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Establishment of the methodology for quantifying lentiviral vector transcriptional read-through rate.

Jiaping HE ; Yudan FANG ; Fan ZHANG ; Fengqiang SUN ; Juan WANG ; Jingzhi ZHANG

Chinese Journal of Biotechnology.2013;29(7):1006-1015.

As an effective vehicle for bio-research and for gene therapy, Lentiviral Vector (LV) has been drawn large attention in recent years. However, transcriptional read-through limits its application. In order to understand the extend of LV read-through in chromosome, a reliable method to assess transcriptional read-through rate is needed. Here, we report the method as follows: 293T cells were transfected with the lentiviral transfer vectors which borne with two LTRs at its two ends in order to mimic the state of "proviral vectors" in chromosome. Using the primers specific for 3'U5 and 3'U3, read-through and total transcripts were reverse transcribed, respectively. These two cDNAs were quantified by realtime PCR using the primers and probe specific for 5'end of 3'U3. Read-through rate was then calculated by the division of the two. Meanwhile, read-through product of green fluorescence protein was also analyzed by Fluorescence Activated Cell Sorter. They both reciprocally proved the principal and confirmed that self-inactivated LV appeared higher read-through rate than the wild type one. The method described in this article, therefore, provides a useful technique to study how to reduce read-through rate, and improve the bio-safety of LV.
Flow Cytometry ; Genetic Therapy ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; HEK293 Cells ; Humans ; Lentivirus ; Transfection

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Expression and purification of hPARP1 by baculovirus system.

Haiyan ZHOU ; Jun MA ; Xueli YANG ; Xiaohai GONG ; Qiuping LI ; Jian JIN

Chinese Journal of Biotechnology.2013;29(7):998-1005.

PARP1 is an important part of DNA repair machinery. In recent years, PARP1 as novel anti-cancer therapeutic target has been broadly explored. In this study, we expressed hPARP1 enzyme in the baculovirus system and tested its activity. We inserted hPARP1 gene into the pFastBac1, a baculovirus transfer vector and then transformed it into DH10Bac containing a shuttle vector of Bacmid. After co-transfecting the recombinant plasmid into Sf9 insect cells, the expressed hPARP1 was purified by 3-aminobezamide affinity chromatography. The expression of hPARPI was visualized by SDS-PAGE and Western blotting; the activity of expressed and purified hPARP1 was confirmed by the reaction of consumption of NAD+ by hPARP1 in vitro. After the purification by 3-aminobezamide affinity column, 3.2 mg protein was obtained and its specific activity was 1.988 nmol/(min x microg).
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; Humans ; Insecta ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; biosynthesis ; Recombinant Proteins ; Sf9 Cells ; Transfection

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Effect of calcium on medium alkalinization induced by salicylic acid in Salvia miltiorrhiza suspension cultures.

Liancheng LIU ; Cong WANG ; Juan'e DONG ; Hui SU ; Zequn ZHUO ; Yaxin XUE

Chinese Journal of Biotechnology.2013;29(7):986-997.

We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.
Calcimycin ; pharmacology ; Calcium ; chemistry ; Calcium Channel Blockers ; pharmacology ; Calcium Ionophores ; pharmacology ; Cell Culture Techniques ; Culture Media ; chemistry ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; metabolism ; Verapamil ; pharmacology

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Expression of human retinol-binding protein 4 in insect baculovirus system and preparation of its polyclonal antibody.

Yuying REN ; Dan CHEN ; Yuzheng GUO ; Hongna SHI ; Juan LIU ; Jingyang BAN ; Yaning LIU ; Xiaofang WU ; Weilong WANG ; Hai CHENG ; Dingfeng LI ; Yong LIU ; Liliang WANG

Chinese Journal of Biotechnology.2013;29(7):974-985.

To prepare recombinant human retinol binding protein 4 (RBP4) by using the baculovirus expression system and to detect its immunogenicity, the fusion DNA fragment of secretory signal peptide SS64 and human RBP4 gene was subcloned into a baculovirus transfer vector pFastBac-dual(pFBd), and the corresponding recombinant transfer plasmid was transformed into E. coli strain DH10bac, after transposition recombinant shuttle bacmid was screened out. The logarithmic phase Sf9 cells were transfected with the recombinant bacmid and then the recombinant baculovirus containing hRBP4 expression box were generated. After amplification of recombinant baculovirus, the recombinant baculovirus seeds were obtained. To express human RBP4, logarithmic phase Sf9 cells were infected with the virus seeds and SDS-PAGE and Western blotting were used to detect and identify the expression. Finally, to prepare a batch of RBP4 protein, logarithmic phase Sf9 cells in suspension culture were infected with recombinant baculovirus seeds and the supernatant was harvested after 120 hours post-infection for purification. Finally for preparation of polyclonal antibody and evaluation of immunogenicity, the recombinant hRBP4 from insect cells and from E. coli were immunized rabbits. Restriction enzyme digestion and sequencing confirmed that the recombinant baculovirus transfer plasmid was constructed correctly, and subsequently recombinant RBP4-bacmid was generated successfully. SDS-PAGE and Western blotting analysis suggested that human RBP4 protein was highly expressed in Sf9 cells with the molecular weight of approximately 23 kDa. The recombinant RBP4 protein could be secreted into the medium efficiently, and the expression level was calculated amount of 100 mg/L. Finally the rabbit antiserum was harvested after recombinant RBP4 immunization, therein the titer of antiserum against baculovirus recombinant RBP4 is 1:100 000 whereas the titer of antiserum against E. coli recombinant RBP4 is only 1:10 000. Overall, human RBP4 was high efficiently expressed successfully with good antigenicity in baculovirus system, and high affinity antiserum was obtained. A solid foundation was laid for the next step of the preparation of human serum RBP4 detection kit.
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Humans ; Immune Sera ; Insecta ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Retinol-Binding Proteins, Plasma ; biosynthesis ; immunology ; Sf9 Cells ; metabolism ; Transfection

