25 April 2013, Volume 33 Issue 4

    

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  Construction and Evaluation of the Mutated Anthrax Toxin Proteins as Drug Deliver System for Targeting Tumor Cells

  LI Bing-juan, LI Yu-xia, LI Bei-ping, LING Yan, ZHOU Wei, LI Wei-dong, LIN Hai-long, LIANG Long, LIU Gang, ZHANG Jin-hai, CHEN Hui-peng

  China Biotechnology. 2013, 33(4): 1-8.

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  Objective: The anthrax toxin proteins constitute a unique membrane translocation system which provide an attractive model system for the development of reagents for cell biology and therapeutics. The mutated anthrax toxin proteins PA and LF were constructed to target to tumor cells. Methods: The recombinant Rosseta(DE3) which harboring three kinds of mutated PA and LF were constructed separately. The cell viability after incubated with mutated PA and LF was detected by cytotoxicity assay. The ability of the mutated PA translocation system targeting to tumor cells was measured by the amount of the luciferase of intra-cellular. Results: The mutated PA can be cleaved by matrix metalloproteinases or uPA in vitro correctly. When the tumor cell incubated with the mutated PA and LF,the cell viability was decreased remarkably. The amount of the luciferase of intra-cellular can correctly reflex the ability of the mutated PA translocation system targeting to tumor cells. Conclusions: These mutated PA proteins showed natural biological activities and had the ability of targeting to tumor cells. These results provide a new insight for the study of the antitumor drugs.
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  Isolation Human Rotavirus Strains and Adaptive Culture

  LI Song, ZHANG Guang-ming, WU Jin-yuan, YIN Na, YI Shan, MI Kai, CAO Ying, LI Yang, SUN Mao-sheng, LI Hong-jun

  China Biotechnology. 2013, 33(4): 9-14.

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  Objective: To investigate separation method,the condition for culture and biological properties of wild rotavirus strain. Methods: To the qualitative detection of samples collected rotavirus use colloidal gold, PCR, PAGE.Process the positive samples according to the conventional method, and then do the separation of tissue culture. To analyze and evaluate the genome, gene sequences and proliferation kinetics of different transfer of culture samples. Using the positive serum of the G1P[8] rotavirus to do the virus neutralizing identification test. Results: By testing, the ZTR-68 rotavirus belong to A group were got, the arrangement of genome is 4:2:3:2,the genotype is G1P[8].The CPE was observed after the adapted culture the rotavirus,proliferated in MA104 cells, in the Vero cells. And the typical rotavirus form can be observed by using electron microscope. The processing is stable, which the strain proliferated in the Vero cells. And the infectious titer reached 7.25 log CCID50/ml, the peak time of proliferation is at 96h.There was not any other virus except the rotavirus in the culture medium of virus neutralizing identification test. Conclusion: The separated strain of human rotavirus, named ZTR-86,which was got from the separation of the diarrhea samples, has a good adaptability to the tissue culture and a good genetic stability. It provides a basis for the further study of its biological properties and the making of corresponding vaccine.
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  Screening and Identification of Mycobacterium tuberculosis Biofilm Formation Related Genes

  LIU Xia, GUO Qing-long, WANG Ruo-jun, WANG Hong-hai, PEI Xiu-ying, ZHANG Xue-lian

  China Biotechnology. 2013, 33(4): 15-21.

