Identification of a novel bombesin bioactive peptide QUB2995 and its insulin secreting activity / 中华内分泌代谢杂志

Objective:To identify a novel bombesin bioactive peptide from the skin secretion of Hylarana Latouchii, and to explore its effect on insulin secretion in islet cells.Methods:The skin secretion from Hylarana Latouchii was extracted by electrical stimulation, and the single chain of bombesin peptide was cloned and sequenced. The peptide QUB2995 was synthesized via solid-phase synthesis, then purified using reversed-phase high performance liquid chromatography (HPLC). Matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was applied to validate. QPCR and ELISA were used to probe the effect of QUB2995 on insulin secretion in MIN6 and INS-1 cells.Results:A novel bombesin peptide named QUB2995 (GAFGDFLKGAAKA GALKILSIAQCKLSGTC) was found in the skin secretion of Hylarana Latouchii through molecular cloning. The bioactive peptide could significantly promote the proliferation and insulin secretion from mouse islet MIN6 cells and rat islet INS-1 cells. The effect reached a climax at the concentration of 10 -5 mol/L. Conclusion:A novel bombesin bioactive peptide named QUB2995 was found from Hylarana Latouchii. It could significantly promote insulin secretion in MIN6 cells of mouse islets and INS-1 cells of rat islets, indicating its potential in the treatment of diabetes.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis / 中国天然药物

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

Construction of human YPEL5 gene eukaryotic expression vector and its expression in esophageal carcinoma cells / 国际检验医学杂志

Objective To construct human yippee-like 5(YPEL5) gene eukaryotic expression recombinant plasmid and to express in esophageal carcinoma EC9706 cells .Methods The cDNA from human normal tissue was taken as a template and amplified to YPEL5 gene coding sequence with 366 bp in length .Then this se-quence was inserted into the multiple cloning site areas of eukaryotic expression vector pCDH-CD513B for ob-taining the eukaryotic expression vector pCDH-CD513B-Flag-YPEL5 .After the bacterial colony PCR identifi-cation ,it was sent to the corporation for testing the sequence .The successfully constructed recombinant plas-mid was transfected into human esophageal carcinoma EC9706 cells .The expression of PEL5 gene in EC9706 cells was detected by QRT-PCR and Western Blot .Results The YPEL5 gene segment with 366 bp in length was successfully amplified .pCDH-CD513B-Flag-YPEL5 recombinant plasmid was obtained by double enzyme digestion ,connection ,conversion and screening .The gene sequencing identification showed that the inserted gene sequence in recombinant plasmid was consistent with that in the GenBank .After 2 d of transfecting into EC9706 cells ,the QRT-PCR and Western Blot revealed that YPEL5 gene expression was significantly up-reg-ulated .Conclusion The pCDH-CD513B-Flag-YPEL5 eukaryotic expression vector is successfully constructed and is expressed in esophageal squamous cancer cell line EC9706 ,thus which lays a foundation for studying its function in the progression of esophageal cancer .

Cloning and Characterization of the Gene Encoding 3-hydroxy-3- Methylglutaryl-coenzyme A (HMG-CoA) Reductase from Fritillaria Cirrhosa D. Don

ABSTRACT The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in Mevalonate (MVA) pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR from Fritillaria cirrhosa (FcHMGR), a bulbous medicinal plant. The full-length cDNA of FcHMGR was 2072 base pair (bp), containing a 1680-bp open reading frame. Bioinformatical analyses revealed that FcHMGR had HMG CoA-binding domains and two NADPH binding domains, which are required for HMGR activity. Quantitative real-time PCR (qRT-PCR) analysis revealed that FcHMGR expressed high in mature bulbs. A truncated version of FcHMGR protein lacking the N-terminal 249-bp GC rich area was expressed in Escherichia coli. The crude cell lysate containing the recombinant protein showed a better HMGR activity than the control and the relative enzyme activity was calculated to be 1.62 U/mg. The cloning, characterization and functional analysis of FcHMGR gene allowed us to further understand the role of FcHMGR involved in steroidal alkaloid biosynthetic pathway in F. cirrhosa at the molecular level.

