Biotechnology Bulletin

Progresses in Studying of Protein Families Involved in Zn/Fe Transporting in Plants

Li Suzhen, Chen Jingtang

2013, 0(2): 8-14.

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Zinc and iron play virtual roles in plants development and involve in many biological functions. Zinc and iron deficiency or excess will cause harmful effects to plants. Therefore, metal transporters are essential for maintaining ion homeostasis. These metal transporters were classified into two groups, metal-uptake proteins and metal-efflux proteins. They are involved in the transmembrane-transporting and translocation of the intracellular zinc and iron, and regulating the homeostatic of zinc and iron. At present, many metal transporters have been identified and their expression profiles associated with zinc/iron accumulation and distribution have been described. The gene expression and protein localization of those zinc/iron-transporter families were reviewed in this article.

Application of Zebrafish in Research of Transgenic Organisms

Liu Lili, Wang Jian, Wu Wei, Wang Haisheng, Yan Yanchun

2013, 0(2): 15-21.

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The recent explosion of transgenic zebrafish lines in the literature demonstrates the value of this model system for detailed in vivo analysis of gene regulation, development and differentiation and morphogenetic movements, as well as drug screening and environment monitoring. After an outline of the application and development history of vital models from Escherichia coli to Musmusculus, the superiority of zebrafish as a model organism will be emphasized. Then this article will review the development history of transgenic technology in zebrafish and the uses of transgenic zebrafish. And finally problems in future development of transgenic zebrafish will be presented.

Advances in the Studies of Ectomycorrhiza in China

Wen Zhugui, Chen Yahua

2013, 0(2): 22-30.

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Studies on the ectomycorrhiza in the past 10 years in China are reviewed in this paper, emphasis on investigations, identification and taxonomy, pure culture, physiological and ecological functions of ectomycorrhizal fungi, as well as application of edible ectomycorrhizal fungi in order to strengthen the utilization of them. The main problems of research and utilization of ectomycorrhiza were analyzed, and the research priorities in future were prospected.

Development of Wastewater Treatment Using Ammoniadegrading Bacteria

Zhao Cuijuan, Song Wenjun, Zhu Gaoxiong, Wei Jiping, Li Bozhi, Zhang Jun

2013, 0(2): 31-34.

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Ammonia nitrogen is the main pollutant in wastewater，and directly reflects the degree of water pollution. Bio-treatment of ammonia-containing wastewater using bacteria has shown great advantages，such as timely degradation，easy operation，no secondary pollution. This aritical review outlines the ammonia-degrading mechanism，separation technique and the application of ammonia-degrading bacteria for wastewater treatment in recent years. And finally discusses the prospects of wastewater treatment using the treatment process.

Progress of Cellulase and Cellulase Gene Research

Zhao Yan, Chen Genghua, Zhou Wei, Hou Yali, Yang Zhonghua

2013, 0(2): 35-40.

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Cellulose is a kind of polysaccharides material existed widely in nature, it can hydrolyze cellulosic materials to monosaccharide effectively, and then ferment to ethanol, H2 and microbial oils. It is useful to ease the shortage of global energy while using cellulose appropriately. In recent decade, cellulase, the tool of cellulose hydrolysis, has been concerned by home and abroad. In this article, combining the relationship of cellulase and bio-energy and the demand of cellulase for market-oriented, development process of cellulase gene were summarized.

Progress in Study and Application of Bacterial Laccase

Ma Yingying, Jia Honghua, Wei Ping

2013, 0(2): 41-48.

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Laccase is widely used in decolorization of dyes, paper as a green polyphenol oxidases. Bacterial laccase has more advantages and potential in the industrial application due to the high temperature and pH stability compared with fungi laccase. In this article, the origination, distribution, molecular structure and fermentation of bacterial laccase were summarized. And the immobilization of bacterial laccase were also mentioned. Meanwhile, the application of bacterial laccase in dyes wastewater, electrochemistry and paper were briefly introduced.

Research Progress on Mycoplasma Lipoprotein and Its Variation Interaction with Host

Ni Bo, Bai Fangfang, Liu Maojun, Feng Zhixin, Xiong Qiyan, Wei Jianzhong, Shao Guoqing

2013, 0(2): 49-54.

