Construction of cDNA library, nucleotide sequence and analysis of entire genome of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 pairs of primers have been designed and synthesized, which cover the entire genome of CSFV strain Shimen. Each cDNA fragment has been amplified by RT-PCR from the anticoagulant blood of strain Shimen infected pig. The PCR products have been cloned respectively and sequenced. Results show that the cDNA library of strain Shimen and its nucleotide sequence have been obtained. The genomic RNA of strain Shimen is 12 298 nucleotides in length, containing a 5′ and a 3′ noncoding region 373 and 231 nt long respectively. The center of genome is a single large open reading frame of 11 697 nt which encodes a polyprotein of 3 898 amino acids. The entire sequence of strain Shimen has also been compared with that of other CSFV strains.     

Molecular cloning and nucleotide sequence of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 paris of partially overlapping primers which span the entire genome of CSFV strain Shimen were designed and synthesized. Each cDNA fragment of strain Shimen was amplified by RT-PCR method from the anticoagulant blood of strain Shimen infected pig. The PCR fragments were cloned into pGEM-T vector respectively and sequenced. The results show that we have obtained the nucleotide sequence of strain Shimen. The viral RNA consists of 12 297 nucleotides including noncoding regions of 373 and 227 bases at the 5′ and 3′ end, respectively, and a single large open reading frame spanning 11 697 nucleotides in the middle, which encodes an amino acid sequence of 3 989 residues with a calculated molecular weight of 437.6×10³. The precisely sequencing of 5′ and 3′ termini is undertaking. Supported by the National Pandeng Project Huang Qianhua: born in 1968. Graduate student     

Construction of cytopathic PK-15 cell model of classical swine fever virus

No cytopathic effect (CPE) can be observed on classical swine fever virus (CSFV) infected cell culture in vitro. This brings an obstacle to the researches on reciprocity between CSFV and host cells. Based on the construction of full-length genomic infectious cDNA clone of Chinese CSFV standard virulent Shimen strain, partial deletion is introduced into genomic cDNA to obtain a 7.5 kb subgenomic cDNA. A new subgenomic CSFV is derived from transfection with the subgenomic cDNA on PK-15 cells pre-infected by CSFV Shimen virus. Typical CPE induced by this subgenomic virus is observed on PK-15 cells. Coexistence of wildtype and subgenomic virus in cytopathic cell culture is demonstrated by RT-PCR detection in cytopathic cells. For conclusion, the construction of cytopathic cell model exploited a new way for researches on the molecular mechanism of CSFV pathogenesis.     

仔猪先天性震颤的病原学调查及其病原特性分析

应用PCR方法对临床收集的8个猪场仔猪先天性震颤病例8例进行CSFV、PCV-2和PPV检测,结果 CSFV阳性率为87.5%,PCV-2阳性率为87.5%,PPV阳性率为37.5%。其中CSFV、PCV-2和PPV混合感染率为25.0%,CSFV和PCV-2混合感染率为50.0%,CSFV和PPV混合感染率为12.5%。序列分析结果显示:7株CSFV的E2基因之间核苷酸序列差异很小,同源性为99.0%～100%,与Alfort株同源性为95.1%～96.2%,与猪瘟兔化弱毒株(HCLV)和石门株(Shimen)的同源性为83.3%～84.7%。5株PCV-2 Cap基因之间核苷酸序列差异很小,分离的同源性为98.9%～100%,与Genbank发布的标准序列的同源性为98.7%～99.4%;3株PPV VP2基因之间核苷酸序列差异也很小,同源性为99.4%～99.6%,与Genbank发布的标准序列的同源性为99.0%～99.8%。PRV动物接种试验结果显示:8个病例的病料组织悬液接种家兔,家兔表现正常,PRV野毒感染试验阴性。     

Molecular cloning and nucleotide sequence of the attenuated hog cholera virus Lapinized Chinese strain

The genomic sequence of the attenuated hog cholera virus Lapinized Chinese strain (HCLV) was determined from overlapping cDNA clones. The viral RNA of HCLV stain comprised 12 310 nucleotide (nt) including 374 nt and 239 nt at the 5′ and 3′-noncoding region, respectively. The complete genome sequence contained one large open reading frame which encoded an amino acid sequence of 3 898 residues with a calculated molecular weight of 437×10³. Although there were mostly only small differences between the sequence of the HCLV strain and the published sequences of strains ALD, GPE⁻, Alfort and Brescia, there was one notable insertion of 12 nucleotides, TTTTCTTTTTTC in the 3′ non-coding region of HCLV strain. Supported by the National Pandeng Project, Genbank accession number AF091507 Wang Jiafu: born in 1972, Ph. D.     