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  Objective:To screen and identify the related genes which may be associated with biofilm formation of Mycobacterium tuberculosis (M. tuberculosis) based on the changes of colony phenotype and biofilm defects. Methods:A random mutagenesis library was constructed by using the MycoMarT7 transposon system. Mutants with colony morphological changes on solid agar plate or with defects in pellicle biofilm formation in liquid culture medium were selected. In order to determine the locus of mutation gene, the flanking fragments of gene were obtained using T-A cloning and resistance marker recovery method. Then, the function of mutated genes was analyzed and predicted by bioinformatics methods. Results: 39 mutants with colony morphological changes or biofilm defects were selected and the loci interrupted of 16 mutants were successfully identified. Among the 16 genes, 5 genes were associated with the regulation of lipid metabolism, 4 were participated in cell wall and cell processes, 2 were related to intermediary metabolism and respiration, the product of 1 gene might be a regulatory protein, 1 was reported as virulence gene in previous studies, 1 was PE/PPE family gene and the function of other genes were unknown. Among these genes, 8 genes might be related to deficient in forming pellicle biofilm. Conclusions: A M. tuberculosis transposon insertion mutant library of approximately 10 000 mutants was successfully constructed, pellicle biofilm formation defects were selected and biofilm formation related genes were identified. All results will be the basis of the further research of biofilm formation mechanism in M. tuberculosis.
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  C-Myc Affects Esophageal Squamous Cancer Sensitivity to Paclitaxel by Regulating BubR1 Expression

  HU Min, SONG Pei-pei, ZHAN Xiao-qin, LÜ Zi-lan, CHEN Chu, SHI Qiong, WENG Ya-guang

  China Biotechnology. 2013, 33(4): 22-27.

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  To explore the relationship between C-Myc and mitotic checkpoint protein BubR1 expression and possible influence of C-Myc on paclitaxel drug effects. Immunohistochemistry assay was explored to detect C-Myc and BubR1 expression in twenty-three clinical esophageal squamous cancer samples. And Western blot was applied to detect and compare C-Myc and BubR1 expression levels in three esophageal squamous cancer cell lines, ECA-109, KYSE150 and KYSE180. The correlation between C-Myc and BubR1 protein expression was analyzed. About 2000bp fragment upstream human BUB1b gene was cloned and inserted into pSEAP2 promoter reporter vector to construct pSEAP2-BubR1-P2000. Then pSEAP2-BubR1-P2000 was transfected into three cell lines for promoter activities detection (ALP activity). ALP activity was detected in ECA-109 cells both overexpressing C-Myc and transfected pSEAP2-BubR1-P2000. Western blot assay was used to detect the effect of C-Myc inhibitor 10058-F4 on BubR1 expression. MTT assay was applied to detect cell viability under gradient paclitaxel exposure after C-Myc suppression. DAPI staining was explored for apoptotic cell count in C-Myc inhibitor group, low concentration paclitaxel (100nM) group and combination of C-Myc inhibitor and paclitaxel, respectively. The results showed that C-Myc expression positively correlated with BubR1 expression both in clinical esophageal squamous cancer tissues and in esophageal squamous cancer cell lines. Cells with high C-Myc expression showed higher BubR1 promoter activity and overexpression of C-Myc in ECA-109 cells can further enhance BubR1 promoter activity. Specific C-Myc inhibitor 10058-F4 can effectively down-regulate BubR1 expression and decrease cell viability under gradient paclitaxel exposure. DAPI staining result exhibited that combination of 10058-F4 and paclitaxel induced significantly more mitotic cells than single usage of 10058-F4 or paclitaxel. In esophageal squamous cancer cells, C-Myc can regulate mitotic checkpoint protein BubR1 expression and related with paclitaxel sensitivity. C-Myc may reduce esophageal squamous cancer response to paclitaxel by up-regulating BubR1 expression.
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  The Disruption of STE13 Gene in Pichia pastoris Improves the Expression and Bioactivity of GGH

  LU Qing-peng, XU Zhen-hong, SI Jin-song, DOU Wen-fang

  China Biotechnology. 2013, 33(4): 28-33.