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Lipoproteins are significant structural of mycoplasma, which play an important role in the interactions with host. In recent years there have been many reports proved that lipoproteins may be importantly contributing in the pathogenesis of mycoplasma. In this article describes the characteristic and function of lipoproteins, the role in adherence, the ability leading to inflammatory response and cell apoptosis. Moreover have a review about phase and antigenic variation under the regulation of gene families through ON/OFF molecular switches, size variation, domain shuffling, horizontal gene transfer, and mycoplasmal antigen modulation. As well as the effect of variation of lipoproteins in virulence and the biological significance of diverse surface of mycoplasma also included.

Roles of POZ-ZF Proteins in Development and Oncogenesis

Mei Zhu, Yang Yutao, Xu Zhiqing

2013, 0(2): 55-60.

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POZ-ZF proteins are highly conserved proteins implicated many biological processes in invertebrate and vertebrate. Members of the POZ-ZF protein contain an amino-terminal POZ domain and a carboxy-terminal DNA-binding domain made of one or more zinc finger motifs and usually act as transcriptional co-repressors to regulate target genes expression. Recent studies showed that POZ-ZF proteins play important roles in development and oncogenesis. In this review, we summarize studies of five POZ-ZF proteins, which is helpful to further understand POZ-ZF proteins.

Progress of Using Nanoparticles as Gene Vector

Lu Yanmin

2013, 0(2): 61-66.

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Nanoparticles have some characteristics such as can combine with big fragment DNA，low immunogenic，good biocompatibility，easy to produce，which can protect DNA against the digestion of nuclease. The nanoparticles have a high stability in organisms and cells，which was difficult to be degraded. Nanoparticles can improve transfection efficiency. As a nonviral gene vector，the application of nanoparticles in gene therapy became research focus. In this article，we mainly discussed the characteristics of nanoparticles， characteristics and transport mechanism of nanoparticles as gene vector，the progress on nanoparticles as gene vector.

Protein Fragment Complementation Assay（PCA）and the Applications

Zhang Jie, Liu Changbai

2013, 0(2): 67-71.

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Protein fragment complementation analysis (PCAs) is a recently developed new technique to assay protein-protein interactions. Typical PCA has been using dihydrofolate reductase, β-lactamase, GFP and luciferases to study protein-protein interactions successfully. This technology can not only analysis the localization of protein molecular interaction dynamically, draw intracellular signal transduction, protein biological chemical network, but also can be applied to protein library and high throughput new drug screening. In this review, we summarized the concept, methodology and the applications of PCA.

Karyotype Analysis of Four Herbaceous Flowers

Qi Yuqing, Li Xinling, Zhang Yanni, Li Feng

2013, 0(2): 72-75.

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In this paper, conven-tional pressed slice method was used to study chromosome number and karyotype of four herbaceous flowers. The results showed that Diranthus chinensis：chromosome number 2n=14, karyotype formula K（2n）=14=12 m+2 sm, belonging to “1A” type; Lavatera trimestris Linn ：chromosome number 2n=14, karyotype formula K（2n）=14=6 m+8 sm, belonging to “2A” type; Capsicum frutescans ：chromosome number 2n=24, karyotype formula K（2n）=24=24 m, belonging to “1B” type; Impatiens balsamina ：chromosome number 2n=14, karyotype formula K（2n）=2x=14=10 m+4 sm, belonging to “1B” type. This is the first report of karyotype of these four flowers, and may help breeding and classification of flowers.

Impact of High Temperature Treatment on the Content of Bt Protein in

Genetically Modified Crop Seeds

2013, 0(2): 76-79.

