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Progress in the tumorigenic potential of iPSCs and methods to reduce it.

Liping ZHANG ; Guanheng YANG ; Jingzhi ZHANG

Chinese Journal of Biotechnology.2015;31(9):1279-1288.

Since Yamanaka successfully reprogrammed murine fibroblasts into iPSCs in 2006, iPSCs technology has drawn much attention worldwide. Although iPSCs provides tremendous possibilities for both basic research and regenerative medicine, it has meanwhile potential risks, e.g. tumorigenicity. Scientists, therefore, have made efforts in clarifying the mechanism of the cause for iPSCs tumorigenicity and the way how to reduce the risk. The results of some researches reveal some of tumorigenic factors, e.g. the partial similarity of gene expression profiles between cancer cells and iPSCs, the accumulation of the genetic damages in the course of reprogramming process, and mutation in the cellular culture. As a consequence, numerous methods for reducing iPSCs tumorigenicity have been explored, such as minimized use of the reprogramming factors at the controlled manner, and the selection of the expression vector or parental cells. In this paper, the cause of iPSCs tumorigenicity and the current achievements on preventing iPSCs tumorigenesis are reviewed.
Animals ; Carcinogenesis ; Cell Culture Techniques ; Cellular Reprogramming ; Fibroblasts ; Genetic Vectors ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Mice ; Mutation ; Transcriptome

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Transcript assembly and quality assessment.

Feilong DENG ; Xianbo JIA ; Songjia LAI ; Yiping LIU ; Shiyi CHEN

Chinese Journal of Biotechnology.2015;31(9):1271-1278.

The transcript assembly is essential for transcriptome studies trom next-generation sequencing data. However, there are still many faults of algorithms in the present assemblers, which should be largely improved in the future. According to the requirement of reference genome or not, the transcript assembly could be classified into the genome-guided and de novo methods. The two methods have different algorithms and implementation processes. The quality of assembled transcripts depends on a large number of factors, such as the PCR amplification, sequencing techniques, assembly algorithm and genome character. Here, we reviewed the present tools of transcript assembly and various indexes for assessing the quality of assembled transcripts, which would help biologists to determine which assembler should be used in their studies.
Algorithms ; Gene Expression Profiling ; Genome ; Genomics ; methods ; High-Throughput Nucleotide Sequencing ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transcriptome

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Collagen quantitation by detection of marker peptides with HPLC-MS.

Kun SUN ; Fan YANG ; Yingjun KONG ; Jiyao KANG ; Wei CAO ; Xiaoyan YANG ; Shenghua ZHA ; Guifeng ZHANG ; Minglin WANG

Chinese Journal of Biotechnology.2015;31(11):1660-1668.

A method for quantitation of collagen was established by detecting marker peptide with high performance liquid chromatography-mass spectrometry (HPLC-MS). Theoretical marker peptides were selected by sequence comparison. Bovine collagen type I was digested with trypsin. Marker peptides typical for collagen type I were identified with HPLC-MS. The relationship between the abundance of marker peptides and collagen concentration was established. The results show that GEAGPSGPAGPTGAR and the other 5 peptides showed high resolution during chromatographic separation and high signal intensity during MS analysis. Peptide signal intensity and collagen concentration showed a good linear relationship in the range from 0.1 to 3 mg/mL. Bovine tendon and collagen sponge were used as actual samples and collagen contents were determined as 90.2% and 93.4% respectively. Quantitation of marker peptides of collagen was a feasible method to identify and quantify collagens in medical device research and development.
Animals ; Cattle ; Chromatography, High Pressure Liquid ; Collagen Type I ; analysis ; Mass Spectrometry ; Peptides ; analysis

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Relationship between interferon-α conformation and its anti-viral activity determined by circular dichroism and flow cytometry.

Maozhi HU ; Wenhua LI ; Qiuxiang YAN ; Yan YANG ; Qing SUN ; Zhiming PAN ; Guiyou CUI ; Xin'an JIAO

Chinese Journal of Biotechnology.2015;31(11):1651-1659.

