In order to differently diagnose avian influenza virus (AIV) subtypes, the HA gene of AIV H9 subtype was cloned, expressed and utilized in an enzyme-linked immunoad sorbent assay (ELISA). HA gene (1683bp) of H9N2 AIV was amplified by RT-PCR from a strain of field isolated H9N2 AIV, and its identity was confirmed by sequencing. The HA gene was subcloned into prokaryotic expression vector pGEX-KG with its secretion signal sequence removed. The expressed HA-GST fusion protein in E. coli BL21 was characterized by SDS-PAGE and western blotting analysis as a 90kD protein with immunogenicity. The fusion protein was present primarily in inclusion bodies and was purified via denaturation and renenaturation. The HA-GST fusion protein was used to establish an indirect ELISA for the detection of antibodies to H9 subtypes of AIV. The assay has 91.57% specificity to H9 AIV, 92.31% sensitivity and excellent reduplication. It could be used to differently detect antibodies to H9 AIV.
Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Hemagglutinin Glycoproteins, Influenza Virus ; biosynthesis ; genetics ; Humans ; Influenza A Virus, H9N2 Subtype ; genetics ; Influenza, Human ; diagnosis ; virology ; Recombinant Proteins ; biosynthesis ; genetics

This study was aimed to establish ELISA for recombinant bovine IFN-gamma (BovIFN-gamma) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-gamma (BovIFN-gamma) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-gamma. The pETBovlFN-gamma was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-gamma as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-gamma were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG1 . Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-gamma, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-gamma specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-gamma, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-gamma paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.
Animals ; Animals, Newborn ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; immunology ; Blotting, Western ; Cattle ; Cells, Cultured ; DNA, Complementary ; genetics ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Hybridomas ; Interferon-gamma ; genetics ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Rabbits ; Recombinant Proteins ; immunology ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Dao-di herbs, which are widely recognized as medicinal materials with a high quality and good efficacy in clinic, are now facing the dilemma of absence of standard. This study focused on a pivotal scientific problem of design and application of quality standard of Dao-di herbs, and systematically illustrated the general rules for the quality standard of Dao-di herbs involving "four rules, six core contents, and three key methods". The quality standard of Dao-di herbs shall be fully based on literatures as well as habitat, planting/breeding, processing, characters, chemical-pharmacological/toxic data. The common requirements for the quality standard of Dao-di herbs contain "clear source, explicit origin, rational indicator, gradable quality, and multiple detection methods". Notably, traditional experiences and modern techniques, quality tracing management system, "quality determination by distinguishing characters" method, rapid detection technology, effective/toxic substances control method, were comprehensively applied in this standard to purse the objectification, automation, and intellectualization of detection technology. Appearance characters, chemical components, and bioactive parameters, unified effective/toxic indicators, quality markers, and pharmacopeial control indicators and reasonable ranges were included in rigorous quality standards for Dao-di herbs. Besides, simple grading method shall be developed to guide the implementation of "high quality-high price" policy. Eventually, the new quality standards for Dao-di herbs will lead international standards and promote the high-quality development of Dao-di herbs.
Drugs, Chinese Herbal ; Medicine, Chinese Traditional ; Plants, Medicinal ; Reference Standards ; Technology

Plants have a memory function for the environmental stress they have suffered. When they are subjected to repeated environmental stress, they can quickly and better activate the response and adaptation mechanism to environmental stress, thus realizing long-term stable reproduction. However, most of the relevant studies are applied to crops and Arabidopsis thaliana rather than medicinal plants about the improvement of plant growth status and the effect on phytoalexin biosynthesis. In this study, yeast extract(YE) was used as an elicitor to simulate biotic stress, and the changes in biomass and the content of some secondary metabolites were measured by giving repeated stresses to Sorbus aucuparia suspension cell(SASC). The results showed that the accumulation levels of biomass and some secondary metabolites in SASC subjected to repeated stress are significantly increased at some time points compared with single stress. A phenomenon that SASC can memorize biotic stress is confirmed in this study and influences phytoalexin accumulation in SASC. Furthermore, the work laid the groundwork for research into the transgenerational stress memory mechanism of medicinal plant.
Cells, Cultured ; Secondary Metabolism ; Sorbus ; Stress, Physiological