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Food safety problem of genetically modified crops have aroused public concern day by day. As crop seed is one of the most important source of food, the research on safety of genetically modified crop seeds need to be strengthened. The present work applied cotton and corn seeds, Bt modified varieties and non-genetically modified varieties, to treat with high temperature（55-130℃）, and then detected the dynamic digestion process of Bt protein with sandwich enzyme link immunoassay（ELISA）. The result of the assay showed that the Bt protein in different crops have different degree of tolerance to high temperature. The capacity of enduring heat stress of Bt protein in cotton seed is better than that in corn seed; the toxin protein significantly decreased within 10 min in the high temperature treatment, followed by slow degradation; all Bt protein in transgenic corn and cotton seeds can obviously inactivation treated with 130℃ or higher temperature.

Cloning and Prokaryotic Expression of Capra hircus Klf4，and Purification of His-Klf4 Fusion Protein

Xin Guiyu, Wang Lixia, Ye Xinhui, Shen Kuikui, Fu Xinhui, Li Gonghe, Zhang Ming, Lu Shengsheng, Lu Kehuan, Zheng Xibang

2013, 0(2): 80-85.

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The paper was to clone Klf4 gene of Capra hircus, and construct recombinant plasmid pET30a-klf4, and finally to prepare the purified His-klf4 fusion protein by means of prokaryotic expression techniques . Total RNA extracted from the genital ridge of fetal Capra hircus, Klf4 cDNA was amplified by RT-PCR and the plasmid pMD18-T-Klf4 was constructed by TA cloning. After restriction endonuclease digestion and sequencing analysis, Klf4 cDNA was subcloned to pET-30a vector to obtain a recombinant plasmid pET30a-Klf4. Transformed into E. coli BL21, the recombinant plasmid pET30a-Klf4 was induced to express by IPTG, which was verified by SDS-PAGE and Western blotting analysis. Finally, the His-Klf4 fusion protein was purified via metal Nickel-chelating affinity chromatography. The results showed that ：（1）the Klf4 gene was successfully cloned from promodial genital ridges of fetal Capra hircus; the open reading frame（ORF）of Capra hircus Klf4 is composed of 1434 nucleutide acids, coding 478 amino acids; comparison of sequence similarity of Capra hircus Klf4 homologue with other vertebrate Klf4 homologues indicated that Capra hircus shared the highest homology（98.5%）with Ovis aries.（2）Signal peptide prediction by SignalP 4.0 online showed that there was no signal peptide in the coding proteins of Capra hircus Klf4.（3）SDS-PAGE and Western blotting assay showed that the recombinant plasmid was expressed in E.coli BL21, and His-Klf4 fusion protein was purified under denature condition.

Optimization of Production Conditions for Dairy Cows Somatic Cell Nuclear Transfer Embryos

Sun Wei, Ba Teer, Guo Jitong, Li Rongfeng, Wang Jianguo, Li Ming, Hu Shuxiang, Wang Chunsheng, Li Xihe

2013, 0(2): 86-92.

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The purpose of the study was to improve the total industrial application efficiency of dairy cow somatic cell nuclear transfer （SCNT）by optimazing the in vitro producting conditions of dairy cow SCNT embryos. In this study, the effect of different factors, such as enucleation method, sources of donor cell different ages cows, serum starvation, and gas component condition, on the efficiency of SCNT were studied. The results demonstrated that there were no significant differences on the rate of enucleation and the rate of blastocyst（100% and 24.83% in the fluorescent-assistant group and 92.44% and 28.26% in the blind-suction group, respectively）（P>0.05）. However, the method of blind-suction was easier to operate and more efficiency. The age of donor cows showed no significant impact on the rate of blastocyst（31.43% and 25.68%, respectively）. There was no significant difference on the rate of blastocyst between serum-starvation and non-starvation group（24% and 29.9%, respectively）, and there was no significant difference on the rate of blastocyst between high-oxygen and low-oxygen group（28.26% and 31.55%, repsctively）. Conclusionly, the results showed that the optimized condition for industrial production of dairy cow SCNT embryos was as follows ：donor cells without serum-starvation could be injected into ooplasm enucleated by blind-suction to construct cloned embryos. Then, fused cloned embrys should be cultured under sealed condition with 5% CO2, 5% O2, and 90% N2 to get a reasonable rate of blastocyst.

Molecular Cloning of Sugarcane ASR Gene（SoASR）and Its Expression Analysis

Huang Xing, Yang Litao, Zhang Baoqing, Song Xiupeng, Li Yangrui, Wang Sheng

2013, 0(2): 93-99.