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  Objective: It is barely found that the bioactivity of recombination protein (GLP-1 A₂G ) ₂ -HSA (GGH) by the cell bioactivity evaluation that related to its protein degradation, which expressed in methylotrophic yeast Pichia pastoris GS115 strain. Therefore, A new P.pastoris GS115 strain with STE13 gene disruption was engineered, helped for the expression of intact GGH, and the bioactivity can be further improved. Methods: The STE13 gene deficient strain GS115/D13 was constructed by the disruption of the P.pastoris homolog of the Saccharomyces cereviaiae dipeptidyl aminopeptidase (STE13 ) gene in Pichia genome by the Cre-Loxp recombination system based on the fusion PCR technology, and the bioactivity of GGH estimated by the cell biological evaluation methods. Results: The recombinant strain GS115/D13 showed the protein degradation much more relieved, apparent bioactivity improved and the yield of about 25.8% increased compared with that in the wild-type strain (GS115/W). Conclusion: The results suggested that STE13 gene disruption offered an expression host for the product of much more intact GGH and helped improve the protein bioactivity.
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  Cloning and Expression of the dCTP Deaminase Gene from Thermus Phage TSP4

  GAO Ting-ting, ZHANG Qi, WEI Yun-lin, JI Xiu-ling, LIN Lian-bing

  China Biotechnology. 2013, 33(4): 34-39.

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  The gene encoding a dCTP deaminase tspdCD from Thermus phage TSP4 was subcloned into pET-32a, generating a recombinant plasmid pET-32a-tspdCD which was further transformed into Escherichia coli Rosetta(DE3) for expression. Analysis of SDS-PAGE showed that target protein was highly expressed as a soluble form after IPTG induction. The expressed protein was purified using Ni-NTA agarose, which was further used to analyze the enzyme activities including optimum activity temperature, pH and substrate, as well as the effects of metal ions and organic solvents on the enzyme activity. The results showed that the specific activity of the expressed tspdCD was 4.12U/mg. Its optimum activity temperature and pH were 60℃ and 7.5, repectively, and its optimum substrate was dCTP. The enzyme activity was stimulated by Ca²⁺ and Mg²⁺ of 2mmol/L, but was inhibited by Ni²⁺ and Cu²⁺ of 2 mmol/L. The results also showed that 10%(V/V) ethyl acetate and isopropanol significantly increased the activity of the expressed tspdCD while 10%(V/V) acetone had a significant inhibitory effect for the same enzyme. The successful expression of the phage tspdCD, in E. coli provides a good basis for further functional study of the purified enzyme.
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  Preparation and Preliminary Characterization of Monoclonal Antibodies against E2 Protein of Bovine Viral Diarrhea Virus

  WANG Shu, ZHU Yuan-mao, CAI Hong, MA Lei, SHI Hong-fei, LÜ Chuang, DONG Xiu-mei, GAO Yu-ran, XUE Fei

  China Biotechnology. 2013, 33(4): 40-45.

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  To prepare monoclonal antibody (MAb) against glycoprotein E2 of bovine viral diarrhea virus (BVDV), BALB/c mice were immunized with purified recombinant E2(rE2) expressed in E.coli and two hybridomas secreting MAb were screened from fusing the SP2/0 cells with the spleen cells of the immunized BALB/c mice by indirect ELISA coated with BVDV.The titers of MAb 4E3 and 1G11 in ascites were 6.21×10⁶,6.83×10⁵, 6.83×10⁵, and 7.5×10⁴ as detected by rE2 and BVDV, respectively. The MAbs secreted by hybridomas 4E3 and 1G11 had highly reactivity and specificity in indirect ELISA, Western blotting, IFA and identified as IgM with a light chain of κ by indirect ELISA.The specific tests indicated that the MAbs 4E3 and 1G11 had no reaction with bovine infectious rhinotracheitis virus,bovine parainfluenza virus type 3 and bovine adenovirus type 3.Meanwhile the MAb 4E3 had no reaction with classical swine fever virus(CSFV)and the MAb 1G11 had reaction with CSFV.Therefore the MAbs 4E3 and 1G11 might be used for differentiating BVDV and CSFV.On the other hand, the MAbs 4E3 and 1G11 could be used for establish diagnosis method for BVDV.
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  Expression, Purification and Characterization of Dimethylarginine Dimethylaminohydrolase

  ZHAO Zhi-jing, WANG Xue-zhong, SHEN Wen-ting, HU Guang

  China Biotechnology. 2013, 33(4): 46-53.

