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.     

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 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.     

Genome sequencing and characterization analysis of a Beijing isolate of chicken coronavirus infectious bronchitis virus

Avian infectious bronchitis virus (AIBV) is classified as a member of the genus coronavirus in the family coronaviridae. The enveloped virus has a positive-sense, single-stranded RNA genome of approximately 28 kilo-bases,which has a 5‘ cap structure and 3‘ polyadenylation tract.The complete genome sequence of infectious bronchitis virus (IBV), Beijing isolate, was determined by cloning sequencing and primer walking. The whole genome is 27733 nucleotides in length, has ten open reading frames:5′-orfla-orflab-s-3a-3b-e-m- 6a-6b-n-3′. Alignments of the genome sequence of IBV Beijing isolate with those of two AIBV strains and one SARS coronavirus were performed respectively. The genome sequence of IBV Beijing isolate compared with that of the IBV strain LX4 (uncompleted, 19440 bp in size) was 91.2% similarity. However, the full-length genome sequence of IBV Beijing isolate was 85.2% identity to that of IBV Strain Beaudette, and was only 50.8% homology to that of SARS coronavirus. The results showed that the genome of IBV has remarkable variation. And IBV Beijing isolate is not closely related to SARS coronavirus. Phylogenetic analyses based on the whole genome sequence, S protein, M protein and N protein, also showed that AIBV Bering isolate is lone virus in group Ⅲ and is distant from SARS coronavirus. In conclusion, this study will contribute to the studies of diagnosis and diseases control on IBV in China.     

Genomic cloning and sequencing of betaine aldehyde dehydrogenase gene in spinach

A genomic library derived from leaves of spinach was constructed with the λGem11_BamHI Arms as the vector. The library was screened using the BADH cDNA of mountain spinach as a probe and six positive clones were obtained through three rounds of screening. One of the positive clones named D, which was hybridized with the 5′600 bp fragment of mountain spinach BADH cDNA, was selected and further analyzed. The size of the insert in clone D was about 12 kb. 8 856 nucleotides of the insert were sequenced which contained 2 459 nucleotides of 5′ noncoding region, 6 111 nucleotides of the complete sequence of the BADH gene, and 286 nucleotides of a 3′ noncoding region. The result of sequence analysis indicated that the BADH gene contained 14 introns and the junction sequences at splicing sites followed the GT_AG rule basically.     

Cloning,sequencing and structural analysis of a pea cDNA encoding EF-1α

A cDNA library with the capacity of 2.0 × 10⁶ was constructed using mRNA extracted, from gibberellic acid (GA)-treated G2 pea seedlings and a 1.6 kb cDNA with 100 nucleotides of 5′ non-coding region and 223 nucleotides of 3′ non-coding region was obtained by random screening. The DNA fragment contains an open reading frame of 1 344 nucleotides and encodes 447 amino acids. Sequence analysis of DNA and deduced polypeptide demonstrated that it encoded for a pea elongation factor 1-alpha (EF-1α). The functional domains of EF-1α is well conserved and EF-1α can be a good candidate for studying molecular evolution.     

Complete nucleotide sequence and genomic organization of Periplaneta fuliginosa densonucleosis virus

We have cloned the replicative form of thePeriplaneta fuliginosa densonucleosis virus (PfDNV) genome and determined its complete sequence. The sequence has 5 454 nucleotides (nt), the genome consists of an internal unique sequence flanked by inverted terminal repeats (201 nt). The first 122 nt at the 5′ end and the terminal 122 nt at the 3′ end of both plus and minus strands can fold into a typical hairpin structure. The genome contains seven major open reading frames (ORFs). The plus strand has 4 ORFs occupying the 5′ half of the plus strand, whereas the others span the 5′ half of the minus strand. Two potential promoters were found at map units (m.u.) 3 and 97. Computer analysis of sequence homologies with other parvoviruses suggests that the plus strand ofPf DNV encodes very likely the nonstructural proteins and the minus strand probably encodes the structural proteins.     

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.     

Thymidine kinase gene mutation leads to reduced virulence of pseudorabies virus

To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK^― mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK^― mutant were sequenced. Analysis of the tk gene sequence of TK^― mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All other nucleotides of TK^―mutant were identical to that of wt virus. Deletion and insertion of the nucleotide sequence resulted in a frameshift and a premature chain termination, and the resultant TK protein was not active. Analysis of the amino acid sequence revealed that TK protein of PRV HB contained the conserved consensus sequence of herpesviral TKs and an additional conserved-DHR-motif. The results of this work also indicated that TK^― mutant was genetically stable. Compared to PRV HB, virulence of TK^― mutant was greatly decreased. Mice vaccinated with TK^― mutant were completely protected against a lethal challenge with virulent PRV (HB).     