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In this study, the full length of SoASR cDNA obtained by homologous cloning and RT-PCR. It consists of 753 bp with an open reading frame of 429 bp, encoding a polypeptide of 142 amino acids, and its GenBank accession number is JX470187. Homology analysis showed that SoASR were clustered into a group with barley, maize and sorghum with homology of 79%, 93% and 88%, respectively. A 32.0 kD heterologous protein was obtained when SoASR gene was expressed in E. coli. The results of quantitative real-time PCR analysis showed that, the mRNA of ASR was first up-regulated and then down-regulated in the sugarcane variety with strong cold resistance, GT28, and down-regulated in the sugarcane variety with weak cold resistance, YL6, under low temperature stress. The expressions of SoASR gene were induced in two varieties. These results suggested that SoASR might be involved in response to cold stress in sugarcane.

Genetic Transformation of Bt（cry1Ab）Gene into Sugarcane（Saccharum officinarum L.）Mediated by Agrobacterium tumefaciens

Li Xiaomei Wang Minxia Qin Tinghao Yang Cui An Qi Zhang Jun

2013, 0(2): 100-105.

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In this research, embryonic callus induced from sugarcane（Saccharum officinarum L.）cultivar “Chuanzhe 23” was selected as recipient material. We successfully transformed anti-insect gene Bt（cry1Ab）into sugarcane mediated by Agrobactenum tumefaciens EHA105 when the explants infected with lower concentration of Agrobactenum tumefaciens（OD ≈ 0.1）and shorter infusion time（1.5 min）. The optimal concentrations of hygromycin for the selection of transformed shoots and roots induction respectively were 20 mg/L and 30 mg/L. The calli cultured for 40 d were optimal for transformation; 65 transformants were obtained after successive hygromycin resistance selection. The hyg-resistant plants were detected by PCR analysis, and 2 plants showed positive, which initially proved that the target gene had been integrated into sugarcane genome．

Cloning and Sequence Analysis of a Osmotin-like Protein Gene from Atriplex canescens and Its Prokaryotic Expression

Wang Shengyang, Zhang Yong, Wang Jian, Liu Yanzhi, Liu Jinliang, Pan Hongyu

2013, 0(2): 106-110.

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A osmotin-like protein gene was isolated based on the library from Atriplex canescens and its EST analysis, and named as AcOLP. The full length of AcOLP contained an open reading frame of 687 bp. It encoded a polypeptide of 229 amino acids, belonging to GH64- TLP-SF superfamily as a kind of PR-5 proteins. The AcOLP had 94% nucleotide sequence homology and 87% amino acid sequence homology to the sequence of osmotin-like protein from Atriplex centralasiatica, respectively. The accession number of AcOLP in GenBank was JN632587.1. Comparison of amino acid sequence with PR-5 proteins from various plants showed AcOLP possessed 16 cysteine residues that were conserved at their invariant and were presumably involved in disulfide bonding. Phylogenic analysis on the amino acid sequence of AcOLP with other plants showed that Atriplex canescens was closely related to Atriplex nummularia. AcOLP was inserted into the prokaryotic expression vector of pET-28a and expressed its fusion protein（about 29 kD）in Escherichia coli BL21（DE3）.

Full-long cDNA Sequence Cloning and Bioinformatic Analysis of Piwi Subfamily Member Giwi in the Gampsocleis gratiosa Gonads

Liu Jing, Zhou Zhijun, Chang Yanlin

2013, 0(2): 111-117.