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  Dimethylarginine Dimethylaminohydrolase(DDAH) is one of the essential enzyme in enzymatic cycling detection of ADMA, a well recognized biomarker for early (acute myocardial infarction) AMI prognosis and diagnosis. To obtain DDAH with high enzymatic activity, the DDAH was cloned from the genome of Pseudomonas aeruginosa(Schroeter), and expressed in E. coli BL21 at high level of 100mg/L. Recombinant DDAH was purified to more than 95% purity from supernatant of disrupted cells with HisTrap Fast Flow and further polished by cation-exchange chromatography with Q column. The optimum reaction temperature, pH, specific activity, Km and Vmax of the purified DDAH was 35℃, 6.0, 0.5 U/mg, 3.04 mM and 8.07mM/h, respectively. No obvious activity losing was observed when the DDAH stored at termperature from 10℃ to 40℃ and pH from 5.0 to 9.0. Furthermore, more than 95% enzymatic activity was preserved after the purified DDAH was exposed to temperature of 37℃ for one week. Dimerization of the recombinant enzyme had no obvious effect on the enzymatic activity. The yield of recombinant DDAH could reach to 0.4 gram/L with high density fermentation, thus are well suitable for application in large-scale manufacturing of clinical test kit of ADMA and drug screening for DDAH.
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  Construction of Co-expression Vector Containing AtCAO and AtHEMA1 Genes from Arabidopsis and Transformation into Tobacco

  LI Cui-ping, PAN Yu, BAI Xiao-ning, CHU Fu-tang, SU Cheng-gang, ZHANG Xing-guo

  China Biotechnology. 2013, 33(4): 54-60.

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  It is a possible way to improve the low light resistance of crops by increasing the amount of chlorophyll, especially that of chlorophyll b. AtHEMA1 and AtCAO from Arabidopsis thaliana were constructed into the binary expression vector pCAMBIA1302. This two genes were introduced into tobacco via Agrobacterium tumefaciens-mediated transformation. Both the amount of chlorophyll b and the rate of photosynthesis were remarkably increased in the transgenic plant lines TL1 and TL2 compared with the wild type tobacco, while the chlorophyll a/b decreased significantly(P<0.01). The growth of the transgenic tobacco was accelerated compared with the WT under low light. The results had laid a foundation for improving crop low light tolerance with those two genes.
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  Cloning and Sequence Analysis of the CkC3H Gene from Caragana korshinskii Kom. and Preliminary Studies of Its Function

  LI Gao, YANG Qi, ZHANG Ye, WANG Rui-gang, LI Guo-jing

  China Biotechnology. 2013, 33(4): 61-67.

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  Coumarate 3-Hydroxylase (C3H) is a key enzyme in lignin biosynthesis pathway. A C3H encoding gene was cloned by rapid amplification of cDNA end technique from Caragana korshinskii Kom. The full length gDNA of CkC3H was 4235bp, contained three exons and two introns. The full length of ORF was 1530bp, and the protein deduced from this cDNA comprised 509 amino acids with a calculated molecular weight of 57.61kDa and an isoelectric point of 7.67. The deduced protein contains a P450 domain and was named as CkC3H (GenBank accession no. KC309408). Phylogenetic analysis indicated that CkC3H shared the closest homology with the C3H from Glycine max. Binary vector carried the cDNA sequence of CkC3H driven by 35S promoter was constructed and transformed into the ref8 mutant of Arabidopsis thaliana. The dwarf and sterile phenotype of the ref8 mutant were partially restored by the transgene. These showed that CkC3H might have at least partially similar function with AtC3H.
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  Cloning and Expression Analysis of Cyt b5 Gene Involved in Electron Transfer in Gossypium hirsuturm

  LIU Jin-zhi, SI Huai-jun, ZHANG Ning, WU Jia-he

  China Biotechnology. 2013, 33(4): 68-73.

