Table of Content

05 September 2013, Volume 0 Issue 9

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Advancement of the Research on Transgenic Sweet Potato (Ipomoea batatas Lam.)

Li Zhiliang, Wu Zhongyi, Wang Yuwen, Xing Haochun, Ye Jia, Zhang Xiuhai, Huang Conglin

2013, 0(9):  1-6. 

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Sweet potato is an important food，forage and industrial crop. It’s also a new high-energy source crop. The research on the establishment of genetic transformation systems of sweet potato is overviewed；meanwhile，the advancement of transgenic sweet potato，including enhancement of the herbicide，abiotic stress，virus，disease, and insect resistance and improvement of nutrient quality were summarized in this paper. Furthermore，the perspective for development of transgenic sweet potato were forecasted.

Progress in Construction of hpRNA Vector for Plant RNAi

Yan Pu Shen Wentao Li Xiaoying Zhou Peng

2013, 0(9):  7-12. 

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RNAi technology plays an important role in the identification of gene functions and the regulation of gene expression. hpRNA vectors have been used widely for RNAi research in plants. The progress in the methods for the construction of plant hpRNA vector was reviewed in this paper, and their advantages and disadvantages were analyzed. It can help to provide a reference to select the appropriate method for the construction of plant hpRNA vector.

Comparison of Equivalence for Micro-Produced Protein with Transgenic Protein

Cui Haoran Lang Zhihong Zhu Li Wang Hai Huang Dafang

2013, 0(9):  13-17. 

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The studies for risk assessment of transgenic crops require amounts of transgenic protein, but it is extremely difficult to prepare the required amount of purified protein from transgenic plants. Nevertheless, ample protein of high purity may be produced by over-expressing the protein in microbes. Using microbial proteins in a study for risk assessment can solve this problem, but the microbial proteins must be equivalent with transgenic plant proteins. In this article, we describe a typical set of methods used to compare the equivalence for micro-protein with transgenic protein, which for the purposes of providing reference for the risk assessment using micro-proteins instead of trans-proteins.

Progress of Streptomycin-Resistance of Pathogenic Bacteria of Plant

Li Jie, Zhong Jie, Huang Jun, Zhao Xiao, Zhu Hongjian

2013, 0(9):  18-26. 

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In order to understand the reasons for the emergence and transfer of streptomycin-resistance gene in pathogenic bacteria of plants and how to delay the development of streptomycin-resistance. The paper reviewed the streptomycin-resistance mechanisms in pathogenic bacteria of plants, distribution and transfer and the detection methods of resistance genes, conducted a preliminary study on the development of resistance causes and impact on the environment and human, and put forward some preliminary proposals and measures on how to delay the development of streptomycin-resistance.

Progress on the Studies and Application of Clostridium butyricum

Zhang Shanting Shi Yan Zhang Shuli Wang Haikuan

2013, 0(9):  27-33. 

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Clostridium butyricum, as a probiotic has been studied widely, is a spore-forming, gram-positive anaerobe that is found in intestines of healthy animals and humans. As a novel spore-forming probiotic, C. butyricum can withstand the low pH, heat and many antibiotic compared with non-spore forming probiotics, and exert different health effects in host, such as regulation of intestinal microbial homeostasis, stabilization of the gastrointestinal barrier function, enzymatic activity inducing absorption and nutrition, immunomodulatory effects, improvement of the animal production performance. So it will be used more widely. In this paper, we reviewed advances in the biological function, the fermentation technology and the application in clinic and feed additive of C. butyricum. Some advices were also put forward for further research of C. butyricum.Key words: Clostridium butyricum Probiotic Fermentation Clinic Feed additive

Progress of miRNA Regulation for Nrf2 and Related Antioxidant Genes

Qin Yu’e Liu Chaoqi

2013, 0(9):  34-37. 

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Nrf2 plays an important role in the anti-oxidation, maintaining the normal physiological function. MicroRNAs（miRNAs）are a kind of endogenous regulators of gene expression, which are eukaryotes non-coding RNA. This article reviewed the recent research progress of miRNAs focused on the antioxidant gene regulation.

TSC1/ TSC2 Heterodimer and Its Role in Cell Growth Regulation

Qin Yi Wang Yanfeng Wang Zhigang

2013, 0(9):  38-42. 