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In order to further research the role of piwi in the stem cell self-renewal and germline development of hemimetabolous insect. To obtain the full-length of piwi homolog giwi, we first picked out piwi homolog fragments in the Gampsocleis gratiosa transcriptome sequences, which had been sequenced by our laboratory and not release to the public database, and then designed specific primers for nested RT-PCR partial amplification and rapid amplification of cDNA ends（RACE）complete 5' and 3'sequence. Sequences analysis showed that the full-long of giwi gene cDNA is 3 462 bp, which consisted of a 2 742 bp open reading frame（ORF）encoding 913 amino acids, a 111 bp 5'-untranslated region（5'UTR）and a 609 bp 3'-untranslated region（3'UTR）. The molecular weight of Giwi has been predicated to be about 102.7 kD and pI 9.55 in theory. Giwi contains the signature motifs of the piwi protein subfamily, including a C-terminal PIWI domain, a centrally located PAZ domain, and an N-terminal variable domain. The homology blast analysis showed that there is a catalytic triad “Asp-Asp-His” motif in the PIWI domain which is similar to the RNase H active center, implying possessing slicer activity of this protein. Phylogenetic analysis showed that appearance of either Piwi or Aubergine in insect might due to an ancient gene duplication event. Transcriptome resources generated based on next-generation sequencing can greatly benefit future gene cloning.

High Expression of Rhizomucor miehei Lipase in Pichia pastoris by Fusion to CBD

Xue Chongchong, Zhang Junhui, Lin Ying, Han Shuangyan

2013, 0(2): 118-123.

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Rhizomucor miehei lipase（RML）, one of the important enzymes applied in industries, has a broad prospect of application. CBD, which is capable of tightly binding cellulose, has been found in diverse hydrolytic enzymes（cellulose, xylanase）. The presence of a CBD is shown to increase the effective concentration of enzyme on substrates, thereby assisting the enhancement of hydrolytic enzyme activity. Here the CBD from Trichoderma harzianum endoglucanase II（THEGII）was connected to the N-terminal of RML through an endogenous linker peptide. Then the fusion genes were cloned into the secreted expression vector pPICZαA to construct recombinant plasmid pPICZαA-CBD-RML. The plasmid was linearized and transformed into Pichia pastoris GS115 to obtain Pichia pastoris recombinant strain. Compared with that of the mature RML without CBD-fusion, secretion of CBD-RML by this fusion construct was about 17% enhanced in addition to similar temperature activity and stability between RML and CBD-RML. Therefore, the CBD from THEGII can be used as a secretion enhancer for secretory production of heterologous protein in Pichia pastoris.

Construction of GAL’s Eukaryotic Expression Vector and Targeting Expression of GAL in N-2a Cells

Rong Man, Zhang Ruihu, Liu Tianfu

2013, 0(2): 124-129.

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The study was designed to construct eukaryotic expression vector pcDNA-PDGF-GAL, and observe the specific expression of GAL in N-2a after transferction. Firstly, the 2.9 kb PDGF-GAL fragment was amplified by PCR, then directedly cloned into pcDNA 3.1 vector, replacing the vector’s CMV promoter and enhancer and finally forming the recombinant vector pcDNA-PDGF-GAL which is neuronspecific expressed. Secondly, pcDNA-PDGF-GAL is transient-transfected into N-2a and the expression of GAL protein is detected by RT-PCR and ELISA. Results showed the sequencings result of pcDNA-PDGF-GAL was blasted on NCBI with standard sequence, in which PDGF-GAL fragment has a high nucleotide sequence identity（99%）, and these results suggested that the construction of recombinant plasmid gets success. By RT-PCR and ELISA, it is confirmed that GAL protein is specifically over-expressed in N-2a. The neuron-specific eukaryotic expression vector was successfully constructed, and GAL protein is successfully over-expressed in N-2a, which established a basis for the preparation of stabletransfected cells over-expressing GAL and further exploring the biological functions of GAL on nervous system.

Heterologous Expression and Characterization of Xylanase XYA6205 from Stachybotrys chartarum

Wang Hongxia, Wang Huaming, Zhang Dalong, Luo Cheng

2013, 0(2): 130-134.

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Xylanase, the main enzyme to hydrolysis hemicellulose, has important application in industry. In this study, a gene xya6205 （with signal peptide）from Stachybotrys chartarum was inserted to an expression vector, eventually an recombinant plasmid pGm-xya6205 was constructed, and then transformed into A. niger G1 mediated by PEG4000. The transformants were confirmed by PCR analysis, and the recombinant xylanase of heterologously expressed protein in SDS-PAGE was about 20 kD. The optimal temperature and pH of the enzyme activity was 50℃ and 5.8, respectively. The recombinant enzyme specific activity reached 392 U/mg by DNS method at optimal condition. The recombinant enzyme remained 83% activity after 18 h in the alkaline buffer.