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  On the basis of salt-stress related EST library from Gossypium hirsuturm L. A partial sequence fragment of cytochrome b5 proteins (Cyt b5) encoding gene was obtained through homologous comparison with other plants. The Cyt b5 gene was isolated by the 3',5'-RACE technology from G. hirsuturm, named GhCyt b5. The full-length cDNA of GhCyt b5 is 810 bp, containing a 402 bp ORF which encodes 134 amino-acid peptide. The relative molecular weight of GhCyt b5 protein is 17 kDa. The coding sequence fragment was amplified by PCR and cloned into vector pET32a to generate the pET32a-GhCyt b5 expression vector, which was transformed into BL21(DE3) for expression of recombinant protein. On the base of optimization of different inducible conditions, the results showed that soluble GhCyt b5 protein under the 28℃ and 1 mmol/L IPTG conditions could be obtained. The expression product of GhCyt b5 was purified by Ni²⁺ affinity column and identified by SDS-PAGE. Using extracted cotton cell material as reaction medium for in vitro electron transfer analysis, it can be found the GhCyt b5 involved in the electron transfer system and accepted electron to become oxidation state from reducing state. Ultimately, the Cyt b5 gene for the first time from G. hirsuturm was cloned and its participating in electron transfer system was confirmed, which provide a base for further dissecting function in biotic and abiotic resistance.
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  Expression and Characterization of ThMSD from Thellungiella halophila in Escherichia coli

  XU Xiao-jing, AN Hui-ling, CHEN Ning-mei, YANG Jing, ZHOU Yi-jun

  China Biotechnology. 2013, 33(4): 74-79.

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  Superoxide dismutases (SODs) are ubiquitous antioxidative enzymes found in eukaryotic and differ by the named metal cofactor, Cu/Zn, Mn or Fe. Plant mitochondria Mn-SOD play a vital role in fight aginst stresses. ThMSD, a gene of Mn-SOD was cloned from Thellungiella halophila with dramatic stresses tolerance in earlier research. ThMSD was ligated to a prokaryotic expression vector pET30a(+) and transferred to E. coli BL21 to get recombinant BL21(pET30a-ThMSD). SDS-PAGE analysis showed an induced expression product band,with a molecular weight of about 32 kDa,which was consistent with the expected value, in all recombinant strains.Western blotting confirmed this band with anti-His monoclonal antibody. The results of SOD activity assay of soluble fractions of total protein from 3 recombinant strains showed that much higher SOD activity than control. The strain with highest expression level of ThMSD was selected and was cultured in large scale and target protein was purified using a Ni²⁺ Chelating column. The effect of temperature on the enzyme activity of ThMSD was tested, the result showed that ThMSD enzyme still had more than 50% residual activities at 55℃. The thermal stability of purified ThMSD was examined at 42℃, the result showed that SOD activity of ThMSD reduced with the time elapse, but it retained more than 60% residual activities after 40 min. In assay of NaCl tolerance of BL21(pET30a-ThMSD), the recombinant strain can grow faster than control in higher concentration of NaCl. From above, the recombinant ThMSD protein had high SOD activity and high thermal stability, and BL21 transformed with ThMSD have increased NaCl tolerance. It was concluded that ThMSD may connect with extreme stresses tolerance of Thellungiella halophila.
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  Cloning and Activity Analysis of Soybean FAD2-1B Promoter

  ZHAO Yan, SHA Wei, ZHANG Mei-juan, YANG Xiao-jie, FAN Zhen-yu, WANG Yan-mei

  China Biotechnology. 2013, 33(4): 80-84.