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Tsc1 and Tsc2 as a tumor suppressor gene are widely expressed in human body, which were located at autosome. Their expression products Hamartin/TSC1 and Tuberin/TSC2 directly interact to form heterodimer（TSC1/TSC2）and become active. Tuberous Sclerosis Complex（TSC）occurred because of the mutations of Tsc1 and Tsc2. It is a kind of autosomal dominant multi-system complications with a high penetrance. The incidence of TSC was 1/8 000 and 1/6 000 in adults and children, respectively. TSC1/TSC2 heterodimer can mediate the growth factors, insulin and various environmental stress signals to target the small G protein Rheb GAP（GTPase active protein, GAP）activity and inhibits the activity of mTORC1（mammalian target of rapamycin complex 1）. TSC1/TSC2 heterodimer regulates cell growth, migration, differentiation and plays key role in metabolism and embryo development. The genetic characterization of Tsc1 and Tsc2 and the function of their products in cell growth regulation were reviewed.

Progress of IGFs and Its Signaling Pathway

Li Cong, Ma Kaili, Liu Zengli, Wang Zhigang,

2013, 0(9):  43-46. 

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Insulin-like growth factors（IGFs）is a multifunctional growth factor family, and plays an important role in cell growth, differentiation and apoptosis. This article reviewed progress in the IGFs research, including component, molecular structure and function, signaling pathway and role in disease development in recent years. Therefore, this review provides an overview of achievements about IGFs and provides some useful data to improve research on IGFs.

Genome-wide DNA Methylation Profiles and Its Progresses in Genetics and Breeding of Animal

Zhao Jingxian Zhang Lupei Gao Huijiang Li Junya Xu Shangzhong Gao Xue

2013, 0(9):  47-53. 

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DNA methylation is one of the important epigenetic mechanisms in eukaryotes. In recent years, more attention was paid upon genome-wide DNA methylation in plant and animal genetics and breeding. The ability to access the methylation status for a large number of genes or the entire genome should greatly facilitate the understanding of the nature of gene regulation in cells, and epigenetic mechanism of interactions between cells and environment. This paper summarized some important aspects of DNA methylation profiles, DNA methylation profiling technologies and the progresses of DNA methylation profiles including animal. Finally, prospect of DNA methylation profiles is given a brief discussion.

Caspase-apoptosis Signal Transduction Pathway and Its Research Advance in Aquatic Invertebrates

He Simei Zhang Lili Wang Yilei Wang Guodong

2013, 0(9):  54-61. 

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Caspases exist in all eukaryotic cells and play important a role in cell apoptosis. They are widely involved in embryonic development, inflammation, organogenesis, metamorphosis, homeostasis and other physiological processes. Caspases transduce signals from outside to the cytoplasm or nucleus by a conserved caspase cascade, hydrolyzing intracellular substrate proteins or activating transcription factors to regulate physiological processes. Currently, most of the knowledge about the mechanisms of the caspase -apoptosis pathway focuses on the mammalian system. So far, basic knowledge of the caspase -apoptosis pathway in aquatic invertebrates is limited. In this article, combined data collected in our laboratory, we reviewed the research advance on caspase-apoptosis pathway in aquatic invertebrates to date.

DNA Molecule Construction by a Modified Overlap Extension PCR

Meng Xiangliang, Wang Ningxin, Niu Lihua, Li Peiguang, Li Yang, Hung Dawei

2013, 0(9):  62-67. 

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In order to assemble large fragments of DNA sequence efficiently, the study modified a advanced method, successive anneal overlap extension PCR, base on the traditional overlap extension PCR. The method of successive anneal overlap extension PCR lengthens the primer sequences corresponding to overlapping region between two fragments and uses successive annealing ranging from 3 to 6℃ in each single PCR cycles of the second PCR process. In this study, we assembled two large DNA fragments, ω3（2）and HCT, using this advanced method. A single band was obtained with the PCR product and the sequence was proved to be right after sequencing repectively, which would contribute to next experiments. This modified method has higher specificity and sensitivity, and is called successive anneal overlap extension PCR.

Cloning and Bioinformatical Analysis of Dihydroflavonol 4-reductase Gene（DFR）from Petunia hybrida with Different Color

Zhu Qilang, Li Xiaobo, Xiao Xiangwen, Li Xueyuan, Zheng Juyun, Ai Xiantao

2013, 0(9):  68-76. 

