structure of the genome, and to investigate whether the 3′ UTR can be used to distinguish HEV genotypes instead of HEV complete genome sequence.METHODS: RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) was employed to obtain the 5′ and 3′ terminal sequences of HEV Morocco strain. The 3′ UTR sequence of the Morocco strain was compared with that of the other 29 HEV strains using the DNAStar software.RESULTS: The 5′ PCR product was obtained only from the RLM-RACE based on the capped RNA template. The 5′ UTR of the Morocco strain had 26 nucleotides, and the 3′ UTR had 65 nucleotides upstream to the polyA. The 5′ UTR between HEV strains had only point mutations of nucleotides.The phylogenetic tree based on the sequences of 3′ UTR was not the same as that based on the complete sequences.CONCLUSION: The genome of HEV Morocco strain was methylated cap structure. The 3′ terminal sequence can not be used for distinguishing HEV genotype for all HEV strains in place of the whole HEV genome sequence.     

Isolation and analysis of genomic DNA clones encoding the third component of mouse complement.

A gene library was constructed with DNA from strain A mice by using the phage lambda vector lambda 1059. By screening with cloned cDNA for the third component of mouse complement, C3, four different C3 genomic clones were isolated from this library. Two of the recombinant phages carry insertions of 14 and 18 kilobase pairs, respectively, which together cover one complete copy of the C3 gene and several hundred nucleotides of its 5' and 3' flanking sequences. The distance from the 5' end of the gene, which includes the hexanucleotide T-A-T-A-A-A and a translation initiation codon, to its 3' end as defined by the poly(A) attachment site is 24 kilobase pairs. From the genomic DNA sequence, a signal peptide of 24 amino acid residues is predicted at the NH2 terminus of the initial translation product. The signal peptide and the next two amino acids are encoded by the first exon of this gene.     

Extensive intragenic sequence homology in two distinct rat lens gamma-crystallin cDNAs suggests duplications of a primordial gene.

The nucleotide sequences of two different rat lens gamma-crystallin cDNA clones, pRL gamma 2 and pRL gamma 3, have been determined. pRL gamma 3 contains the complete coding information for a gamma-crystallin of 173 amino acids whereas pRL gamma 2 is incomplete in that it lacks the codons for the first three amino acids of a separate but very homologous gamma-crystallin of identical length. Both rat gamma-crystallins are homologous to the known amino acid sequence of bovine gamma-crystallin II which is only a single amino acid longer. The length of the region downstream the coding sequence to the A-A-T-A-A-A polyadenylylation signal sequence is 40 nucleotides in each clone. In pRL gamma 3 the poly(A) signal sequence is followed at 14 nucleotides by a remnant of the poly(A) tail which indicates that this clone contains a complete 3' noncoding region. pRL gamma 2 has only seven nucleotides following this signal sequence and no poly(A) tail, suggesting an incomplete 3' end. The cDNA clones show an overall nucleotide sequence homology of 85%. The mutual homology at the amino acid level is 73% whereas their amino acid homology with bovine gamma-crystallin II is about 70%. The nucleotide sequence of each clone also reveals a high intragenic homology and seems to be duplicated in itself. We suggest that the gamma-crystallin genes have arisen by multiple duplications of a primordial gene which consisted of about 120 nucleotides.     

Cloning of complimentary deoxyribonucleic acid encoding follicle-stimulating hormone and luteinizing hormone beta subunit precursor molecules in Reeves's turtle (Geoclemys reevesii) and Japanese grass lizard (Takydromus tachydromoides)

Reptilia is the only vertebrate class in which cDNA for the gonadotropin beta subunit precursor molecule has not been cloned. We have isolated the full-length cDNA clone encoding the LH beta subunit precursor molecule and a partial cDNA clone encoding the FSH beta subunit precursor molecule from a pituitary cDNA library of Reeves's turtle. We further clarified the nucleotide sequence of the remaining part of the turtle FSH beta cDNA and that of full-length cDNA encoding the LH beta subunit precursor molecule of the Japanese grass lizard, by means of the 5' rapid amplification of cDNA end (RACE) and 3' RACE. The nucleotide sequence of the turtle FSH beta cDNA we determined was 584 bp long and contained the coding sequence, 5' untranslated region (UTR) and 3' UTR of 396, 34, and 154 bp, respectively. The nucleotide sequence of the turtle LH beta we isolated was 498 bp long and contained the coding sequence, 5' UTR and 3' UTR of 420, 7, and 71 bp, respectively. The nucleotide sequence of the lizard LH beta we determined was 537 bp long and contained the coding sequence, 5' UTR and 3' UTR of 441, 35, and 61 bp, respectively. Amino acid sequences deduced from coding regions of the turtle FSH beta, LH beta and the lizard LH beta were 131, 139, and 146 residues, respectively. Referring to the amino acid sequences of the bullfrog FSH and LH beta subunit molecules determined chemically, we deduced the amino acid sequences of mature peptide. Amino acid sequences of mature peptides of the turtle FSH, turtle LH, and the lizard LH were 111, 112, and 112 residues, respectively. Amino acid sequences of the mature peptides were compared with those of other vertebrates. The amino acid sequence of the turtle FSH beta subunit molecule was 84.7-85.6, 67.8-71.4, and 61.3-62.2% identical to the FSH sequence of birds, mammals, and amphibians, respectively. The amino acid sequence of the turtle LH beta subunit molecule was 51.6-54.6, 36.2-48.7, and 56.3-57.5% identical to the LH sequence of birds, mammals, and amphibians, respectively. The amino acid sequence of the lizard LH beta subunit molecule was 39.1-47.1, 32.9-43.0, and 46.0-47.3% identical to the LH sequence of birds, mammals, and amphibians, respectively. These identity values suggest that the turtle or reptilian FSH beta subunit molecule is more closely related to avian and mammalian FSH beta subunit molecules than to amphibian FSH beta subunit molecules but reptilian LH beta subunit molecules are more closely related to amphibian LH beta subunit molecules than to avian and mammalian LH beta subunit molecules. This discrepancy in the molecular similarity relationship found in the reptilian FSH and LH beta subunit molecules can be interpreted by assuming that evolution speed was not the same among hormone species and also among vertebrate groups.     

Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication.

An oligo(dT)-primed cDNA copy of the mRNA coding for the human gastrin precursor was constructed from poly(A)-containing RNA from a human pancreatic, gastrin-producing tumor (a gastrinoma). The cDNA was inserted into the Pst I endonuclease site of plasmid pBR322 by the use of the poly(dC) and poly(dG) tailing procedure. Clones containing gastrin sequences were selected by hybridization to a purified single-stranded 32P-labeled gastrin cDNA probe. This probe was constructed with gastrinoma mRNA as template. As primer for the cDNA synthesis, we used a synthetic oligonucleotide mixture, d(AG-A-A-AG-T-C-C-A-T-C-C-A), corresponding to the gastrin-specific amino acid sequence Trp-Met-Asp-Phe. In this way we determined the nucleotide sequence of the entire coding region (303 nucleotides), the entire 3' untranslated region (102 nucleotides), and 8 nucleotides of the 5' untranslated region. A striking homology between parts of the coding region suggests that evolution of the gastrin gene has involved a gene duplication.