Analysis of microbial diversity in Shenqu with different fermentation times by PCR-DGGE

Abstract Shenqu is a fermented product that is widely used in traditional Chinese medicine (TCM) to treat indigestion; however, the microbial strains in the fermentation process are still unknown. The aim of this study was to investigate microbial diversity in Shenqu using different fermentation time periods. DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) profiles indicated that a strain of Pediococcus acidilactici (band 9) is the predominant bacteria during fermentation and that the predominant fungi were uncultured Rhizopus, Aspergillus oryzae, and Rhizopus oryzae. In addition, pathogenic bacteria, such as Enterobacter cloacae, Klebsiella oxytoca, Erwinia billingiae, and Pantoea vagan were detected in Shenqu. DGGE analysis showed that bacterial and fungal diversity declined over the course of fermentation. This determination of the predominant bacterial and fungal strains responsible for fermentation may contribute to further Shenqu research, such as optimization of the fermentation process.

Molecular cloning and characterization of porcine ribosomal protein L21

Ribosomal protein L21 (RPL21) is a structural component of the 60S subunit of the eukaryotic ribosome. This protein has an important role in protein synthesis and the occurrence of hereditary diseases. Pig is a common laboratory model, however, to the best of our knowledge, its RPL21 gene has not been cloned to date. In this study, we cloned and identified the full-length sequence of the pig RPL21 gene for the first time. In addition, we examined its expression pattern and function by using overexpression or knockdown approaches. As a result, we obtained a 604 bp segment that contains a 483 bp open reading frame encoding 160 amino acids. The pig RPL21 gene is located in the “+” strand of chromosome 11, which spans 2167 bp from 4199792 to 4201958. Pig RPL21 protein has nine strands and two helices in its secondary structure. Pig RPL21 is predominantly expressed in ovary and lung, at lower levels in kidney, small intestine, and skin, and at the lowest levels in heart and liver. Furthermore, RPL21 expression is closely connected with cell proliferation and cell cycle arrest. The results are intended to provide useful information for the further study of pig RPL21.

Cloning short gene fragment and constructing recombinant plasmid based on restoration of antibiotic resistance / 生物工程学报

Molecular cloning is one of the most important and widely used technologies in molecular biology research. Generally, the target DNA fragment and the vector are separately digested by restriction enzyme, then purified and recovered, and then ligated with DNA-ligase. For some very short gene fragments (<300 bp), the recovery efficiency of the purified fragment is very low after digestion and cleavage, leading to the difficulty in its inserting into the expression vector. To address this issue, we developed a cloning method based on restoration of antibiotic resistance in constructing recombinant plasmid, which proved highly efficient in cloning very short gene fragments.

Preparation of standard molecular weight fragment mixture based on molecular cloning / 中国法医学杂志

Objective To prepare the standard molecular weight fragment mixtures. Methods Primers were designed to prepare clones which contained different sizes of standard molecular weight fragments. The template used for amplification of insert fragments was the pMD18-T vector. Bacteria culture and plasmid extraction were used to obtain abundant target fragment. Unlabeled DNA fragments were prepared by double digestion of the recombinant plasmids, and the fluorescent adaptor was prepared by annealing with two partial reverse complimentary DNA fragments. The unlabeled fragments and fluorescent adaptor were connected by DNA ligation reaction assisted with T4 DNA ligase. In this way, different sizes of standard molecular weight fragments were prepared. Standard molecular weight fragment mixture was finally prepared by mixing all the fragments together before purification. Results Ten standard molecular weight fragments of different sizes were prepared. The sizes of each fragment are 80bp, 124bp, 194bp, 224bp, 254bp, 304bp, 349bp, 399bp, 424bp and 454bp. The internal standard could accurately determine the size of PCR products amplified with the DNATyper15 kit. Conclusion Using this method, the standard molecular weight fragment mixture which meet the requirements of research and laboratory use was prepared, perfectly providing a new method for preparation of the DNA molecular weight standards. The peaks and the size of the prepared DNA internal lane standard are correct, which can be used to calculate the DNA fragments size in capillary electrophoresis.