The relationship between the conformation of interferon-α (IFN-α) and its anti-viral activity were analyzed by circular dichroism (CD) and flow cytometry (FCM) techniques. The recombinant human IFN-α (rIFN-α2b and rIFN-α2a) were used. CD spectra from 190 nm to 240 nm indicated that two the IFN-α showed stable secondary structure at 65 degrees C, but unstable when the temperature was above 65 degrees C, and the change was irreversible. FCM data of the anti-viral activity of IFN-α indicated that the change of its secondary structures partly weakened its anti-viral activity. The rIFN-α2b and rIFN-α2a showed the same phenomenon. These data indicated that the conformation of IFN-α is one of the factors to influence its anti-viral activity and the combination of CD and FCM is a good method to analyze the relationship between the conformation of protein drugs and their biological activities in single cell level.
Antiviral Agents ; chemistry ; Circular Dichroism ; Flow Cytometry ; Humans ; Interferon-alpha ; chemistry ; Protein Structure, Secondary ; Recombinant Proteins ; chemistry

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Application of reverse phase C18 tip in pretreatment of small amount complex proteomic samples.

Danqi WANG ; Wei SUN ; Lili ZOU ; Zhao WANG

Chinese Journal of Biotechnology.2015;31(11):1643-1650.

To develop a timesaving and easy operating Reverse Phase (RP) chromatography method, we adopted Thermo Pierce RP C18 Tip to separate small amount hippocampus peptide mixtures and to compare with high performance liquid chromatography (HPLC). According to the separation performance of 4 ACN gradient optimization methods, we determined the best ACN concentration gradient. The results showed that, the experiment took only 10 min by separating with eight ACN concentration gradient, which accounted 1/4 for HPLC. But as for the identified proteins, RP C18 Tip accounted 85.5% for HPLC. ACN gradient of 5%, 15%, 20% and 90% had best repeatability (P = 0.429) and result for separating 30 μg peptides. This method is easy to operate, timesaving and has low cost. It could be used into pretreatment of small amount complex proteomic samples.
Chromatography, High Pressure Liquid ; Chromatography, Reverse-Phase ; methods ; Peptides ; Proteins ; isolation & purification ; Proteomics ; methods

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Expression, purification and characterization of recombinant onconase expressed in Pichia pastoris.

Ganggang YANG ; Chengkai MA ; Quanyi ZHANG ; Shihui SHI ; Ze WANG ; Zhongyuan LÜ ; Xuyang WANG ; Xiaoya XU ; Qingqing CUI ; Jihong ZHANG ; Ruigang ZHANG ; Cunshuan XU

Chinese Journal of Biotechnology.2015;31(11):1632-1642.

Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.
Antineoplastic Agents ; metabolism ; Bioreactors ; Cell Line, Tumor ; Codon ; DNA, Complementary ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; Ribonucleases ; biosynthesis

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Prokaryotic expression of Staphylococcus aureus Clumping factor B and evaluation of the antiserum-mediated opsonic activity.

Lili ZHANG ; Shanshan CAO ; Yifeng ZHANG ; Chunling XUAN ; Youqiang WU ; Songhua HU

Chinese Journal of Biotechnology.2015;31(11):1623-1631.

Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.
Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Escherichia coli ; Flow Cytometry ; Immune Sera ; Immunoglobulin G ; blood ; Opsonin Proteins ; immunology ; Phagocytosis ; Rabbits ; Staphylococcal Infections ; immunology ; Staphylococcus aureus

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Cloning and expression profile of Bmyan in the silkworm (Bombyx mori) and experimental validation as one target of microRNA 7.

Shiping LIU ; Yaxi HUANG ; Jiyun YIN ; Xiaoyan WU ; Lanting ZHOU ; Wei WANG ; Qingyou XIA

Chinese Journal of Biotechnology.2015;31(11):1612-1622.

