Molecular Cloning,DNA Sequence Analysis,and Expression of cDNA Sequence of RNA Genomic Segment 6 (S6) that Encodes a Viral Outer Capsid Protein of Threadfin Aquareovirus (TFV)

The genome segment 6 (S6) of threadfin reovirus (TFV) was cloned and sequenced. The entire S6 nucleotide sequence is 2056 bp long with an open reading frame that encodes a protein of 653 amino acids. Sequence analysis of the TFV S6 genome revealed that the 5-terminal sequence, GTTTTA and the 3-terminal sequence, ATTCATC of the plus strand is common to other genome segments of TFV. The pentanucleotide, TCATC, at the 3-terminal of the plus strand was also conserved in other reported isolates of Aquareovirus such as chum salmon reovirus (CSV), striped bass reovirus (SBR), grass carp reovirus (GCRV) and golden shiner reovirus (GSV) as well as to the 10 genome segments of mammalian reovirus (MRV). Blast results indicated that the TFV S6 gene segment sequence had high identity towards the CSV S6 gene sequence, which codes for the CSV outer coat protein. This implied that the TFV S6 gene segment codes for an outer capsid protein (OCP) of the virus. Amino acid sequence analysis of this TFV OCP sequence revealed the presence of a putative conserved asparagine-proline (Asn–Pro) protease cleavage site, which was found in all reported isolates of Aquareovirus as well as in the MRV 1 protein. N-terminal sequencing of the corresponding S6 native protein obtained from purified TFV particles verified the presence of this cleavage site. Phylogenetic analysis of the TFV S6 protein revealed that TFV was closely related to CSV, from Aquareovirus species, ARV-A. Cloning of the TFV S6 gene sequence into an Escherichia coli expression host produced a recombinant protein that corresponded to the predicated size of the OCP of TFV. Immunization of mice using this recombinant outer capsid protein (rOCP) revealed that the protein was able to elicit an antibody response, thus indicating that the rOCP of TFV was immunogenic.     

Cloning and Sequence Analysis of the Nucleocapsid Gene of Porcine Epidemic Diarrhea Virus Chinju99

The nucleocapsid (N) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to establish the information for the development of genetically engineered diagnostic reagents. Also, sequences of the nucleotides and deduced amino acids of the Chinju99 N gene were analyzed by alignment with those of CV777 and Br1/87. The nucleotide sequence encoding the entire N gene open reading frame (ORF) of Chinju99 was 1326 bases long and encoded a protein of 441 amino acids with predicted M _r of 49 kDa. It consisted of 405 adenine (30.5%), 293 cytosine (22.1%), 334 guanines (25.2%) and 294 thymines (22.2%) residues. The Chinju99 N ORF nucleotide sequence was 96.5% and 96.4% homologous with that of the CV777 and Br1/87, respectively. The Chinju99 N protein revealed 96.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained seven potential sites for threonine (T)- or serine (S)-linked phosphorylation by each protein kinase C and casein kinase II.     

Cloning and Sequence Analysis of the Spike Gene of Porcine Epidemic Diarrhea Virus Chinju99

The spike (S) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to aid in the development of genetically engineered vaccines and diagnostic reagents against PEDV. The nucleotide sequence encoding the entire S gene open reading frame (ORF) of Chinju99 was 4152 bases long encoding 1383 amino acids. It consisted of 1001 adenine (24.1%), 849 cytosine (20.4%), 877 guanine (21.1%) and 1425 thymine (34.3%) residues. The Chinju99 S ORF nucleotide sequence was 94.5% homologous with that of the Br1/87 and CV777 strains, respectively. The Chinju99 S protein had 92.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained 27 potential sites for asparagine (N)-linked glycosylation and there was a stretch of highly hydrophobic residues at position 1325–1350.     