人核蛋白P68 cDNA基因克隆及全序列测定

靠人合成４个混合聚核苷酸探针，从一个人盘λｇｔ１１ ｃＤＮＡ库筛选到含有ｐ６８ ｃＤＮＡ基因的阳性噬菌斑。经次克隆得到含ｐ６８ ｃＤＮＡ基因的ｐＢＲＢｔ７－ｐ６８质粒。用末端终止法对此ｃＤＮＡ基因进行了全序列测定。分析结构证明得到的ｐ６８ｃＤＮＡ基因含有２２８７３个苷酸，具有一个可阅读框架，共编码６１４个氨基酸。其５＇端非编码区含有１３０个碱基。３＇端非编码区含有３１５个碱基，比已发表的３＇端非     

从EST出发克隆人基因组Xq13区段一个候选基因的初步研究

人类基因组表达序列筛选是寻找候选基因的重要路线之一，外显子陷阱法，ｃＤＮＡ直接筛筛选法，它们可分别根据表达序列的结构及表达特点进行筛选，ＥＳＴ是表达图的位标，它们是一些位点专一的表达序列位标，根据ＥＳＴ的特征，在国内首次建立了一种从ＥＳＴ出发的筛选候选基因的新方法，用睦方法已在人Ｘ染色体Ｘｑ１３区段筛选得到了一个新的ｃＤＮＡ，总测序徇的１３９８ｂｐ包含了完整的３末端。     

Cloning and sequence analysis of a gene encoding polygalacturonase-inhibiting protein from cotton

Polygalacturonase-inhibiting proteins (PGIP) play important roles in plant defense of pathogen, especially fungi. A pair of degenerated primers is designed based on the conserved sequence of 20 other known pgip genes and used to amplify Gossypium barbadense cultivation 7124 cDNA library by touch-down PCR. A 561 bp internal fragment of the pgip gene is obtained and used to design the primers for rapid amplification of cDNA ends. A composite pgip gene sequence is constructed from the products of 5′ and 3′ RACE, which are 666 bp and 906 bp respectively. Analysis of nucleic acid sequence shows 69.2% and 68.7% similarity to Citrus and Poncirus pgip genes, respectively. Its open reading frame of the gene encodes a polypeptide of 330 amino acids, in which 10 leucine-rich repeats arrange tandemly. A new set of primers is designed to the 5′ and 3′ ends of the gene, which allows amplification of the full-length gene from the cotton cDNA library. Genomic DNA analysis reveals that this gene has no intron.     

Genomic sequence determination of Classical Swine Fever Virus persistent infection strain

Full genomic sequence of a newly isolated persistent infection strain of classical swine fever virus was firstly determined. It was demonstrated by sequence analyses that nucleotides homologies of this strain compared with virulent Shimen and vaccine HCLV were 89.7% and 87.7%, and homologies of amino acids were 94.8% and 93.3%, respectively. The sequencing results primarily suggest a tighter relationship between this persistent infection strain and virulent Shimen strain than vaccine HCLV strain. Foundation item: Supported by National Basic Research Developmental Project (G199911900). GenBank NO.: AF407339 Biography: Wu Hai-xiang (1976-), Ph.D. candidate, research direction: virus genetics.     

Cloning of a novel gene encoding human thioredoxin-like protein

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A1 .2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5' untranslated sequence and a 209 bp 3' untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).     

Sequencing and rescuing a highly virulent classical swine fever virus： Chinese strain cF114 from a full-length cDNA clone

Classical swine fever is an economically important, highly contagious disease of pigs caused by the classical swine fever virus (CSFV), as referred to as hog cholera virus. CSFV belongs to Pestivirus within the family of Flaviviridae. The virus contains a positivestranded RNA of approximately 12.3 kb in length[1]. The genome is composed of a 5′ non-coding region, a single large open reading frame (ORF) encoding the viral polyprotein with 3898 amino acid residues and a 3′ non-coding reg…     

蚯蚓纤溶酶在大肠杆菌中的克隆与表达

根据蚯蚓纤溶酶最佳组分的N-末端氨基酸序列设计简并引物,以蚯蚓cDNA为模板,获得该组分蛋白质的部分编码序列(AF432224,GenBank 登陆号).BLAST相似性分析表明,该序列与U25643和U25648的部分序列相似性达91%, 根据5' 和3' 末端保守序列设计引物,经PCR获得全长cDNA编码序列.将该序列插入pTBY-11质粒,转化大肠杆菌,表达蛋白以包含体形式存在.     

Cloning and characterization of vernalization-related gene (ver203F) cDNA 3' end

Based on the cDNA fragment sequence of vernalization-related gene verc203 cloned by differential screening in our lab, the 5' primer has been designed. The cDNA 3' end of ver203 gene (1 197 bp) has been cloned by the RACE method. And it is identified by Northern blotting that its expression is special in vernalization treatment. After comparing the sequence in the nucleotide sequence databases of Genbank, EMBL and DDBJ, the gene has homology with Hordeum vulgare jesmonate-induced protein gene. It is suggested that this gene might be related to the signal transduction mediated by jamonate.     

