cDNA cloning of a novel lectin that induce cell apoptosis from Artocarpus hypargyreus / 中国天然药物

We isolated a novel lectin (AHL) from Artocarpus hypargyreusHance and showed its immunomodulatory activities. In this study, the amino acid sequence of AHL was determined by cDNA sequencing. AHL cDNA (875bp) contains a 456-bp open reading frame (ORF), which encodes a protein with 151 amino acids. AHL is a new member of jacalin-related lectin family (JRLs), which share high sequence similarities to KM+ and Morniga M, and contain the conserved carbohydrate binding domains. The antitumor activity of AHL was also explored using Jurkat T cell lines. AHL exhibits a strong binding affinity to cell membrane, which can be effectively inhibited by methyl-α-D-galactose. AHL inhibits cell proliferation in a time- and dose-dependent manner through apoptosis, evidenced by morphological changes, phosphatidylserine externalization, poly ADP-ribose polymerase (PARP) cleavage, Bad and Bax up-regulation, and caspase-3 activation. We further showed that the activation of ERK and p38 signaling pathways is involved for the pro-apoptotic effect of AHL.

Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia / 中国实验方剂学杂志

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.

Cloning and expression analysis of squalene synthase gene in Tripterygium wilfordii / 药学学报

In this paper, we cloned the full-length cDNA of TwSQS from Tripterygium wilfordii suspension cells (GenBank:KR401220) and performed the bioinformation and mRNA expression analysis. The expression after methyl jasmonate (MJ) treatment of the gene was detected by RT-PCR. The full-length cDNA of TwSQS was 1800 bp containing a 1242 bp open reading frame (ORF) encoding a polypeptide of 413 amino acids. The theoretical isoelectric point (pI) was 7.94 and the calculate molecular weight was about 47.20 kD. The relative expression level of TwSQS was deduced by MJ and reached the highest at 4 h after the treatment. The gene information we got in this study enriched the biosynthesis pathway of triterpenoids in Tripterygium wilfordii and laid foundation for further studies.

Expression and Purification of recombinant soluble human BAFF / 中国生化药物杂志

Objective To construct pET21a-sBAFF by cloning the extracellular regions of 134-285 amino acids of BAFF, a member of human TNF family, and then express the gene in prokaryotic cells and purify the expressed product.Methods cDNA of K562 cell line was used as the template to amplify sBAFF gene to construct pET21a-sBAFF.Expression of sBAFF in E.coli BL21 was induced by IPTG, and the expressed proteins were assayed by SDS-PAGE.The bacteria were analyzed by sonication, and the target proteins mainly existed as inclusion bodies.Then sBAFF was purified by Ni2 +-IDA affinity chromatography.SDS-PAGE electrophoreses displayed that the expressed sBAFF migrated with a relative molecular weight of 18000.ResuIts The induction parameters such as temperature and inducing time were optimized.The target protein accounted for 38.59%of the total bacterial proteins.After refolding, 38.14% of sBAFF proteins were polymerized as an active trimer.The dimer form of sBAFF, which is representative of wrongly refolded product, accounted for very few.ConcIusion The expression and purification of BAFF which formed active trimer after refolding pave the way for its further function study and provide a novel approach for the development of BAFF-targeted therapeutics for autoimmune diseases.

Molecular cloning, characterization and expression of lipoxygenase 2 (lox-2) isozyme from Indian soybean [Glycine max (L.) Merrill] cv. Pusa 16.

The consumption of soybean is limited worldwide, despite being highly nutritious and having versatile uses, due to the presence of grassy, beany and rancid off-flavour. The lipoxygenase-2 (LOX-2) is the key enzyme responsible for the production of volatiles released from the beans, which cause off-flavour in soy products. In this study, a 2.6-kb full-length lox2 gene (NCBI accession No. JQ929619.1) was isolated and cloned from soybean (Glycine max L. Merril) cv. Pusa 16. The cloned cDNA sequence of lox2 gene showed the complete open reading frame (ORF) of a putative protein, having 866 amino acids with start codon present at the foremost position and stop codon at the end. The theoretical pI of predicted protein was 6.22. A hydropathy profile calculated from the amino acid sequence resembled those of dicot LOXs, suggesting conservation of the secondary structure of these enzymes. The LOX-2 showed conserved six Histidine residues within a span of 520 to 590 amino acid position, a signature element for the enzyme activity. The lox2 gene was expressed using pET vector in prokaryotic expression system. The recombinant LOX-2 protein was purified after induction with IPTG (isopentyl thiogalactoside). A prominent band of 97 kDa was observed, when affinity purified fractions were analyzed by SDS-PAGE. The purified protein was characterized for the enzyme activity, substrate preference and Km. Inhibitor studies with natural antioxidant molecules present in soybean revealed α-tocopherol to be the most effective inhibitor of LOX-2.