The Development and Identification of Rift Valley Fever Virus-like Particles

Deng Junhua, Lin Xiangmei, Zhang Yongning, Wang Caixia, Feng Chunyan, Wu Shaoqiang

2013, 0(2): 135-139.

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It was to develop Rift valley fever（RVF）virus-like particles（VLPs）to effectively offer positive material for molecular biological detection. pGEM-T-RVFV plasmid and p-MS2 were individually digested with both Pst I and Hind Ⅲ , and then purified ligated to generate recombinant plasmid p-MS2-RVFV. The plasmid was cotransformed into E. coli. DH5α cell and induced with IPTG, incubated with RNase A and DNase I, and then subsided with salt to obtain the rough-wrought virus-like particles. The method of gradient centrifugation was used to prepared rarefied virus-like particles which was determined for characteristic property. The puried particles were verified by Real-time RT-PCR. Results showed that the rarefied virus-like particles were able to endure RNase A, saved at any rate one month under subzero climatic condition, which indicated that the virus-like particles were stable and were simple transported；it indicated that the VLPs could attain a minimum detectable limit of 4.25×102 copies. The construction of virus-like particles could provide positive material for RVF molecular detection.

Cell Model Construction of Regulated Hepatitis B Virus X Gene Expression

Bai Helu, Liu Rui, Zhu Naishuo

2013, 0(2): 140-146.

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An HBx inducible expression plasmid carrier was constructed by Lentil-XTM Tet-On Advanced Inducible Expression System, and rt-TA gene virus particles and HBx gene virus particles were synthesized by lentiviral packing cell line HEK293T cells, with the titer reaching 108 LPs/mL. When HepG2 cell lines were infected by the two virus particles and selected by G418 and puromycin antibiotics, stable cell line with HBx gene could be constructed. After adding Dox into the stable cell line, the inducible expression of HBx gene could be proved by RT-PCR and Western blot. Our study provides an ideal experiment model for further research on the roles of HBx in HBV infection and the host immune tolerance.

Primary Investigation of Improved Subtractive Hybridization that Can High Efficiently Screen Differentially Expressed Small ncRNA

Wang Yan, Peng Liping, Chen Jinzhen, Liu Xing, Luo Zhenming, Zhou Tianhui

2013, 0(2): 147-150.

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The purpose of this paper is to build a new method that can detect the differences of small non-coding RNAs in breast cancer cells. This paper adopt the methods of isolating the total RNA from breast cancer cell lines MCF7 and nontumorigenic cell line HBL100, then adopting the RNA Isolation Kit to isolate the special size of RNAs（18-100 nt）, which were added poly（A）trails at the 3'-end and then reversing into the first strand of cDNA. After the subtractive hybridization of single stranded cDNA and RNAs, the differences of expressed small non-coding RNAs can be obtained with the method of using SA-PMPS Isolation System and the final result of hybridization can been tested by the internal reference of U6. Eventually, the products of hybridization were amplified through Nest PCR and PCR products were then lighted into T vector for sequencing. Results showed that after isolation, some special bands will be shown within 100 nt in the 10% polyacrylamide gel; the result of subtractive products PCR, which adopts the internal reference of U6, only shows visible band after 33 PCR circles. There were obvious bands within the size of 100 nt after Nest PCR and electrophoresis for the products of subtractive hybridization concluding the differences of expressed small non-coding RNAs were obtained. In this study, the improved subtractive hybridization can effectively identify the differences of expressed small non-coding RNAs in different cells.

Establishment of the Screening Oligonucleotide Microarray of Apscaviroid in Genus Level

Zhang Yongjiang, Xin Yanyan, Zhu Shuifang, Li Shifang

2013, 0(2): 151-156.