抗人CD3单链抗体基因的构建及序列分析

本文在已克隆抗人ＣＤ３抗体ＶＨ和ＶＫ基因的基础上，设计并合成了ＰＣＲ引物。两个外侧引物分别含有ＥｃｏＲＩ和ＳａｌＩ酶切位点及起始码和终止码序列，４个内侧引物各含部分连肽基因序列，回收后混合退火。     

中国旱獭IL-10分子的克隆和序列分析

目的克隆旱獭白细胞介素-10(IL-10)全长cDNA序列进行序列分析,为IL-10分子的表达和在土拨鼠肝炎病毒(woodchuckhepatitisvirus,WHV)感染中的应用奠定基础。方法根据Genbank的土拨鼠IL-10cDNA序列,在5′非编码区和3′非编码区设计特异性引物,提取旱獭脾组织总RNA作为模板,RT-PCR扩增旱獭IL-10cDNA。PCR产物纯化后连接至T载体(pMD18-T),构建重组质粒pMD18-T-cmIL-10。对重组质粒进行酶切鉴定,选择阳性克隆测序。对所获得的序列应用分析软件进行分析。结果RT-PCR扩增产物为685bp。重组质粒pMD18-T-cmIL-10经EcoRⅠ与PstⅠ双酶切提示含目的基因片段。序列分析提示旱獭IL-10的编码序列为537bp,与土拨鼠IL-10的同源性为100%。结论成功克隆旱獭IL-10分子,并构建重组质粒pMD18-T-cmIL-10。     

The Non-structural Leader Protein Gene of Foot-and-Mouth Disease Virus is Highly Variable between Serotypes

Aphthoviruses are unique among picornaviruses in that they alone encode a functional L proteinase as the first component of the viral polyprotein. The L genes of a few Indian foot-and-mouth disease viruses were sequenced and compared with those available to study the extent of variation in this gene. Besides the two in-frame start codons present in all FMDV L genes, the Asia-I vaccine virus had an additional in-frame AUG (start) codon, at codon position 3. Amino acid sequence comparison revealed that 39.8% of positions were capable of accepting replacements, yet the residues of the catalytic dyad were totally conserved. Sequence comparison at the C-terminus of the protein indicated that K/RGAGQS is sufficient for L/P1 cleavage. Phylogenetic analysis based on the L gene sequences did not reveal any serotype-specific clustering. The probable implications of the observed high variability in this non-structural gene is briefly discussed.     

Cloning and Sequence Analysis of the M gene of Porcine Epidemic Diarrhea Virus LJB/03

Porcine epidemic diarrhea virus (PEDV) LJB/03 was isolated from the fece of piglets infected with PEDV on a pig farm, Heilongjiang province, China. The M gene of LJB/03 was amplified from the RNA extracted directly from the fece samples by RT-PCR and cloned into pMD18-T vector. The M gene cDNA was sequenced and encompasses an open reading frame of 681 nucleotides, encoding a 226-amino acid protein. The LJB/03 M gene has a base composition of 152 adenines (22%), 153 cytosines (23%), 161 guanines (24%), and 214 thymines (31%). Sequence comparison with other PEDV strains selected from GenBank revealed that the LJB/03 M gene has a high sequence homology to those of other PEDV isolates, 97.80% with JMe2, 96.92% with KPEDV-9 (Korean field isolate), 97.36% with KPEDV-9 (Korean), 97.80% with Br1/87, and 97.94% with CV777. The encoded protein shared 97.79% amino acid identities compared with CV777, 97.35% with Br1/87, 97.79% with JMe2, 96.90% with KPEDV-9 (Korean field isolate), 96.46% with KPEDV-9 (Korean). Sequence analysis of the M gene, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, showed that all other PED viruses analyzed fell into three groups, and the LJB/03 itself branched in an independent group. These data revealed that the M gene nucleotide sequence of LJB/03 has some mutations in comparison with the other PED viruses.     

犬复孔绦虫ITS及5．8 SrDNA的PCR扩增、克隆及序列分析

目的以从我国广东广州和湛江犬小肠中采集的2条犬复孔绦虫作为研究对象。以保守引物NC5及NC2扩增犬复孔绦虫的ITS-1，5．8S及ITS-2rDNA片段并进行序列分析。方法将PCR扩增出的片段纯化后克隆至pGEM-TEasy载体，重组质粒通过菌落PCR和酶切鉴定后，对阳性菌落进行序列测定。结果来自广州和湛江的2条犬复孔绦虫ITS及5．8 S rDNA序列总长分别为1536bp、1385bp，2条犬复孔绦虫的ITS-1、ITS-2序列相差较大，分别为20．80％、27．17％，而5．8S序列相差较小（1．49％）。结论由于犬复孔绦虫ITS序列复杂，种内存在的差异大，故不适于作为犬复孔绦虫种的遗传标记。     