Cloning, prokaryotic expression,purification and sequence analysis of 20S proteasome subunit gene from T.harzianum

The gene encoding the 20S proteasome subunit（PR29） was cloned from cDNA library of Trichoderma harzianum and expressed in Escherichia coli BL21 （D3） using a pET-28a expression system. The molecular weight of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on gels. The target protein was insoluble when induced at 22℃ with 0.4 mmol/L IPTG, while dissoluble if induced at 37℃ with 0.8mmoL/L IPTG. The expressed product was purified through Ni-magnetic beads His Bind. The purity of the fusion protein reached above 80%. The entire eDNA sequence consisted of 1094 bp with 173 and 135 bp in 5＇ and 3＇ untranslated regions respectively. The gene encoding 261 amino acids has no signal peptide sequence. These results could provide a basis for validating the func-tions of PR29. It also provided a preliminary indication for further study of the mechanism and function of proteasome, and more information of proteasome mechanism in T.harzianum could be obtained.     

Nucleotide sequence of RNA2 and polyprotein processing sites of a Chinese isolate of broad bean wilt virus 2

RNA2 of broad bean wilt virus 2 (BBWV2) isolate B935 is composed of 3601 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end. Only one of the six possible reading frames has a long open reading frame, which extends from nt 231 to nt 3428 in the polarity of encapsidated RNA, and encodes a polyprotein of 119 kD. The N-terminus of the large coat protein (LCP) is located at 599 nt and the small coat protein (SCP) at 197 nl from the C-terminus of the 119 kD protein, which suggests that the coat proteins are released from the polyprotein by cleavages of a glulamine-glycine (Q-G) and a glutamine-alanine (Q-A) bond respectively. The sequence comparison of B935 with fabaviruses shows that B935 has very high sequence homology with other BBWV2 isolates and with patchoul' mild mosaic virus, but has lower homology with BBWV1 isolates. B935 has a similar genomic organization, but a low sequence homology to RNA2 molecules of comoviruses and nepoviruses.     

Reiteration frequency of the gene for tissue-specific histone H5 in the chicken genome.

Chicken erythroid cells contain a tissue specific histone known as H5 in addition to the five major histone species found in other organisms. The mRNA coding for this histone has been isolated by indirect immunoprecipitation from immature, non-dividing reticulocytes in which this is the only histone synthesised. The mRNA has been modified by the enzymatic addition of a 3' polyadenylic acid tract, and transcribed into complementary DNA (cDNA) using the RNA-dependent DNA-polymerase from avian myeloblastosis virus. Studies on the hybridisation of this cDNA indicate that the gene coding for the H5 histone is reiterated 10 times in the chicken genome.     

Primary structure, gene organization and polypeptide expression of poliovirus RNA

The primary structure of the poliovirus genome has been determined. The RNA molecule is 7,433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2,207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.     

Candidate Multi-Peptide-Vaccine Against Classical Swine Fever Virus Induces Strong Antibody Response with Predefined Specificity

IntroductionClassical swine fever virus(CSFV) is a pestiviruswhich causes significantmortality and morbidity inpigs.An epidemic of CSFV in a high pig densityarea can result in devastating financial losses,because few infected pigs can be effectively cured.In many countries in Europe and Asia,classicalswine fever (CSF) is controlled by vaccinationwith commercially available vaccine strains,suchas the Chinese vaccine strain(C-strain,i.e.,hogcholera lapinized virus) orsome modified-live vir…     

Nucleotide sequence of cloned cDNA of human c-myc oncogene

Like other transforming genes of retroviruses, the v-myc gene of the avian virus, MC29, has a homologue in the genome of normal eukaryotic cells. The human cellular homologue, c-myc, located on human chromosome 8, region q24 leads to qter (refs 1, 2), is translocated into the immunoglobulin heavy-chain locus on human chromosome 14 (ref. 3) in Burkitt's lymphoma, suggesting that c-myc has a primary role in transformation of some human haematopoietic cells. In addition, c-myc is amplified in the human promyelocytic leukaemia cell line, HL60 (refs 6, 7) which also contains high levels of c-myc mRNA. Recently, Colby et al. reported the nucleotide sequence of the human c-myc DNA isolated from a genomic recombinant DNA library derived from human fetal liver. This 4,053-base pair (bp) sequence includes two exons and one intron of the myc gene, and the authors have suggested the existence of a human c-myc mRNA of 2,291 nucleotides that has a coding capacity for a protein of molecular weight (Mr) 48,812. We have approached the problem of accurately defining the characteristics of the human c-myc mRNA and c-myc protein by determining the sequence of the c-myc cDNA isolated from a cDNA library prepared from mRNA of a clone of the K562 human leukaemic cell line. K562 cells are known to contain c-myc mRNA which is similar in size to the c-myc mRNA of other human cell types. We report here the sequence of 2,121 nucleotides of a human c-myc mRNA and demonstrate that its 5' noncoding sequence does not correspond to the sequence of the reported genomic human sequence. However, our data confirm that the intact human c-myc mRNA can encode a 48,812-Mr protein with a sequence identical to that reported by Colby et al.