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In this study, through homologous gene cloning technology, three complete CDSs encoding dihydroflavonol 4-reductase（DFR）, a key enzyme in the pathway of anthocyanin biosynthesis, were cloned from the corolla with white, red and blue color of petunia hybrid planted in Xinjiang respectively, and named PhDFR1, PhDFR2 and PhDFR3. Homology alignment indicated that the nucleotide similarity of the three DFRs is 97.79 %、96.59% and 97.99% with another DFRA gene from Petunia hybrida（GenBank access number：X15537）. All these DFR CDSs encoded a polypeptide composed of 380 amino acid residues. The amino acid residues similarity of the three DFRs is 95.53 %、94.21% and 95.79% with DFRA protein（GenBank access number：CAA33544）. DFRs of the three Petunia hybrida strains with different flower color contain a highly conserved NADP（H）-binding site and substrate specificity site which belong to NADB-Rossmann superfamily. They are stable proteins without signal peptide and are hydrophilic proteins probably located in cytoplasm. All of them have two transmembrane domains. α-helix and β-sheet are primary secondary structural components of DFR. The β-α-β-α-β structures forming Rossmann folding are symmetrically distributed on the whole. The phylogenyetic analysis of DFR proteins from different species revealed that three DFRs have closer relationship with the DFR from Dianthus caryophyllus than from the other plant species. Key words: Petunia hybrida Dihydroflavonol 4-reductase Homologous gene cloning Bioinformatical analysis

Cloning and Expression Analysis of Small GTP-binding Protein Gene from Dunaliella salina（DsRab）Under Salt Stress

Yu Zhujun Chai Xiaojie Zhang Xiaolin Zhang Ting Xue Fei

2013, 0(9):  77-83. 

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Dunaliella salina small GTP-binding Protein Gene（GenBank Accession No.JN989548）was cloned by RT-PCR and RACE technology, named DsRab, the bioinformatics-analysis was performed, and the DsRab gene expression pattern was studied under 3 mol/L NaCl by Real-time quantitative RT-PCR method. The results showed that the full length of cDNA for DsRab Gene was 1 299 bp, which contains 78 bp 5'UTR, 609 bp 3'UTR and 612 bp ORF.The ORF codes a 203 amino acids protein with four GTP/GDP binding conserved domains, one effector domain, a cysteine residues at the COOH-terminus, and five common domain of Rab sub-family；Secondary structure prediction indicated that the α-helix, β-strands and random coil in it were 32.02% of, 23.65% and 44.33%. Protein homologue analysis showed that the DsRab shared high homology with Ypt/Rab from other species. The real-time quantitative PCR（RT-PCR）revealed that the expression of DsRab gene was increased by 4.9-fold under 3 mol/L NaCl comparising with that under normal level（including 1.5 mol/L NaCl）（P<0.01）. Key words: Dunaliella salina Small GTP-binding protein gene RACE Bioinformatics-analysis QRT-PCR

Genetic Diversity of Wild Populations of Conyza blinii Lévl. and Extraction of Total Flavonoids

Liu Shan, Sun Rong, Tang Zizhong, Gao Jinglei, Hu Hongli, Chen Hui

2013, 0(9):  84-88. 

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The genetic relationship of Conyza blinii Lévl. was analyzed in molecular level and the distribution characteristics of total flavonoid constituents. The RAPD method was applied to the study on Conyza blinii Lévl. from different germplasms by use of 10 random primers about 10 bp, and the parameters were calculated by NTSYSpc2.1 and the relationship was constructed based on UPGMA. Use UV spectrophotometry to test total flavonoids in each sample. 10 primers screened out from 100 primers were used for RAPD amplification. A total of 71 bands were generated, of which 68 bands were polymorphism bands. According to RAPD analysis, 18 germplasms of Conyza blinii Lévl. were classified into two categories. The genetic diversity of the 18 species in Conyza blinii Lévl. is high and the total flavinoids was related of geographic distribution.Key words: Conyza blinii Lévl. RAPD Cluster analysis Total flavonoids

Effects of Different Factors on Recovery of Strawberry Vitrification Shoots

Zhou Shuli, Tang Haoru, Chen Qing, Zhang Xiaonan, Yu Dingqun

2013, 0(9):  89-93. 