Molecular Cloning and Sequence Analysis of the Phosphoprotein (P) Gene of the Lapinized Rinderpest Virus

We determined the nucleotide sequence of the coding region for the phosphoprotein (P) gene of the L strain of rinderpest virus (RPV). The gene encodes two overlapping open reading frames of 1521 and 531 nucleotides. Use of the first ATG would produce a P polypeptide of 507 amino acids, while use of the second ATG would produce a C polypeptide of 177 amino acids. In addition, the insertion of an extra G residue at the editing site generates an alternative mRNA potentially encoding the V protein of RPV. Homology comparisons of the P, C and V proteins among various viruses suggest that RPV is closer to measles virus (MV) than to canine distemper virus (CDV). Alignment of the sequences unique to the V protein revealed that the cysteine residues are well conserved among RPV, MV and CDV, and form a zinc finger-like motif.     

Cloning and Sequence Analysis of Banana Streak Virus DNA

Banana streak virus (BSV), a member of the Badnavirus group of plant viruses, causes severe problems in banana cultivation, reducing fruit yield and restricting plant breeding and the movement of germplasm. Current detection methods are relatively insensitive. In order to develop a PCR-based diagnostic method that is both reliable and sensitive, the genome of a Nigerian isolate of BSV has been sequenced and shown to comprise 7389 bp and to be organized in a manner characteristic of badnaviruses. Comparison of this sequence with those of other badnaviruses showed that BSV is a distinct virus. PCR with primers based on sequence data indicated that BSV sequences are present in the banana genome.     

Sequence Analysis of the RNA Polymerase Gene of Foot-and-Mouth Disease Virus Serotype Asia1

The complete nucleotide (nt.) sequence of the RNA polymerase (3D) gene and 81 nt. in the 3-untranslated region of foot-and-mouth disease virus (FMDV) serotype Asia1 (IND63/72) was determined and compared with the sequence of other FMDV serotypes. The 3D genomic region was 1410 nt. long encoding 470 amino acids with an inframe stop codon (TAA) at nt. position 1411–1413. The deduced amino acid sequence of the protein showed 8 conserved motifs as reported in other picornaviruses, 2 of which are 100% identical across the serotypes. Antigenic regions in the polymerase protein were predicted and found to be located at the N-terminus of the protein. The phylogenetic analysis showed that the FMD viruses were segregated into different clusters based on geographical origin; the Asia1 virus did not cluster tightly with any of the geographical groups.     

Cloning and Sequence Analysis of Envelope Glycoprotein E1 Gene of Rubella Virus, JR23 Strain

Rubella virus (RV), the only member of the Rubivirusgenus in the family Togaviridae, is a single stranded, pos itive sense RNA virus. The structural gene in the 3′endof the genomic RNA encodes 3 virion proteins: the capsidprotein (C), and two envelop glycoproteins, E1 and E2.E1 gene is 1484 bp in length, encoding the hemagglutina tion activity and immune antigenic sites of RV. Studies in dicate that the degrees of variance in E1 gene sequence aresimilar to that of the complete gen…     

Sequence Analysis of the NSP4 Gene from Human Rotavirus Strains Isolated in the United States

Two major and one minor genotype of the rotavirus NSP4 gene have been described. The sequences of 29 NSP4 genes from rotavirus isolates obtained in the United States during the 1996–1997 rotavirus season (types P[8]G1, P[8]G9, P[4]G2 and P[6]G9) and 10 strains isolated during previous rotavirus seasons (types P[8]G1 and P[4]G2) were determined. All NSP4 genes from strains with short E types (6 P[4]G2, 4 P[6]G9) belonged to genotype NSP4A, whereas all 19 strains with long E types (16 P[8]G1, 3 P[8]G9) had NSP4 genes of genotype NSP4B. Genetic variation within genotypes was low (2.3% for both NSP4A and NSP4B), confirming that the NSP4 genes are highly conserved. Nonetheless, at least two distinct sub-lineages could be detected within each genotype: strains isolated in the same year, regardless of geographic location, were more closely related or even identical at the deduced amino acid level; strains isolated in different years were more distinct. Thus, geographic distance did not affect genetic distance. Northern hybridization analysis with NSP4A and NSP4B total gene probes failed to detect any unusual combinations of the VP6 and NSP4 genes in 31 additional isolates from the 1996–1997 rotavirus season.     

