Objective To construct eukaryotic expression vector of human antimicrobial peptide LL-37 pPIC9-LL-37, and transform the plasmid pPIC9-LL-37 into P.pastoris GS115 to obtain the recombinant P.pastoris strains.Methods The full-length of antimicsobial peptide LL-37 gene was artificially synthesized by overlap extension method and was fused to pPIC9 and then the fused plasmid was transformed into E.coli DH5?.After analysis by PCR and sequencing,the plasmid pPIC9-LL-37 was transformed into P.pastoris.The colonies exhibiting the phenotype of His+Mut+ or His+Mut-were screened by means of MM and MD plates and the insertion was confirmed by PCR.Results The results of PCR and sequencing confirmed that the LL-37 gene was correctly inserted into pPIC9. The colonies of 10 His+Mut+ and 9 His+Mut-were obtained by means of MM and MD plates screening and were confirmed by PCR.Conclsion The recombinant P.pastoris strains containing LL-37 were successfully obtained.

In order to develop clinical diagnostic tools for rapid detection of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.
Antigens, Viral ; immunology ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Genome, Viral ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; SARS Virus ; genetics ; immunology ; Yeasts ; genetics

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and alpha-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.
Amino Acid Sequence ; Base Sequence ; Cluster Analysis ; Codon ; genetics ; Gene Components ; Genome, Viral ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Phylogeny ; Protein Conformation ; SARS Virus ; genetics ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; genetics ; metabolism

We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To investigate its immunogenicity, we synthesized eight oligopeptides covering 69.2% of the entire ORF and screened them by using ELISA (enzyme-linked immunosorbent assay) with sera from SARS patients. The results confirmed our predictions on antigenic sites.
Amino Acid Sequence ; Base Sequence ; Cluster Analysis ; Enzyme-Linked Immunosorbent Assay ; Immunoassay ; Molecular Sequence Data ; Mutation ; genetics ; Oligopeptides ; Phylogeny ; Protein Structure, Tertiary ; SARS Virus ; genetics ; Sequence Alignment ; Sequence Analysis, DNA ; Viral Matrix Proteins ; chemistry ; genetics ; immunology

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.
Genome, Viral ; Haplotypes ; Humans ; Mutation ; Open Reading Frames ; Phylogeny ; SARS Virus ; genetics

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.
Base Sequence ; China ; Cluster Analysis ; Gene Components ; Genetic Variation ; Genome, Viral ; Genotype ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; genetics