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  The soybean oleate desaturase gene(FAD2-1B ) has previously been shown to be expressed specifically in soybean seeds. The 5'-flanking upstream sequence of FAD2-1B gene, named FP, was isolated from the genomic DNA of soybean by PCR method. Sequence analysis by PLACE revealed that this fragment contains a series of motifs related to seed-specific promoters, such as Skn-1 motif,AACACA,SEF4 motif,E-box,ACGT. Replacing CaMV35S promoter of pCAMBIA1301 with FP fragment, the binary expression vector pCAM-FP was constructed. Transient expression in soybean tissues by Agrobacterium tumefaciens mediated method, the results of histochemical GUS analysis showed that there were little or not GUS activities in roots, stems and leaves, but there was higher activity in soybean seeds. It is inferred that FP promoter possess the function driven downstream gene expression exclusively in soybean seeds.
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  Search for Optimum Substitutive Neutralizing Agent and pH Control Strategy in Fumaric Acid Fermentation by Rhizopus oryzae

  CHEN Chen, TAI Chao, LI Shuang

  China Biotechnology. 2013, 33(4): 85-91.

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  Four different neutralizing agents (CaCO₃, Na₂CO₃, NH₃稨₂O, NaOH) were chosen to examine the effects of neutralizing agents on fumaric acid fermentation by Rhizopus oryzae ME-F12 which is the mutant of R. oryzae ATCC 20344. It was found that fumaric acid yield and productivity in the fermentation using Na₂CO₃ as neutralizing agent were the closest to which in the traditional CaCO₃ case. Then the effects of different pH values (3.5, 4.5, 5.5, and 6.5) on fumaric acid fermentation using Na₂CO₃ as neutralizing agent were investigated. Based on the analysis of three kinetic parameters, a two-stage pH control strategy, aimed at achieving high concentration, high yield and high productivity of fumaric acid simultaneously, was proposed. pH was controlled at 5.5 at the first 24 h, and then switched to 4.5 till the end of the fermentation. Finally, the maximum concentration of fumaric acid reached 40.5 g/L with the yield of 0.55 g/g and the productivity of 0.61 g/L/h, which were 8.3%, 10.0% and 17.3% higher than the best result of constant pH control process. Its productivity was even 3.4% higher than which in CaCO₃ case. With the internal advantages of reducing power consumption and simplifying downstream processing, using Na₂CO₃ as neutralizing agent under two-stage pH control strategy successfully take place of CaCO₃.
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  Detection of Individualized Treatment and Test of Helicobacter pylori Infection through Visualized Gene Chip Technology

  YAO Xue, LIU Qi-qi, ZHAO Qing, CHEN Su-hong

  China Biotechnology. 2013, 33(4): 92-100.

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  Objective: To establish a method of visualized gene chip detection that can simultaneously measure the gene polymorphism of CYP2C19 that is related to clarithromycin and quinolones resistance mutation in Helicobacter pylori infection and to the metabolism of proton pump inhibitor. Method: Through analysis the sequence of human CYP2C19^*2, CYP2C19^*3 and the targets related to resistance to clarithromycin and quinolone, a gene chip to test the resistence of Helicobacter Pylori was established. The specificity, sensitivity and repeatability of the gene chip was evaluated in the optimized PCR system, with hybridization reaction and visualized detection. Result: The results of detection of 196 clinical samples using gene chips were consistent with that by sequencing. The detection sensitivity of the chip for Hp was 10³CFU/ml, and that for human genomic DNA was 2ng/μl.Conclusion: This method was quick, highly specific, sensitive, and economic. With its clinical application potential, it is promising in providing medication guidance for Hp elimination in clinical use and individualized treatment.
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  Preparation of a Novel Self-assembly Nanoparticle Based on Amphiphilic γ-Polyglutamic Acid Derivatives as a Protein Carrier

  CHEN Kuan-ting, YAO Jun, RUAN Wen-hui, WEI Qin-jun, LU Ya-jie, CAO Xin

  China Biotechnology. 2013, 33(4): 101-105.