乙型肝炎病毒核心基因的酶切图谱及核苷酸序列分析

对慢性乙型肝炎患者的２２份肝穿刺活检组织及其中配对的６份血清用ＰＣＲ扩增出乙型肝炎病毒（ＨＢＶ）的ＰｒｅＣ／Ｃ基因，选用ＡｖａⅡ、Ｓａｕ３ＡⅠ、ＸｍｎＩ，ＢｓｔＮＩ及ＴａｑＩ５种限制性内切酶消化后，对Ｃ基因进行酶谱分析。发现有６份肝组织及１份血清出现异常酶谱。对Ｓａｕ３ＡⅠ酶解异常标本进行分子克隆及核苷酸序列分析，发现２例患者因２１３７位核苷酸点突变出现了Ｓａｕ３ＡⅠ的新酶切点。类似变化在肝癌组织的ＨＢＶＣ基因中也曾发现，其意义待进一步研究。     

Sequence Analysis of the Gene Encoding the Capsid Protein of the Snow Mountain Human Calicivirus

Snow Mountain virus (SMV) is the reference strain for serotype 3 as determined by immune electron microscopy of the human caliciviruses that are associated with epidemic gastroenteritis. In order to establish the genetic relationship of its capsid protein with those from other human caliciviriuses, the sequence of the open reading frame 2 (ORF2) encoding the SMV capsid protein was determined. The SMV ORF2 sequence was 1626 nucleotides in length and the deduced protein of 542 amino acids had a calculated molecular weight of 59.2 kD. The SMV capsid sequence showed approximately 48 and 77% amino acid sequence identity with the capsid proteins of the Norwalk (serotype 1) and Hawaii (serotype 2) human calicivirus reference strains, respectively, a finding consistent with its serotypic distinctiveness. Furthermore, the predicted amino acid sequence of the SMV capsid was found to share highest sequence identity (98%) with the Melksham human calicivirus in database searches. This revised version was published online in July 2006 with corrections to the Cover Date.     

五株隐孢子虫18S rRNA部分基因克隆和序列分析

设计Nested PCR引物扩增牛源微小隐孢子虫Cryptospordium parvum、羊源微小隐孢子虫C. parvum、牛源安氏隐孢子虫C. andersoni、鸡源贝氏隐孢子虫C. baileyi及猪源隐孢子虫C. suis 18S rRNA基因突变区,PCR产物经克隆测序,其片段大小分别为212、213、213、213和210 bp.将测得的序列用DNAStar软件分析并与NCBI数据库中相同与相近种株序列进行相似性比较, 进行相似性分析并用TREECON软件绘制系统发育进化树.结果表明测得的5株隐孢子虫与各自相同种相似性为98.1%～100%,与其他种相似性为90.1%～98.6%.分析显示在此突变区设置特异酶切位点能区分开C. parvum, C. andersoni, C. baileyi与C. suis,位点分别是TaqI、BstUI、MseI.本研究为我国隐孢子虫分类、分子流行病学研究提供了新的方法.     

Identification and Sequence Analysis of Potato yellow vein virusCapsid Protein Minor Gene

Potato yellow vein virus (PYVV) is a whitefly-transmitted (Trialeurodes vaporariorum) closterovirus (WTC) with an as yet unidentified genome composition. PYVV dsRNA preparations consist of three high molecular weight dsRNA species (dsRNAs 1, 2 and 3) 8.0, 5.5 and 4.0kbp in size respectively, as well as two low molecular weight dsRNA species of 2.0 and 1.8kbp (denoted x and y). The PYVV capsid protein minor (CPm) gene was identified on the dsRNA 3 species, and was subsequently cloned and sequenced. The PYVV CPm gene is 2022 nucleotides long and putatively encodes a protein with estimated size 77.5kDa. The PYVV CPm gene product is considerably larger than the equivalent proteins encoded by the bipartite criniviruses, Lettuce infectious yellows virus (LIYV) and Cucurbit yellow stunting disorder virus (CYSDV) (52 and 53kDa, respectively). The PYVV CPm possesses a centralized domain which is absent from both the LIYV and CYSDV CPm counterparts. Pairwise comparisons as well as phylogenetic analysis based on the available amino acid sequences of the CPm of various WTCs, showed that PYVV is closely related to LIYV, CYSDV and also Beet pseudo-yellows virus.