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Apscaviroid is a significant plant viroid genus and has not screening method available at present. For the establishment of the screening oligonucleotide microarray of Apscaviroid in genus level, thirty-five genus specific probes from the sequences of Apscaviroid viroid were designed. Microarray was constructed based on these probes and identificated with Apple scar skin viroid and Citrus dwarfing viroid standard samples. Results showed a high specificity for detection of Apscaviroid viroid and the same detection sensitivity with RT-PCR. The microarray can be used to screen and provide technical support of quarantine and control for Apscaviroid viroid.

Advantages of the Dynamic Model in Real-time PCR Data Processing

Liu Yanli, Li Xu, Su Zhencheng, Xu Mingkai, Zhang Huiwen

2013, 0(2): 157-162.

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In recent years, Real-time PCR with its advantages in the quantitation of gene is widely used, the development of equipment ensure the accuracy of raw data, however the reliability of quantitative result highly depends on the PCR efficiency and the mathematical model on which the quantitative methods are based, The quantitative result processed with different mathematical models will be quite different. In this paper, the sample was 5-fold diluted for gaining the raw data of quantitative PCR, and the same raw data was processed by three kinds of mathematical models, including 2 －△△ Ct model, relative standard curve model, dynamic model. And the results demonstrate the dynamic model has a good prospect.

Antioxidant Activity of Solvent Extracts of Xinjiang Propolis and Determination of Quercetin and Chrysin Propolis

Buwihelqem Ababekri, Mutallip Amet Aminigul, Mamat Nizam Ehet, Yimit Rahman

2013, 0(2): 163-171.

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To study the DPPH free radical scavenging activity and anti-lipid peroxidation activity of Kuqa and Yili, and determine the content of the Quercetin and Chrysin in Xinjiang Yili propolis. Different extracts of raw propolis were extracted by four kinds of solvents, taking tea polyphenol as comparison; measure Kuqa and Yili, Xinjiang propolis extracts antioxidant activity in removal of DPPH（1, 1-Diphenyl- 1-picrylhydrazyl）free radicals and antioxidant properties of linoleic acid peroxidation. With 70% ethanol and methanol as solvent to extract Quercetin and Chrysin from Xinjiang Yili propolis, using HPLC（high performance liquid chromatography）to determine the content of the extracted Quercetin and Chrysin, the detection wavelength was 370 nm and 268 nm, respectively. Result showed that propolis different extracts in different concentrations has different scavenging free radical DPPH capacity, in partial experimental groups’ scavenging capacity is even surpassed to same concentration of tea polyphenol group; has antioxidant activity to linoleic acid, in which Yili propolis has higher antiperoxidation activity. The content of Quercetin was the lowest in the without defatted ethanol extracts（2.2950 mg/g）, the highest in the defatted methanol extracts（2.8150 mg/g）；The content of Chrysin was the lowest in the without defatted methanol extracts（60.72 mg/g）, the highest in the defatted methanol extracts（74.37 mg/g）. The Kuqa and Yili propolis has a certain extent of antioxidant capacity, this DPPH method is fast, sensitive and simple, this results showed that the Xinjiang propolis is a natural antioxidant, and waiting for further study and utilization; HPLC analysis showed that Quercetin and Chrysin the active ingredients of propolis samples from Xinjiang Yili were detected.

Preparation and Primary Application of Enterobacter sakazakii Colloidal Gold Test Strip

Zhou Hefeng, Shao Min, Li Changfu, Ge Zhenglong

2013, 0(2): 172-176.

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To develop and test its performance of colloidal gold immunochromatographic test strip for rapid detection of Enterobacter sakazakii（ES）. The colloidal gold particles were prepared by the sodium citrate reduction method. Purified ES monoclonal antibodies were labelled with the colloidal gold particles. The gold-labeled antibody was coated on the gold conjugate pad using the immune double sandwich method. The another mAb of ES was coated on the surface of nitrocellulose filter membrane（NC）as the test line（T line）, while the secondary antibody（goat anti-rabbit IgG antibody）was coated on the surface of NC as the control line（C line）. Then the test strip was assembled with sample pad, absorbing pad, and dorsal shield. Its sensitivity, specificity, stablility and reproducibility were evaluated. It can detect the sample content of ES by means of a simple enrichment process. This whole time will take 6 h. The test strip’s sensitivity reached 1×104 CFU/mL, and displayed perfect specificity without cross reactions with food-borne pathogens. All these results showed that the strip was simple, sensitive, specific, stable, and rapid for detection of ES.