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Regulatory effect of 11 kDa protein of parvovirus B19 on NF-kappaB pathway in Hela cells.

Yanming DONG ; Yu HUANG ; Jianxin PENG ; Yi LI

Chinese Journal of Biotechnology.2013;29(7):965-973.

The 11 kDa protein, a small nonstructural protein of parvovirus B19, may play important roles in viral replication cycle. To investigate the effect of 11 kDa protein on the NF-kappaB signaling pathway, we first prepared the poly-antiserum using GST-11 kDa fusion protein purified via prokaryotic expression system, and demonstrated that the 11 kDa protein mainly localized in cytoplasm when expressed in Hela cells. Meanwhile, luciferase activity assay and Western blotting assay showed that 11 kDa up-regulated the transcriptional activity of NF-kappaB and induced the degradation of IkappaB-alpha in Hela cells. Moreover, the 11 kDa protein activated the IL6 promoter, which is probably through the NF-kappaB pathway. Taken together, these results suggested that 11 kDa protein may contribute to activating inflammatory factors through participating in the cell signaling pathway.
HeLa Cells ; Humans ; I-kappa B Proteins ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Parvovirus B19, Human ; Promoter Regions, Genetic ; Signal Transduction ; Transcriptional Activation ; Viral Nonstructural Proteins ; metabolism

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Anti-tumor efficacy of P53 with 9R cell-penetrating peptides.

Yuan LIU ; Rui CHEN ; Nan ZHANG ; Xianlong YE ; Yin BAI ; Yuquan WEI ; Guiping REN ; Deshan LI

Chinese Journal of Biotechnology.2013;29(7):955-964.

To enhance the penetration of P53 into tumor cells by fusion it with the cell penetrating peptide 9R. The fusion gene of 9R-p53 was cloned into the expression vector. The fusion protein, CPPs-P53, was expressed and purified. We detected the rate of cell growth inhibition and apoptosis by MTT and Annexin-V-FITC/PI double stained method respectively for measuring its effect on tumor cells. CPPs-P53 and P53 were successfully expressed and purified, the purity of both proteins reached up to 90%. MTT assay showed that the cell growth inhibition by CPPs-P53 was more efficient than P53, and the rate of cell growth inhibition is dose-dependent. The apoptosis experiment showed that P53 could induce apoptosis of tumor cells. Compared with the P53, CPPs-P53 had a more significant effect in inducing cell apoptosis (**P < 0.01). The CPPs-P53 shows more significant effects than P53 in inhibiting cell growth and inducing apoptosis on tumor cells.
Apoptosis ; Cell Line, Tumor ; Cell-Penetrating Peptides ; pharmacology ; Humans ; Tumor Suppressor Protein p53 ; pharmacology

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MiR-24 improves beta-like globin gene expression through targeting Sp1.

Yanni MA ; Bin WANG ; Bei GONG ; Fang WANG ; Hualu ZHAO ; Junwu ZHANG ; Jia YU

Chinese Journal of Biotechnology.2013;29(7):946-954.

We studied the function and mechanism of miR-24 in regulating beta-like globin gene expression. We first detected the expression of miR-24 during erythroid differentiation and also detected the globin gene expression in miR-24 overexpressing K562 cells through q-PCR. Dual-luciferase reporter assay and Western blotting were used to identify target genes of miR-24. "Rescue experiment" was further used to investigate the regulation of miR-24 on globin gene expression whether depending on targeting Sp1 or not. We found that miR-24 increased during hemin-induced K562 cells and EPO-induced HPCs (hematopoietic progenitor cells) erythroid differentiation. Overexpression of miR-24 in K562 cells promoted the epsilon- and gamma-globin gene expression during hemin-induced erythroid differentiation through targeting the negative globin regulator Sp1. These results suggested that miR-24 can improve the expression of beta-like globin gene through targeting Sp1.
Cell Differentiation ; Gene Expression Regulation ; Hematopoietic Stem Cells ; metabolism ; Humans ; K562 Cells ; MicroRNAs ; genetics ; Sp1 Transcription Factor ; genetics ; epsilon-Globins ; genetics ; gamma-Globins ; genetics

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Knockout of the ptsG gene in engineered Escherichia coli for homoethanol fermentation from sugar mixture.