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  A novel amphiphilic graft copolymer composed of γ-polyglutamic acid (γ-PGA) as the hydrophilic backbone and cholesterol as the hydrophobic segment was synthesized. The cholesterol-bearing γ-PGA (γ-PGA-Graft-CH) was used to form self-assembly nanoparticles (γ-PGA-Graft-CH NPs) with an inner hydrophobic core and an outer hydrophilic shell via the ultrasonic probe method. The obtained nanoparticles showed low cytotoxicity and a narrow size distribution (PDI=0.17) with a mean diameter 299.6+5.4 nm. OVA-loading γ-PGA-Graft-CH NPs was also successfully prepared, with drug loading content of 118.8μg/mg, and entrapment efficiency of 33.5%. The experimental results also showed that OVA continuously released from γ-PGA-Graft-CH NPs in the phosphate buffered saline (PBS) solutions,and its release was sensitive to the pH of the release medium.
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  Rapid Construction of GPR126 Conditional Gene-targeting Vector

  YE Xiang-li, LI Da-li

  China Biotechnology. 2013, 33(4): 106-113.

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  Generation of specific gene knockout mouse model is a reliable technique for studying the role of genes in mammalian model organism. One of the key steps to acquire a gene knockout mouse is to construct a targeting vector for homologous recombination in mouse embryonic stem cells. For some genes, the mutants will die in uteri owing to their critical roles in embryonic development, or the mutant mice may have different phenotypes according to the period of development and types of tissues. It is difficult to study these genes by the conventional knockout approaches. Thereby the conditional knockout strategy had been developed for its advantages to circumvent the embryonic lethality problem and to investigate gene function temporally and spatially. Using modified Red homologous recombineering system, the two LoxP sites were inserted into the target position accurately and the GPR126 conditional gene-targeting vector was rapidly constructed. The successful generation of GPR126 conditional knockout construct will be helpful for the subsequent production of knockout mouse model and exploration of the function of GPR126 in mice.
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  A Simple and Cost Effective Process for Large-scale Production of Long Oligoribonucleotides

  ZHANG Ping-jing, LI Tie-jun, ZHOU Song-feng, ZHU Yuan-yuan, CHEN Jian-xin, LU Yi-xiang, WEN Feng

  China Biotechnology. 2013, 33(4): 114-120.

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  The length and stable secondary structure of RNA molecule are general obstacles in RNA synthesis because of current technological bottlenecks. A simple and cost effective process for large-scale preparation and purification of long oligoribonucleotides with stable secondary structure was presented. High homogeneous RNAs are transcribed in vitro with T7 RNA polymerase using linear 2'-Ome modified DNA templates, which were prepared by primer extension instead of PCR amplification or linearized plasmid DNA transcription to reduce contamination. The crude transcripts are then directly subjected to an anion-exchange HPLC using source 15Q to separate T7 RNA polymerase, unincorporated rNTPs, small abortive transcripts, endotoxin and DNA templates from pure RNA products. The novel process does neither require tedious phenol/chloroform extraction nor denaturation of RNA, which is especially useful for larger RNAs preparations.
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  Expression of XRN1 Protein and Optimization of Fermentation Medium with Response Surface Method

  WU Ying-chun, MA Qin-qin, DING Xian-feng, ZHANG Kai, LIU Li-li, GUO Jiang-feng

  China Biotechnology. 2013, 33(4): 121-128.

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  Response surface method (RSM) was used to optimize the fermentation medium of XRN1 protein expression. The Plackett-Burman design was used to evaluate the effects of eight factors, including yeast extract, calcium sulfate, MgSO₄?7H₂O, glycerol, potassium sulfate, phosphoric acid, ammonium sulfate and potassium hydroxide. The statistical analysis revealed that the key factors influencing XRN1 protein expression were yeast extract, magnesium sulfate and potassium sulfate. The path of steepest ascent was used to define optimal response region for these three factors, then the optimal level combinations of factors were obtained through Box-Behnken design, the optimal conditions were as follows: yeast extract 0.45%, magnesium sulfate 0.38%, and potassium sulfate 1.4%. Under optimal conditions, biomass of yeast could increase by 0.5 fold and expression of XRN1 protein could increase by 40%.
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  Development of Gene Therapy for Parkinson’s Disease And Alzheimer’s Disease

  FAN Fu, CHEN Jian-guo, REN Hong-wei

  China Biotechnology. 2013, 33(4): 129-135.