Cloning and expression stability analysis of actin gene in Eleutherococcus senticosus / 中草药

Objective: To clone the actin (ACT) gene of Eleutherococcus senticosus, and to make the gene a valuable internal gene. Methods: Part of the sequence of ACT gene was cloned by real-time PCR (RT-PCR), and the sequence was used as internal control gene for analyses of semiquantitative PCR and RT-PCR. Results: The ACT gene (1031 bp) of E. senticosus was cloned, coding 343 amino acids. To compare the amino acid sequence of E. senticosus ACT gene with those of Betula luminifera, Gossypium hirsutum, and Camellia sinensis, the amino acid homology was 99.42%, 98.83%, and 98.54%. The expression of ACT in different organs of E. senticosus during various growing periods was constant. The expression of ACT gene in different organs and during different growth and development stages was basically constant, and when the sequence acted as internal control gene, the semiquantative PCR and RT-PCR have good amplification effect and reproducibility. Conclusion: The ACT sequence in E. senticosus is firstly separated and reported, it could act as an internal control gene, and its reaction system of RT-PCR is established.

A preferable approach to clone hLIF cDNA from the genomic DNA

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the research of life science, but it is difficult to obtain by traditional reverse transcription. In this study, we employed a novel strategy to clone the human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA directly isolated from the mucous membrane of mouth. The hLIF sequence can be acquired within a few hours by means of amplification of each exon and splicing using overlap-PCR. Thus, the new approach developed in this study is simple, time- and cost-effective, and it is not limited to particular gene expression levels of each tissue.

Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus)

RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH) cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases / 现代生物医学进展

Background & Objective:Background &Objective: The class Ⅰ Alcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism. Human ADH is encoded by at least seven genes, and three class Ⅰ ADH genes-ADH1, ADH2 and ADH3, which encode the α, β, and γ subunit respectively, had been isolated and mapped on chromosome 4q21-q25. This experiment tends to clone the human class Ⅰ ADH and investigate its role in the hepatic alcohol catabolism. Methods: A pair of primers were designed and the full-length cDNAs encoding human Class Ⅰ ADH were cloned at one time. Class Ⅰ ADH cDNAs were amplified with RT-PCR from total RNA extracted from fetal human liver and kidney, and cloned into pGEM-T vector. To identify cDNA segments, a pair of differential primers was designed. By using them, a portion of the ADHs which encodes the segment from -4 to 296 was cloned. These cDNA segments then were detected directly when being digested with Kpn Ⅰ and Pst Ⅰ, respectively. Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E. Coli. Stably. Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340 nm. Results: Here we had successfully the human class Ⅰ ADH cloned and the full-length cDNAs expressed in E.col.I stably. The relative activity of recombinant enzymes metabolizing ethanol was 0.81 ～1.31 U/mg,0.09 ～0.15 U/mg and 0.76～1.11 U/mg, respectively. Conclusions: In the paper, the full-length cDNAs encoding human class Ⅰ AD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isolated from liver.

Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases / 现代生物医学进展

Background & Objective:Background &Objective: The class Ⅰ Alcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism. Human ADH is encoded by at least seven genes, and three class Ⅰ ADH genes-ADH1, ADH2 and ADH3, which encode the α, β, and γ subunit respectively, had been isolated and mapped on chromosome 4q21-q25. This experiment tends to clone the human class Ⅰ ADH and investigate its role in the hepatic alcohol catabolism. Methods: A pair of primers were designed and the full-length cDNAs encoding human Class Ⅰ ADH were cloned at one time. Class Ⅰ ADH cDNAs were amplified with RT-PCR from total RNA extracted from fetal human liver and kidney, and cloned into pGEM-T vector. To identify cDNA segments, a pair of differential primers was designed. By using them, a portion of the ADHs which encodes the segment from -4 to 296 was cloned. These cDNA segments then were detected directly when being digested with Kpn Ⅰ and Pst Ⅰ, respectively. Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E. Coli. Stably. Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340 nm. Results: Here we had successfully the human class Ⅰ ADH cloned and the full-length cDNAs expressed in E.col.I stably. The relative activity of recombinant enzymes metabolizing ethanol was 0.81 ～1.31 U/mg,0.09 ～0.15 U/mg and 0.76～1.11 U/mg, respectively. Conclusions: In the paper, the full-length cDNAs encoding human class Ⅰ AD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isolated from liver.

Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases / 现代生物医学进展

Background & Objective:Background &Objective: The class Ⅰ Alcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism. Human ADH is encoded by at least seven genes, and three class Ⅰ ADH genes-ADH1, ADH2 and ADH3, which encode the α, β, and γ subunit respectively, had been isolated and mapped on chromosome 4q21-q25. This experiment tends to clone the human class Ⅰ ADH and investigate its role in the hepatic alcohol catabolism. Methods: A pair of primers were designed and the full-length cDNAs encoding human Class Ⅰ ADH were cloned at one time. Class Ⅰ ADH cDNAs were amplified with RT-PCR from total RNA extracted from fetal human liver and kidney, and cloned into pGEM-T vector. To identify cDNA segments, a pair of differential primers was designed. By using them, a portion of the ADHs which encodes the segment from -4 to 296 was cloned. These cDNA segments then were detected directly when being digested with Kpn Ⅰ and Pst Ⅰ, respectively. Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E. Coli. Stably. Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340 nm. Results: Here we had successfully the human class Ⅰ ADH cloned and the full-length cDNAs expressed in E.col.I stably. The relative activity of recombinant enzymes metabolizing ethanol was 0.81 ～1.31 U/mg,0.09 ～0.15 U/mg and 0.76～1.11 U/mg, respectively. Conclusions: In the paper, the full-length cDNAs encoding human class Ⅰ AD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isolated from liver.

cDNA cloning and prokaryotic expression of human neural RNA binding protein HuC / 基础医学与临床

Objective To clone human neural RNA binding protein HuC cDNA,express and purify the recombinant human HuC(Hu antigen C)protein in E.coli.Method Human HuC cDNA was cloned by RT-PCR.HuC cDNA was inserted into the prokaryotic expression vector pGEX-4T-3.The recombinant protein HuC was expressed in E.coli BL-21,and purified by the GST Sepharose 4B affinity column.Results The(62 ku) recombinant GST-HuC fusion protein was obtained.Conclusion The recombinant human HuC protein was successfully prokaryotic expressed and purified.

Advances in Herpesviruses-encoded MicroRNAs / 中国生物工程杂志

MicroRNAs (miRNAs) are small (approximately 22 nt) RNAs that exhibit a diversity in sequence,structure,abudance,and expression profile. Bioinformatic approaches and direct cloning methods have identified 3518 miRNAs from various species. MiRNAs play a pivotal role in the regulation of genes involved in diverse processes such as development,differentiation,and cellular growth control. Recently,many viral-encoded miRNAs have been discovered, most from the herpesviruses viral family. Virus-encoded miRNAs seem to evolve rapidly and regulate both the viral life cycle and the interaction between viruses and their hosts. The detailed study on the virus-encoded microRNAs and the role they play in the progress of viral infection,replication and expression will be beneficial to understand viral molecular biology,and also provide new strategies for the prevention and treatment of virus.

Molecular Cloning of Human 14-3-3 cDNA with CD3 Zeta Peptide Containing Phosphosering Tag / 대한면역학회지

14-3-3 proteins are highly conserved proteins of about 29 kDa and have a minimum of seven isoforms. 14- 3-3 proteins interact with many signalling proteins by the recognition of phosphoserine. For the identification of proteins which react with ITAM (immunoreceptor tyrosine-based activation motif) of CD3 zeta chain, labeled synthetic peptides representing the CD3 zeta chain structual motifs (ITAMs) with a tag of PKC substrate sequence were used for western blotting. One major protein band of approximately 29 kDa was identified in lysate of Jurkat T cell, B cells and HeLa cells. Screening of lamda gt 11 library derived from HeLa cell gave two clones of 14-3-3 protein cDNA. Inspection of their nucleotide sequences identified these two full length cDNA clones as the 29 kDa human homologue of rat 14-3-3 gamma and the human 14-3-3 zeta protein. The human 14-3-3 gamma isoform also showed high homology with other species in amino acid and nucleotide sequence. Although 14-3-3 proteins are phosphoserine-binding proteins, there may be another way of interaction between ITAMs of CD3 and 14-3-3 proteins.