Cloning and analysis of rDNA ITS sequences from seven kinds of medicinal plants in Goodyera L / 中草药

Objective: To identify the medicinal plants in Goodyera L. at molecular level, cloning and sequence analysis of seven internal transcribed spacer (ITS) sequences from seven plant species of Goodyera L. were performed. Methods: ITS sequences were cloned using PCR amplification, and sequence analysis, evaluation of genetic distance as well as construction of phylogenetic tree were conducted by bioinformatics software. Results: Full ITS sequences of seven kinds of plants in Goodyera L. were 700-702 bp in length. The length of ITS1 and ITS2 were 239-240 bp and 298-300 bp, respectively, while 5.8S sequences were more conserved with identical length of 162 bp. Thirty-four parsimony information sites among 107 variable sites were detected in the sequence. Genetic distances among seven plant species of Goodyera L. were 0.006-0.110. The greatest genetic distances were observed between V. grypoceras and V. diffusa var. brevibarbata indicating the farthest of their relative distinct relationship, and the smallest existed between V. diffusa and V. acuminata revealing their closest relationship. Conclusion: ITS sequences of seven kinds of medicinal plants in Goodyera L. are obtained with a result of rich information sites which could be used to distinguish these seven species completely.

Development and significance of RAPD-SCAR markers for the identification of Litchi chinensis Sonn: by improved RAPD amplification and molecular cloning

Background Analysis of genetic diversity is important for the authentication of a species. Litchi (Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species.

Antigenic properties and virulence of foot-and-mouth disease virus rescued from full-length cDNA clone of serotype O, typical vaccine strain

We cloned the full-length cDNA of O Manisa, the virus for vaccinating against foot-and-mouth disease. The antigenic properties of the virus recovered from the cDNA were similar to those of the parental virus. Pathogenesis did not appear in the pigs, dairy goats or suckling mice, but neutralizing antibodies were raised 5-6 days after the virus challenge. The utilization of O Manisa as a safe vaccine strain will increase if recombinant viruses can be manipulated by inserting or removing a marker gene for differential serology or replacing the protective gene from another serotype.

Cloning and analysis of rDNA ITS sequences from 11 medicinal plants in Viola L / 中草药

Objective: To provide molecular evidences for identification of medicinal plants in Viola L. by determining and analyzing the rDNA internal transcribed spacers (ITS) sequences of 11 species in Viola L. Methods: ITS sequences were isolated using PCR amplification, and sequence analysis, evaluation of genetic distance as well as construction of phylogenetic tree were conducted by bioinformatics software. Results: Full ITS sequences of 11 plants in Viola L. were varied from 612 to 638 bp in length. The lengths of ITS1 and ITS2 were 251-265 bp and 198-211 bp, respectively, while 5.8 S sequences were more conserved with identical length of 163 bp. Parsimony information sites of ITS1, ITS2, and 5.8 S were 50, 23, and 3, respectively. Genetic distance among 11 species in Viola L. varied from 0.025 to 0.137, and the greatest genetic distances were observed between V. grypoceras and V. diffusa var. brevibarbata, indicating their relative distinct relationship, and the smallest existed between V. diffusa and V. acuminata, revealing their closest relationship. Conclusion: ITS sequences of 11 medicinal plants in Viola L. were obtained with a result of rich information sites, which provideds a foundation for molecular identification.

Molecular cloning and expression pattern of duck Six1 and its preliminary functional analysis in myoblasts transfected with eukaryotic expression vector.