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  Alzheimer’s and Parkinson’s diseases represent the most prevalent neurodegenerative disorders worldwide. Current pharmacological or surgical treatments provide symptomatic benefits, but none can delay or stop the progression of these diseases. In recent years, the development of molecular biology and medicine has benefited for the understanding of Alzheimer’ s and Parkinson’ s diseases and prompted the research of gene therapy for them.An overview of the current efforts in the field for the treatment of Alzheimer’s and Parkinson’s diseases is presented. Against the pathogenesis of Parkinson’s disease and Alzheimer’s disease, there are several effective gene therapy strategies. It is no doubt that, gene therapy, as a novel treatment, is of great significance for understanding the causes, as well as comprehensive treatment for Parkinson’s disease and Alzheimer’s disease.
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  Development Present Situation,Problems and Solve Method of Somatic Cell Nuclear Transfer

  LÜ xin, DU Wei-hua, ZHU Hua-bin

  China Biotechnology. 2013, 33(4): 136-142.

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  Somatic cell nuclear transfer (SCNT)is widely used in animal hunsbandry.However, the low efficiency restricted its application severely. Almost all the cloned animals have the hypertrophic placenta and aberrantly epigenetic inheritance which also result into the low efficiency of the cloning technology. The current progresses of SCNT were summarized and a new method, tetraploid compensation, to improve the placenta hypertrophy and the efficiency cloning by epigenetic modification were disccussed.
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  DNA Barcoding Used in the Identification of Ginseng

  SUN Tao, TENG Shao-na, KONG De-ying, SONG Yun, XU Jin, LI Ying-guo, WANG Yu, LI Ming-fu

  China Biotechnology. 2013, 33(4): 143-148.

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  Ginseng (Panax ginseng C.A.Meyer), known as "the King of Herbs",which is endangered famous,precious chinese herbal and senior tonic, and in urgent need of resources protection. With the internationalization of Chinese herbal,more and more pseudo mix products appeared.So,the right identification become the chief condition of resources protection. Identification and sustainable use of the medicinal plant resources in ginseng have been extensively studied by scholars from domestic and abroad. DNA barcoding is the latest development in molecular identification, it is a method of rapid and accurate species identification and recognition using a short, standardized DNA region. DNA barcoding has become one of hotspots of biodiversity research, shows the broad application prospects in terms of species identification. The scope and limitations of the traditional identification methods were analyzed,and the features and application of new DNA barcoding technology and its analysis mothod used in the identification of ginseng were emphatically induced.
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  Low Level Presence of Genetically Modified Crops: Policies and Implications

  HUANG Xue-tao, YANG Jun, DONG Wan-lu, WANG Xiao-bing

  China Biotechnology. 2013, 33(4): 149-155.

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  Biotech crops, also known as genetically modified (GM) crops have been in rapid development worldwide in recent years. However, a problem of low level presence (LLP) of GM products is emerging because of asynchronous approvals among importing and exporting countries, and low tolerance level for adventitious presence of GM products unapproved by importing countries. Major concerns have been raised about LLP leading to trade conflict and even trade disruption. The insights into the definition of low level presence, emphasizing its specificity and inevitability from a technical perspective are provided. It also enumerates LLP policies of several important countries, illustrating the potential adverse effects of strict LLP policies on agricultural trade. According to the findings, it points out that mutual trust and information exchange mechanisms should be built up among importing and exporting countries. Meanwhile, it is very critical to shorten the time to get the import safety approval and set up the science-based LLP threshold.