microRNAs (miRNAs) are an extensive class of -22-nucleotide (nt) endogenous noncoding RNAs regulating life activities ofmetazoans through binding to 3'-untranslated regions (3'-UTRs) of their target genes. This work aimed to identify yan gene in the silkworm, reveal its expression profile and confirm if it is one target of bmo-miR-7 and, as such, have potential for contributing to better understanding of the molecular mechanisms involved in the metamorphosis of silkworm. Based on homolog searching and PCR amplification, we cloned the coding sequence (CDS) of Bmyan, which encodes 476 amino acid residues and contains SAM-PNT and ETs domains. Quantitative PCR (q-PCR), RT-PCR and microarray data revealed high expression of Bmyan in the head, body wall and ovary of day-3 fifth instar larval silkworm, low or no expression in other tissues. It was lowly expressed in the early larval stages, but highly expressed from late spinning to day 4 pupa. The 3'-UTR of Bmyan was obtained by rapid-amplification of cDNA ends (3'RACE) and predicted to contain two potential recognition sites of bmo-miR-7. The luciferase reporter vector containing the 3'-UTR of Bmyan was constructed and co-transfected into BmE cell line with the mimic of bmo-miR-7 and the decreased relative activity of luciferase showed that Bmyan is one target of bmo-miR-7. This work helps further functional analysis of bmo-miR-7 and Bmyan in the silkworm.
3' Untranslated Regions ; Animals ; Bombyx ; genetics ; Cell Line ; Cloning, Molecular ; Female ; Genetic Vectors ; Insect Proteins ; genetics ; Larva ; Metamorphosis, Biological ; MicroRNAs ; genetics ; Pupa

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Detection of cadmium by a double-promoters based Escherichia coli biosensor.

Panpan LI ; Fanglan XIAO ; Xijuan YAN ; Binbin LU ; Weiwei LIN ; Qingqing XU ; Zhenzhen ZHANG ; Wu WANG ; Jianxin LÜ

Chinese Journal of Biotechnology.2015;31(11):1601-1611.

To detect cadmium ions, we constructed a specific microbial sensor and screened detecting cassettes and different fluorescence proteins. Blue fluorescence protein mTagBFP2 was selected as a reporter and a double-promoters model was used in the construction of the fusion reporter vector Pmer::merR-m-Pmer::mTagBFP2-pMD19-T. The reporter vector was then transformed into Escherichia coli MC4100 wild type strain. The medium, incubation time, initial density for induction, and the optimal detection range were determined. The specificity of the biosensor was also checked. The biosensor responded specifically to cadmium irons with low background, and the linear concentration range detection ranged from 0.1 to 75 μmol/L at the initial OD600 = 0.1 with 2 h incubation in IHMM medium. Thus we successfully constructed a specific biosensor to detect cadmium irons and provided useful strategies for development and optimization of microbial sensors to detect heavy metals.
Biosensing Techniques ; Cadmium ; analysis ; Escherichia coli ; Genetic Vectors ; Promoter Regions, Genetic

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Overexpressing 3-ketosteroid-Δ1-dehydrogenase for degrading phytosterols into androst-1,4-diene-3,17-dione.

Lele ZHANG ; Xian ZHANG ; Minglong SHAO ; Rongrong CHEN ; Zhiming RAO ; Hu LI ; Zhenghong XU

Chinese Journal of Biotechnology.2015;31(11):1589-1600.

We constructed plasmid pMTac to overexpress 3-ketosteroid-Δ1-dehydrogenase (KSDD) in Mycobacterium neoaurum JC-12 for improving androst-1,4-diene-3,17-dione (ADD) production. To construct pMTac, pACE promoter on pMF41 was replaced by tac promoter, and then four recombinants were constructed, which were M. neoaurum JC-12/pMF41-gfp, M. neoaurum JC-12/pMTac-gfp, M. neoaurum JC-12/pMF41-ksdd and M. neoaurum JC-12/pMTac-ksdd. Fluorescence detection results show that much more green fluorescent protein (GFP) was expressed in M. neoaurum JC-12/pMTac-ksdd than M. neoaurum JC-12/pMF41-ksdd. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/pMTac-ksdd, 6.53-fold as that of M. neoaurum JC-12 and 4.36-fold as that of M. neoaurum JC-12/pMF41-ksdd. In shake flask fermentation, ADD production of M. neoaurum JC-12/pMTac-ksdd was 5.94 g/L, increased about 22.2% compared to the original strain M. neoaurum JC-12 and 12.7% to M. neoaurum JC-12/pMF41-ksdd. AD (4-androstene-3,17-dione) production of JC-12/pMTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M neoaurum JC-12/pMF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/pMTac-ksdd was improved to 10.28 g/L. pMTac is favorable for expressing KSDD in M. neoaurum JC-12, and overexpression of KSDD has beneficial effect on ADD producing, and it is the highest level ever reported using fermentation method in M. neoaurum.
Androstadienes ; metabolism ; Fermentation ; Industrial Microbiology ; Mycobacterium ; Oxidoreductases ; genetics ; metabolism ; Phytosterols ; metabolism ; Plasmids

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