Objective: To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis. METHODS: The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry. RESULTS: rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids. CONCLUSIONS An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.
Acrosome ; immunology ; Animals ; Antibodies ; analysis ; Blotting, Western ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; immunology ; Escherichia coli ; Humans ; Immunohistochemistry ; Male ; Muramidase ; genetics ; immunology ; Plasmids ; Recombinant Proteins ; genetics ; Semen ; immunology ; Spermatozoa ; immunology ; Testis ; immunology

OBJECTIVETo develop an assay for cDNA cloning and haplotype sequencing of KIR2DL1 framework gene and determine the genotype of an ethnic Han from southern China.

METHODSTotal RNA was isolated from peripheral blood sample, and complementary DNA (cDNA) transcript was synthesized by RT-PCR. The entire coding sequence of the KIR2DL1 framework gene was amplified with a pair of KIR2DL1-specific PCR primers. The PCR products with a length of approximately 1.2 kb were then subjected to cloning and haplotype sequencing.

RESULTSA specific target fragment of the KIR2DL1 framework gene was obtained. Following allele separation, a wild-type KIR2DL1*00302 allele and a novel variant allele, KIR2DL1*031, were identified. Sequence alignment with KIR2DL1 alleles from the IPD-KIR Database showed that the novel allele KIR2DL1*031 has differed from the closest allele KIR2DL1*00302 by a non-synonymous mutation at CDS nt 188A>G (codon 42 GAG>GGG) in exon 4, which has caused an amino acid change Glu42Gly. The sequence of the novel allele KIR2DL1*031 was submitted to GenBank under the accession number KP025960 and to the IPD-KIR Database under the submission number IWS40001982. A name KIR2DL1*031 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee.

CONCLUSIONAn assay for cDNA cloning and haplotype sequencing of KIR2DL1 has been established, which has a broad applications in KIR studies at allelic level.

Alleles ; Base Sequence ; China ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Haplotypes ; Humans ; Male ; Mutation, Missense ; Receptors, KIR2DL1 ; genetics ; Sequence Analysis, DNA ; methods

OBJECTIVETo construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector.

METHODSFirst, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells.

RESULTSThe construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69.

CONCLUSIONThe construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.

Animals ; Antigens, CD ; genetics ; Antigens, Differentiation, T-Lymphocyte ; genetics ; DNA, Complementary ; Genetic Vectors ; Genotype ; Green Fluorescent Proteins ; genetics ; Lectins, C-Type ; genetics ; Mice ; Mice, Transgenic ; Plasmids ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transfection

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.
Amino Acid Sequence ; Animals ; Clonorchis sinensis/*enzymology ; Cluster Analysis ; Conserved Sequence ; DNA, Complementary/genetics ; Energy Metabolism ; Gene Expression Profiling ; Mitochondria/metabolism ; Models, Molecular ; Molecular Weight ; Phylogeny ; Protein Conformation ; Sequence Homology, Amino Acid ; Ubiquitin-Protein Ligases/chemistry/*genetics/*metabolism

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.
Amino Acid Sequence ; Animals ; Clonorchis sinensis/*enzymology ; Cluster Analysis ; Conserved Sequence ; DNA, Complementary/genetics ; Energy Metabolism ; Gene Expression Profiling ; Mitochondria/metabolism ; Models, Molecular ; Molecular Weight ; Phylogeny ; Protein Conformation ; Sequence Homology, Amino Acid ; Ubiquitin-Protein Ligases/chemistry/*genetics/*metabolism

Molecular mechanisms governing peritonitis caused by the presence of aseptic gauze have remained unclear. To identify the genes involved, sterile gauze-exposed omentum was collected at 0, 6, 12, 24, and 48 h intervals, and analyzed by differential display RT(reverse transcription)-PCR. Among over 1,200 bands, 230 bands were found differentially expressed. These bands represented the fragment sizes of approximately 200 to 1,500 bp. The eight fragments were expressed differentially in the treatment group but not in the control. The sequences of two bands were similar to those of genes associated with the inflammatory process and a band was related to repair and regeneration process. Another one was related with spermatogonia and the rest four were unknown. Additionally, amplicons corresponding to the full-length sequences of two inflammatory gene fragments were synthesized by rapid amplification of cDNA end PCR. One showed 99% similarity to the major histocompatibility complex class II dog leukocyte antigen-DR beta chain and the other was canis familiaris proteasome beta type 3. Results of the present study suggested that sterile gauze induced the differential expression of genes in the omentum involved in inflammation and healing process.
Animals ; *Bandages ; Base Sequence ; DNA, Complementary/analysis ; Dogs/*genetics/metabolism ; Gene Expression Profiling/veterinary ; Gene Expression Regulation ; Histocompatibility Antigens Class II/*genetics/metabolism ; Molecular Sequence Data ; Omentum/*metabolism ; Proteasome Endopeptidase Complex/*genetics/metabolism ; RNA, Messenger/analysis ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; *Wound Healing

OBJECTIVEIn order to obtain functional genes, a normalized stems cDNA library was constructed from medicinal plant Dendrobium officinale.