Tao YAN ; Jinfang ZHAO ; Wenhui GAO ; Jinhua WANG ; Yongze WANG ; Xiao ZHAO ; Shengde ZHOU

Chinese Journal of Biotechnology.2013;29(7):937-945.

To realize the simultaneous fermentation of xylose and glucose, ptsG (one of the glucose-PTS genes) was deleted from the engineered ethanologenic Escherichia coli SZ470 (deltapflB, deltafrdABCD, deltaackA, deltaldhA), resulting in loss of glucose effect in the mutant SZ470P (deltaptsG). When tested in 5% mixture of glucose (2.5%) and xylose (2.5%), SZ470P simultaneously used glucose (13 g/L) and xylose (20 g/L) whereas the parent strain SZ470 sequentially used glucose (25 g/L) then xylose (5 g/L). Upon completion of the fermentation, both strains achieved similar product yield of 89%. SZ470P produced 15.01 g/L of ethanol, which was 14.32% higher than that produced by SZ470 (12.86 g/L). Deleting ptsG gene enabled the mutant strain SZ470P to simultaneously use both glucose and xylose and achieve better ethanol production.
Escherichia coli ; enzymology ; genetics ; Ethanol ; chemistry ; Fermentation ; Glucose ; chemistry ; Phosphoenolpyruvate Sugar Phosphotransferase System ; genetics ; Xylose ; chemistry

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Effects of mixed carbon sources on glucose oxidase production by recombinant Pichia pastoris.

Yina SHEN ; Lei GU ; Juan ZHANG ; Jian CHEN ; Guocheng DU

Chinese Journal of Biotechnology.2013;29(7):927-936.

Glucose oxidase (GOD) is an important industrial enzyme with many potential applications. In order to increase the production and productivity of GOD by recombinant Pichia pastoris GS115, we investigated the feeding strategies of mixed carbon sources during induction phase, based on results of the optimization of initial cell and methanol concentration on GOD production. The optimal initial cell and methanol concentration were 100 g/L and 18 g/L. During induction phase, the mixed-carbon-sources strategies showed that glycerol, sorbitol or mannitol co-feeding with methanol could enhance GOD production. With mannitol co-feeding (20:1(W/W)), the maximum GOD production and maximum GOD productivity reached 711.3 U/mL and 4.60 U/(mL x h) after an induction period of 156 h. Compared to the control, the enhancements of GOD production and productivity were 66.3% and 67.9%, respectively. Meanwhile, we found an appropriate mannitol co-feeding strategy that would not inhibit the expression of promote. The activity of alcohol oxidase was 8.8 U/g, which was enhanced by 69.2% compared to the control (5.2 U/g). We can use the same optimization process to improve the production of other proteins from recombinant Pichia pastoris by changing the fermentation parameters.
Carbon ; metabolism ; Fermentation ; Glucose Oxidase ; biosynthesis ; Glycerol ; metabolism ; Industrial Microbiology ; Mannitol ; metabolism ; Methanol ; metabolism ; Pichia ; metabolism ; Sorbitol ; metabolism

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Promoter detection and transcriptional analysis of the spinosad biosynthetic gene cluster.

Xiaozhou FENG ; Weishan WANG ; Xiaohui REN ; Xinli LIU ; Xiangzhao MAO ; Keqian YANG

Chinese Journal of Biotechnology.2013;29(7):914-926.

Spinosad represents a new class of insecticides produced by Saccharopolyspora spinosa. To understand the transcription of the spinosad biosynthetic gene cluster, two promoter detection plasmids based on different reporter genes were constructed and used to detect 9 promoters in the spinosad biosynthetic gene cluster. In addition, the temporal transcriptional profiles of the corresponding genes controlled by the 9 promoters, together with 4 genes outside of the spinosad cluster but are required for the synthesis of sugars in spinosad, were examined by real-time PCR. The results indicate that the 9 spinosad biosynthetic genes were highly expressed at the stationary phase, which coincides with the accumulation of spinosad in the fermentation broth. Of particular note is that the transcription of the 4 sugar synthetic genes showed higher level at the exponential phase, suggesting the expression of sugar synthetic genes is not correlated with the spinosad synthetic genes. The data suggest that spinosad biosynthesis could be improved by engineering the expression pattern of the sugar synthetic genes that lie outside the spinosad gene cluster.
Drug Combinations ; Insecticides ; metabolism ; Macrolides ; metabolism ; Multigene Family ; Plasmids ; Promoter Regions, Genetic ; Real-Time Polymerase Chain Reaction ; Saccharopolyspora ; genetics ; metabolism

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