Research of Reversal Effect on K562/A02 Cell Line with a Novel Marine Bioactive Substances

Zhang Yuxia, Liang Qiong, Yong Guoxin

2013, 0(2): 177-183.

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This test was designed to study the effects of FAT on multidrug resistance（MDR）in human leukemia K562/A02 cells. MTT method was used to observe the drug sensitivity and the effect of FAT on the drug resistance; Flow cytometer was used to measure the intracellular drug accumulation; Cellular GSH concentration was examined by biochemical analyses with DTNB. Results showed that FAT did not exhibit an inhibitory activity to proliferation in K562 cell line and K562/A02 cell line. FAT decreased IC50 of ADM in K562/A02 cell line. And it had no remarkable effect on K562 cell line. FAT increased the intracellular Rho-123 concentration. Cellular GSH concentration in K562/ A02 cell line was higher than thatin K562 cell line. FAT decreased cellular GSH concentration in K562/A02 cell line. One of the mechanisms of multidrug resistance reversal by FAT was decrease of cellular GSH concentration in K562/A02 cell line.

Identification of BSNK-5 and Optimization of the Fermentation Conditions

Li Shuying, Nie Ying, Du Huan, Zhao Zhonglin, Yuan Chao, Li Yan, Song Xiaoyan, Tang Xuanming

2013, 0(2): 184-189.

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Nattokinase production bacteria BSNK-5 was preliminarily identified. The effects of liquid fermentation on nattokinase activity produced by BSNK-5 were studied in order to improve production yield. BSNK-5 is a Bacillus subtilis. Different carbon sources, nitrogen sources, pH, inoculate size；temperature, and time were optimized. Two optimum culture conditions were obtained. The nattokinase activity was 1 160 U/mL under the optimum culture conditions, which was 283% times of the former culture conditions.

A Modified Method for Quantification of L-lysine

Jiang Anna, Cao Mingfeng, Li Qinggang, Zheng Ping, Yang Hongjiang, Sun Jibin

2013, 0(2): 190-194.

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A modified method was established to determine lysine concentration using ninhydrin-ferric reagents. Unlike the traditional method which measures the total amino acids, our modification showed high specificity and favorable linearity to lysine（0-200 mmol/L）. In our protocol the characteristic absorption at 480 nm was measured after the reaction mixture was boiled for 35 minutes in disodium hydrogen phosphate-citrate buffer at pH2.2. The method not only successfully excludes the interference of impurities including proline, ornithine, glycine, arginine and histidine, but offers the accurate and reliable determination of the lysine in the fermentation broth directly.

Investigation of Microbial Species in Domestic Refrigerator

Yang Songzhen, Feng Guangda, Yao Qing, Wang Yonghong, Yao Yuxin, Zhu Honghui

2013, 0(2): 195-200.

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It was to understand microbial species and their pathogenic risk in domestic refrigerator. Five domestic refrigerators were randomly selected, from different positions of which bacteria and fungi were isolated using media of nutrition agar（NA）, potato dextrose agar （PDA）and Gause’s No.1. The isolates were identified by 16S rRNA gene, 26S rRNA gene or ITS sequences analysis and morphological characteristics observation using the scanning electron microscopy（SEM）. Total of 84 bacterial and 74 fungal strains were isolated. Most of bacteria belonged to the genera of Bacillus, Staphylococcus, Kocuria and Pseudomonas. Fungal strains mainly belonged to the genera of Cladosporium, Aspergillus and Penicillium. Pathogenic microbes in domestic refrigerators were mainly composed of the strains in Staphylococcus, while others distributed randomly. Variety of bacteria and fungi existed in domestic refrigerators. Domestic refrigerators were mainly contaminated by Staphylococcus. The safe problem of domestic refrigerator should be taken seriously enough.

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