METHODSMART (switching mechanism at 5' end of RNA transcript) cDNA synthesis combined with DSN (duplex-specific nuclease) normalization was applied to construct the normalized full-length cDNA library of D. officinale.

RESULTThe titer of cDNA library was about 1.3 x 10(6) cfu x mL(-1) and the average insertion size was about 1.5 kb with high recombination rate (93.9%). Random selected 163 positive clones were sequenced at single side. Bio-information analysis indicated that 147 from 150 high-quality unique sequences matched corresponding homologous proteins, and they participated in various biological processes based on GO (gene ontology). There were 8 clones with complete coding sequence, which presumed to be full-length genes.

CONCLUSIONThese results showed preliminarily that we successfully constructed a normalized full-length cDNA library of D. officinale which could be used to screen the functional genes related to metabolic pathways of medicinal ingredients.

Base Sequence ; Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; Dendrobium ; genetics ; Gene Library ; Molecular Sequence Data ; Plants, Medicinal ; genetics ; RNA, Messenger ; genetics ; metabolism ; Sequence Analysis, DNA ; methods

OBJECTIVEThe study aimed to clone the open reading frame of chalcone synthase (CHS) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene.

METHODOne unique sequence containing CHS domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of CHS was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. The AsCHS1 expression in calli was analyzed with histone gene as an internal control gene under wound condition by qRT-PCR technique.

RESULTOne unique sequence of CHS, named as AsCHS1, was cloned from A. sinensis. The full length of AsCHS1 cDNA was containing a 1 192 bp ORF that encoded 397 amino acids. The result of qRT-PCR displayed that the highest expression level was at 12 h, which indicated that it was possibly involved in early-stage response to wound.

CONCLUSIONCloning and analyzing AsCHS1 gene from A. sinensis provided basic information for study the function and expression regulation of AsCHS1 in the flavonoids biosynthesis.

Acyltransferases ; genetics ; Base Sequence ; Cloning, Molecular ; Computational Biology ; DNA, Complementary ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Drugs, Chinese Herbal ; Flavonoids ; metabolism ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; genetics ; Plant Stems ; chemistry ; enzymology ; genetics ; Plants, Medicinal ; Protein Structure, Tertiary ; RNA, Messenger ; genetics ; RNA, Plant ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Thymelaeaceae ; chemistry ; enzymology ; genetics

OBJECTIVETo analyze the differentially expressed genes in the testicular tissues of men with unilateral cryptorchidism using cDNA gene chips.

METHODSProbes were prepared with the mRNA extracted from the testes of 6 patients with unilateral cryptorchidism and 3 normal fertile men. Then the differential gene expression profiles of the two groups were detected with cDNA gene chips containing 45 034 genes. The differentially expressed genes were analyzed with Pathway and GO in the MAS system.

RESULTSBased on the ratio of > 3.0 or < 0.33, 346 differentially expressed genes were detected in the testis tissues of the patients with unilateral cryptorchidism, among which 60 were up-regulated and 286 down-regulated. The up-regulated genes were distributed mainly on chromosomes 1, 15, 5 and 19, associated with cell cycles, sperm motility, flagellar movement, DNA replication, and chromatin modification, while the down-regulated genes, mainly on chromosomes 1, 19, 16 and 11, related with spermatogenesis and anti-apoptosis.

CONCLUSIONUnilateral cryptorchidism involves the variation of the expressions of multifunctional genes. The establishment of gene expression profiles of unilateral cryptorchidism in human testes may provide a new theoretical basis for analyzing the genetic factors of unilateral cryptorchidism and investigating the etiology of spermatogenic failure.

Adult ; Case-Control Studies ; Cryptorchidism ; genetics ; DNA, Complementary ; genetics ; Humans ; Male ; Oligonucleotide Array Sequence Analysis ; RNA, Messenger ; genetics ; Testis ; chemistry ; Transcriptome ; Young Adult

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.
Bunyaviridae Infections ; diagnosis ; virology ; Cell Line ; DNA Ligases ; metabolism ; DNA, Complementary ; analysis ; genetics ; DNA-Directed DNA Polymerase ; metabolism ; Dengue ; diagnosis ; virology ; Dengue Virus ; genetics ; isolation & purification ; Genome, Viral ; genetics ; Humans ; Phlebovirus ; genetics ; isolation & purification ; RNA, Viral ; analysis ; genetics ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Load