Cloning and Nucleotide Sequence Analysis of HLA - DRA * 0101 and DRB1 * 0405 Alleles / 대한면역학회지

No abstract available.

Cloning of cDNAs Encoding Extra-cellular Domain of Human IL-6, IL-10, IL-13 and SCF / 中国肿瘤生物治疗杂志

Human IL-6,IL-10,IL-13 and SCF cDNAs encoding extra - cellular domain were isolated by reverse transcription polymerase reaction (RT-PCR). These cDNA fragments were inserted into expression vector PBV220 which contains PRP_(L), promoters and SD sequence. Then the E. coli DH5? were tronsformed with the recombinant plasmids. After screening, the clones carrying those recombinant plasmids were identified. SDS-PAEG and biological assay indicated that human IL-6, IL-10 and IL-13 were expressed in E.coli. The expression level of rhIL-6 was as high as 30% of the total bacterial protein.

Cloning and expression of mouse canstatin cDNA in E.coli / 中国病理生理杂志

AIM: To clone and express mouse canstatin (m canstatin)cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21.

Cloning and Sequence Analysis of a Novel Stage-Specific cDNA from Adult Trichinella spiralis / 中国寄生虫学与寄生虫病杂志

Objective To clone a stage-specific novel cDNA from 5 day-old adult worm (ADS) of Trichinella spiralis. Methods The cDNA library of AD5 was screened by an AD5 stage-specific cDNA probe labeled with digoxigenin (DIG). The positive clones were sequenced and analysed. Results The positive clone contained a cDNA insert of 1 132 bp in length with a full length open reading frame (ORF) of 1 032 bp. The cDNA encoded a polypeptide of 343 amino acid residues(aa) with a molecular weight of 35.1 kDa and an isoelectric point (IP) of 4.8. InterProScan analysis showed that the 117 - 120 aa (SGYG) was a glycosaminoglycan attachment site, 27- 86 aa was nematode cuticle collagen N-terminal domain and 153-228 aa was collagen repeat (G-x-y) domain. Signal PV2.0 analysis indicated that the region of 1-43 aa was a singal peptide. Blastn homology analysis in Genbank revealed that the cDNA had no obvious homology to any other known gene sequence. Blastp analysis revealed high homology to cuticle collagen with identities more than 40 % . Conclusion A novel ADS stage-specific cDNA encoding a full length ORF was cloned and sequence analysis showed this gene encoded cuticle collagen of Trichinella spiralis.

Cloning expression purification and cytotoxic assay of sTRAIL in A549 cell line / 中国药理学通报

Aim To clone,express sTRAIL gene,then purify sTRAIL(114-281 amino acid) and assay its cytotoxic activity in human A549 cell line.Methods The intact full human TRAIL gene was amplified using PCR method from the human placenta and lung cDNA library.The full human TRAIL cDNA gene was inserted into pUC19 vector and sequenced.The extracellular DNA fragment was amplified using PCR,which was cloned to pET-11a vector and transformed into E.coli BL21.The denatured and refolded sTRAIL was purified and cytotoxic activity of sTRAIL was assayed using crystal violet staining and fluorescence-activated cell sort(FACS) in A549 cell line.Results The full length TRAIL cDNA gene was amplified from the human placenta and lung cDNA library,which was identical to the published TRAIL sequence.The extracellular DNA fragment was cloned to pET-11a.The expression level reached 50% of the total protein of BL21.The purity of sTRAIL was about 98%,while IC_(50) was about(24?5.2) ?g?L~(1) in TRAIL-treated A549 cells with crystal violet staining method.The time-dependent relationship of sTRAIL-induced apoptotic death in A549 cells was significant with FASC analysis.Conclusion sTRAIL gene has been cloned and successfully expressed.The process of refolding and purification of sTRAIL has been established.sTRAIL demonstrated cytotoxicity in A549 cell line.