Skeletal muscle development is regulated by Six1, an important myogenic transcription factor. However, the functional analysis of duck Six1 has not been reported. Here, we cloned the coding domain sequence (CDS) region of the duck Six1 gene using RT-PCR and RACE methods. Bioinformatics analysis revealed that duck Six1 CDS region comprised of 849 bp and encoded 282 amino acids and had a high degree of homology with other species, suggesting that the functions of duck Six1 gene are conserved among other animals. Real-time PCR used to determine the mRNA expression profiles of duck Six1 in different tissues and different developmental stages showed that Six1 was highly expressed in skeletal muscle and the embryonic stage. Furthermore, the eukaryotic expression vector pEGFP-duSix1 was constructed and transfected into the duck myoblasts; the MTT assay revealed an obvious increase of cell proliferation after transfection. The expression profiles of Six1, Myf5 and MyoD showed that their expression levels were significantly increased. These results together suggested that pEGFP-duSix1 vector was constructed successfully and overexpression of duck Six1 in the myoblasts could promote cell proliferation activity and significant up-regulate expression of Myf5 and MyoD.

Cloning and expression analysis on 3-hydroxy-3-methylglutaryl-coenzyme A reductase from aquilaria sinensis / 中草药·英文版

Objective: To clone the full-length cDNA of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) from Aquilaria sinensis (AsHMGR1) and to analyze its expression profile in different tissues and in response to different treatments. HMGR is the first rate-limiting enzyme for sesquiterpene synthesis in the mevalonate pathway. Methods: RT-PCR and RACE were used to clone the full-length cDNA of HMGR from A. sinensis based on the conserved HMGR gene fragments. The bioinformatic analysis was performed on its nucleic acid and protein sequence. The expression profile of AsHMGR1 in different tissues and in response to different treatments was analyzed by quantitative RT-PCR. Results: The full-length AsHMGR1 cDNA was 2026 bp, containing a 1719 bp open reading frame which encoded a protein of 572 amino acids. Amino acid sequence homology alignment and phylogenetic analysis demonstrated that AsHMGR1 belonged to the HMGR gene family. The detection of tissue expression patterns showed that AsHMGR1 was mainly expressed in the stem, followed by roots and branches. AsHMGR1 could be stimulated by methyl jasmonate and H2O2 to varying degrees in a time-dependent manner. Conclusion: These data will provide a foundation for further investigation on AsHMGR1 functions and regulatory mechanisms in sesquiterpene synthesis in A. sinensis. © 2013 Tianjin Press of Chinese Herbal Medicines.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Molecular cloning and characterization of calcium-dependent protein kinase genes in Dendrobium officinale / 中国药学杂志

OBJECTIVE: To clone and characterize calcium-dependent protein kinases (CDPKs) genes in Dendrobium officinale Kimura et Migo. METHODS: Reverse transcription PCR(RT-PCR) and rapid-amplification of cDNA ends (RACE) methods were used to isolate CDPKs genes from the leaf cDNA of D. officinale. Characteristics including the molecular weight, theoretical pI (isoelectric point), conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced proteins were analyzed using serials of bioinformatics algorithms. The analyses of multiple alignment and phylogenetic tree were respectively performed using DNASTAR and MEGA4. Tissue specific expression patterns were determined using real-time quantitative PCR (qPCR) analyses. RESULTS: Two full length genes DoCPK2 and DoCPK3 (GenBank accessions JX219469 and JX219470), 2119 and 2418 bp in length, respectively, were obtained. DoCPK2 was deduced to a 541 aa (amino acid) protein with a molecular weight of 60.46·103 and a pI of 6.14, while DoCPK3 encoded a 536 aa protein with a molecular weight of 60.30·103 and a pI of 6.32. The two deduced proteins, without signal peptide, both contained the conserved caniocal serine/threonine- protein kinase catalytic domain, Ca2+ binding EF hand motifs, and a 19 aa transmembrane structure at 248-266 and 243-261 aa position, respectively. They were highly homologues (67%-81%) to the plant CDPKs genes, and were mostly close to monocots CDPKs genes from wheat and rice. DoCPK2 and DoCPK3 were constitutively expressed among the five tissues. DoCPK2 transcripts were more abundant in the roots and PLBs (protocorm-like bodies), while the transcription levels of DoCPK3 were repressed in the seeds and stems. CONCLUSION: Two calcium-dependent kinases genes DoCPK2 and DoCPK3 are identified from the valuable herb D. officinale, which will be useful for further functional determination of the genes involving in the growth, development, biotic and abiotic responses in D. officinale.