Objective: To obtain the glycosyltransferase gene involved in modification reaction of phytoalexin from Sorbus pohuashanensis suspension cell,and conduct sequence analysis and prokaryotic expression analysis. Method: Based on the transcriptome data,specific primers were designed to obtain 2 cDNA sequences of SaUGTs genes,construct prokaryotic expression vector HIS-MBP-pET28a-SaUGTs and induce the expression of recombinant SaUGTs protein. Result: SaUGT1 and SaUGT2 sequences were cloned and obtained from glycosyltransferases,then bioinformatic analysis of the sequence and prokaryotic expression analysis were conducted. SaUGT1 gene contained 1 458 bp open reading frame (ORF),encoding a polypeptide of 485 amino acids,with a relative molecular weight of 54.27 kDa and theoretical isoelectric point (pI) of 5.50.SaUGT2 gene contained 1 431 bp ORF,encoding a polypeptide of 476 amino acids,with a relative molecular weight of 53.49 kDa and theoretical pI of 5.63. Bioinformatics analysis indicated that SaUGT1 and SaUGT2 protein had no signal peptide,and the conserved domains of glycosyltransferase family were detected. Phylogenetic results showed that SaUGT1 and SaUGT2 proteins had the closest relationship with the UGT85 family of A. thaliana. Differential expression analysis revealed that the relative expression levels of SaUGT1 and SaUGT2 were increased significantly after being induced by yeast extract (YE), with the highest expression level found at 24 h and 12 h. The recombinant SaUGT1 and SaUGT2 proteins were successfully expressed in Escherichia coli DE3 cells and finally,the recombinant SaUGT1 and SaUGT2 proteins were purified through Ni²⁺ affinity chromatography. Conclusion: The glycosyltransferase gene was cloned from the S. aucuparia for the first time,and the prokaryotic expression vector was successfully constructed,laying foundation for further study of the function of this gene.

In this study, the fatty acid desaturase gene FAD2 was cloned from Coix lacryma-jobi L. and its molecular structure and function were studied. The results showed that the full-length cDNA sequence of FAD2 gene was 936 bp encoding 311 amino acid residues. Bioinformatics prediction results showed that the protein encoded by the FAD2 gene was an alkaline hydrophilic unstable protein with a molecular weight of 34.87 kDa. It contained three transmembrane helix domain, and did not contain the signal peptide splicing site, and was most likely to be located in plasmid membrane. Compared with other similar genes in plants, it has only a histidine conserved site, His Box Ⅲ histidine site (HXXHH), suggesting its activity may be reduced. Phylogenetic tree analysis showed that FAD2 was closely related to monocotyledonous plants, especially Maize and Oryza sativa japonica Group, but farther from dicotyledonous plants. Therefore, it was inferred that FAD2 might have similar functions with similar genes in Maize and Oryza sativa japonica Group. In addition, the expression of FAD2 gene could be detected in Coix lacryma-jobi L. with high oil content, but not in low oil content of Coix lacryma-jobi L. In order to clarify the function of FAD2, the gene was heterologously expressed in sporomyces cerevisiae. The results showed that the protein encoded by FAD2 gene did not catalyze the formation of C18∶1 unsaturated fatty acid into C18∶2 unsaturated fatty acid. Therefore, it was speculated that the deletion of histidinine conserved site of FAD2 gene might lead to the decrease of protein activity or even inactivation. This study provides reference value for further understanding the molecular structure characteristics of fatty acid desaturase. At the same time, it laid a foundation for elucidating the biosynthetic pathway of Coix lacryma-jobi L.

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 proprietary Chinese medicines (APCMs). Multi-type dosage forms (, tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 μL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography-mass spectrometry (LC-MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.

BACKGROUND: Desminopathy, a hereditary myofibrillar myopathy, mainly results from the desmin gene (DES) mutations. Desminopathy involves various phenotypes, mainly including different cardiomyopathies, skeletal myopathy, and arrhythmia. Combined with genotype, it helps us precisely diagnose and treat for desminopathy. METHODS: Sanger sequencing was used to characterize DES variation, and then a minigene assay was used to verify the effect of splice-site mutation on pre-mRNA splicing. Phenotypes were analyzed based on clinical characteristics associated with desminopathy. RESULTS: A splicing mutation (c.735+1G>T) in DES was detected in the proband. A minigene assay revealed skipping of the whole exon 3 and transcription of abnormal pre-mRNA lacking 32 codons. Another affected family member who carried the identical mutation, was identified with a novel phenotype of desminopathy, non-compaction of ventricular myocardium. There were 2 different phenotypes varied in cardiomyopathy and skeletal myopathy among the 2 patients, but no significant correlation between genotype and phenotype was identified. CONCLUSIONS We reported a novel phenotype with a splicing mutation in DES, enlarging the spectrum of phenotype in desminopathy. Molecular studies of desminopathy should promote our understanding of its pathogenesis and provide a precise molecular diagnosis of this disorder, facilitating clinical prevention and treatment at an early stage.
Animals ; Asian Continental Ancestry Group ; Cardiomyopathies ; genetics ; pathology ; Desmin ; genetics ; Electrocardiography ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Muscular Dystrophies ; genetics ; pathology ; Mutation ; genetics ; Pedigree ; Phenotype