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 of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

Molecular characterization of a K+ channel blocker in the scorpionButhus martensii Karsch

K⁺ channel blockers of scorpion venoms are of important value in studying pharmacology and physiology of specific K⁺ channel of cells. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca²⁺-activated K⁺ channel blocker, two “back to back” degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpionButhus martensii. The cDNA is composed of 3 parts: 5′ UTR, ORF and 3′ UTR. The flanking sequence of translation initiation codon ATG is AAAATGA, which is highly conserved in scorpion Na⁺ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3′ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is significantly different from that of the determined scorpion Na⁺ channel toxin. The deduced amino acid sequence of mature peptide is completely consistent with BmP01 previously determined by primary structure analysis.     

Expression of human cytomegalovirus DNA polymerase in insect cells using baculovirus expression system: Purification and biochemical characterizations

Human Cytomegalovirus (HCMV) DNA polymerase gene was overexpressed in insect cells using the baculovirus transfer system. A6. 2 kb HCMV Rsr II-EcoRI DNA fragment with intact HCMV pol gene coding sequence was engineered into NheI site of vector pBlueBac under the control of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPV carried HCMV pol gene was generated by cotransfection of Spodoptera frugiperta cell (SF21) with AcNPV DNA and baculovirus transfer vector with HCMV pol gene. Infection of SF21 cell with recombinant virus lead to the expression of 140 kD peptide of HCMV specific DNA polymerase at the level approximately 2 mg per 10⁸ cells. The polypeptide was purified from the infected SF21 cells by a series of column chromatography to homogeneity. The purified enzyme had a molecular weight of 140 kD and reacted with antiserum specific for HCMV DNA polymerase. It exhibited both 3′–5′ and 5′–3′ exonuclease activities. This enzyme is also sensitive to phosphono acetate. Ye Linbai: born in Feb. 1948. Professor. Current research interest is in Vitology and Molecular Biology Supported by Public Health Service Grants CA21773, CA15036 and AI12717 from the National Institutes of Health     

Synthesis and characterization of novel bridged porphyrins

The reaction of 5-(4-hydroxyphenyl)-10,15,20-triphenyl porphyrin with 2,6-dibromomethylpyridine and 4,4‘-dicarboxyl-2,2‘-bipyridine respectively gave 2,6-bis-[5,10,15-triphenyl-20-( 4-phenoxymethyl )-porphyrin-yl]-pyridine(3) and 4, 4‘-di-[5,10,15-triphenyl-20-( p-phenoxycarbonyl)-porphyrin-yl]-2,2‘-bipyridine (4). 5-[4-(4‘- Bromobutoxy) phenyl]-10, 15, 20-triphenyl porphyrin reacted with 2,6-dihydroxymethyl pyridine to give 2,6-bis-[5,10,15-triphenyl- 20-(4-(p-phenoxy)-butoxymethyl)-porphyrin-yl]-pyridine(5). Those new compounds have been identified by ^1H NMR, IR, MS and UV-visible spectra, and elemental analysis.     

Spectroscopic studies of the interaction between methylene blue and G-quadruplex

The telomere is a complex of simple guanine-rich repeating sequences of single-stranded DNA and pro-tein at the ends of eukaryotic chromosomes[1―3]. Hu-man telomere consists of the sequence, 5′-TTAGGG-3′, with the length ranging from 5 to 15 kb. The gu…     

Approach to synthesis of novel chiral 3-chloro-2(5H)-furanone and its application in asymmetric reactions

The synthetic method of the novel chiral synthon, 5-l-menthyloxy-3-chloro-2-(5H)-furanone 5a and its application in asymmetric reactions were investigated. 5a is easily obtained in highly optical purity, and acts as a stable acceptor of Michael addition with oxygen nucleophiles in tandem double Michael addition / internal nucleophilic substitution to offer the spiro-cyclopropane derivative containing four stereogenic centers 8, which it is difficult to obtain by routine methods. The synthetic methods for 5a and 8 are reported in detail and the new compounds are identified on the basis of their analytical data and spectroscopic data, such as UV, IR,¹H NMR,¹³C NMR, MS and elementary analysis. The absolute configuration of the interesting spiro-cyclopropanes, spiro [1-chloro-4-(l-menthyloxy)-5-oxo-6-oxa-biscyclo[3.1.0]hexane-2,3′-(4′-/-menthyloxy-5′-l-menthyloxybutyrolactone)] 8 was established by X-ray crystallography. This result can provide important synthetic strategy in synthesis of some complex molecules containing spiro-cyclopropane skeleton with multiple chiral centers.     

Synthesis of a new type tetraamides bridged calix[4]arene

Two new tetraamides bridged calix[4]arenes were synthesized by the condensation reaction of 1,3-bis-chlorocarbonylmethyl p-tert-butylcalix[4]arene with 1,2-bis (2′-amino-2′-methylpropanamido) benzene or 1,2-bis (2′-amino-2′-methylpropanamido)-4,5-dichloro benzene, respectively. The new compounds were characterized by¹H-NMR, MS-FAB, and elemental analysis; macrocyclic polyamine. Foundation item: Supported by the National Science Foundation of China (20072029) Biography: Xiao Yuan-jing (1974-), male, Doctor, research direction: supramolecular chemistry.     

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.