Molecular identification of viral pathogens in Crocus sativus / 中草药

Objective: To investigate the viral pathogens in cultivated Crocus sativus. Methods: Viral pathogen identification was carried out by the observation of virus particle morphology and cytopathology as well as the detection of DAS-ELISA, RT-PCR, and sequencing. Results: Linear virus particles of 600-900 nm in length were observed in C. sativus by negative staining under transmission electron microscope (TEM). Bundles of linear virus particles, cylindrical inclusion bodies of subdivision II, and amorphous inclusion bodies were observed in the cells of C. sativus under TEM after ultrathin-section. These observations resembled the cytopathology of infectious Bean yellow mosaic virus (BYMV). Positive result of DAS-ELISA was obtained from the leaves of C. sativus by using monoclonal antiserum against BYMV capsid protein. Positive result of RT-PCR induced by the Potyvirus specific primers (Sprimer and M4) was also obtained. Sequencing after RT-PCR revealed that the viral sequence in this diseased C. sativus had a homology of 99% with the BYMV sequence. Conclusion: The pathogenic virus of this C. sativus disease is identified as BYMV.

Cloning of β-amyrin synthase gene from Panax quinquefolius and its bioinformatics analysis / 中草药

Objective: To obtain the full-length cDNA of β-amyrin synthase (bAS) involved in triterpene saponin biosynthesis in Panax quinquefolius and provide the reference for saponin biosynthesis and the regulation of secondary metabolism in P. quinquefolius. Methods: Based on large-scale ESTs sequencing and RACE technology, the full-length cDNA of P. quinquefolius bAS (PqbAS) was obtained. Results: The full-length cDNA of PqbAS (GenBank No. JX185490) was 2 309 bp which included an open reading frame (ORF) code of 631 amino acid peptide. Common conserved domains of oxidosqualene cyclases (OSCs) were found in PqbAS including active sites and conserved sequence. Singal P4.0 analysis showed that PqbAS was a non-secreted protein. Tmhmm 2.0 analysis showed that PqbAS was a non-transmembrane protein. Real-time fluorescence quantitative PCR analysis showed PqbAS gene expressed in various organs, higher in flowers and stems while relatively lower in roots and leaves. Conclusion: The full-length of bAS is first cloned, which could provide the foundation for the investigation on expression characteristics and its role in synthesis of saponin.

Cloning and analysis of rDNA ITS sequences from plants in Rubus L / 中草药

Objective: To provide the evidences for molecular identification as well as genetic diversity studies, sequencing and comparison of rDNA ITS sequences from 15 plants in Rubus L. were performed. Methods: Full length rDNA ITS sequences were isolated from leaf genomic DNA by PCR method with universal primers, and these sequences was analyzed using bioinformatic softwares. Results: The length of ITS1, ITS2, and 5.8 S sequences for the 15 plants in Rubus L. were 255-258, 208-211, and 164 bp, respectively. Total 138 variable sites were found in ITS1 and ITS2 sequences with 41 parsimony information ones, and some transitions, transversions, and deletions were observed. The 5.8 S sequences were more conserved and there were only four variable sites with no parsimony information ones. The genetic distance of the 15 plants in Rubus L. ranged from 0.1390 to 0.0081, and the smallest genetic distance was observed between R. irenaeus and R. reflexus. Conclusion: The rDNA ITS sequences of the 15 plants in Rubus L. are obtained, which would provide the evidences for molecular identification and genetic diversity studies.