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The objective of this study is to explore the prevention and recovery measures of strawberry vitrification shoots. Using method of orthogonal design, different combinations of activated carbon, PVA, calcium ion（calcium chloride）were added to subculture medium of the vitrification shoots of ‘Toyonoka’ strawberry to study the effects of strawberry recovery rate. Meanwhile, the physiological and biochemical indexes among recovery plantlets, vitrification shoots and normal plantlets were compared. There were significant differences in recovery rate of vitrification shoots among 9 treatments. The optimum one was No.9（1g/L activated carbon + 2 g/L PVA + 166 mg/L calcium ion）and the recovery rate was 89.07%, followed by the 4^{th treatment（0.5 g/L activated carbon + 166 mg/L calcium ion）, 81.05%, and the 8th treatment（1 g/L activated carbon + 1 g/L polyvinyl alcohol）, 73.87%, respectively. Analysis of variance showed that calcium ion concentration was the most significant impact factor on strawberry vitrification recovery, the second was calcium ion, and polyvinyl alcohol affected least. Significant different physiological and biochemical indexes between the recovery plantlets and vitrification shoots were observed but not between recovery plantlets and normal plantlets. Combined with the recovery rate of vitrification shoots, physiological and biochemical indexes, and the reproduction coefficient, the best recommended recipe for vitrification shoots recovery was the 9th .}

Cloning and Sequence Analysis of cDNA of Cashmere Goat rsb66 Gene

Bu Xianglong, Hu Tianyuan, Luo Fenhua, Wang Ke, Wu Yingji

2013, 0(9):  94-98. 

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Spermatids are essential cells in testes of male animals during spermatogenesis. The rsb66 gene has been reported expressing in spermatids specifically. However, the DNA sequence of rsb66 gene in Cashmere goat have not been reported. In this study, we designed primers according to the reported rsb66 gene cDNA sequences from other species in the GenBank. The coding sequence was amplified by RT-PCR. The PCR fragment was inserted into the T-A cloning vector pMD19-T. The sequencing result showed that the coding sequence of Cashmere goat rsb66 gene possesses 97.0% homology compared with that of bos Taurus. Such highly homology between Cashmere goat and Bos taurus suggests that this gene is conserved in the evolution.

Single Nucleotide Polymorphism of MDFI Gene and Association with Body Size Traits in Haimen Goats

Wang Chunxia, Lin Jiajuan, Liu Xuanxuan, Li Yuan, Fang Xingtang

2013, 0(9):  99-104. 

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Genetic variations of MDFI gene were detected by PCR-SSCP and DNA sequencing in 116 Haimen goats at 1.5 years old. Association analyses were conducted to evaluate the effect of genotypes of polymorphic loci on body size traits of the breed. The results showed that two SNPs of Haimen goats in MDFI gene were found in extron 3 and extron 4, and caused one nonsense mutation and one missense mutation. The genotypes of P4 and P5 loci had significant associations with the body height, circumference of cannon bone and chest circumference of Haimen goat（P<0.05）. The associations were appropriate for the auxiliary mark of molecular breeding.

cDNA Cloning and Bioinformatic Analysis of Musca domesitca 14-3-3

Guo Guo Wu Qinyi Wu Jianwei Fu Ping Zhang Yong

2013, 0(9):  105-109. 

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The aim of this study is to clone the cDNA sequence of Musca domestica 14-3-3（MD14-3-3），analysis the gene and encoded protein sequnece of MD14-3-3 by the bioinformatics methods in the following aspects, such as general physical and chemical properties, hydrophobicity, signal peptide, secondary structure and subcellular localization. Results showed that the open reading frame of the MD14-3-3 was 771 bp that encoded a putative protein with 257 amino acids. The protein, with predicted molecular weight 29.35 kD and pI of 5.78, had one active site of family 14-3-3, without signal peptide. It was a hydrophilicity acidic protein which was mainly located in cell nucleus possible, containing many potential modified sites. The secondary structures were mainly composed of αcoiling and random coil. The gene coding for MD14-3-3 was amplified by polymerase chain reaction（PCR）, then the PCR product was transformed into the E.coli DH5α through being linked with pET 28a（+）vector. As demonstrated by PCR, double enzyme digestion and DNA sequencing, it was confirmed that the recombinant expression plasmid was construction succeed. Key words: Musca domestic 14-3-3 Gene cloning Bioinformatics

Gender Determination in Sheep Preimplantation Embryos by Amplifying Chromosome X and Y Sequence of Amelogenin（AML）Gene

Ma Youji, Li Haijing, Zhu Huabin, Li Fadi

2013, 0(9):  110-113. 

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Based on the multiple base deletions on the fifth intron of the sheep AMELX/Y（sAMELX/Y）gene，a pair of specific primers was designed to amplify the different sequences of AMEL gene on X and Y chromosome of sheep blood, fibroblasts and embryos by two-temperature -PCR in this test. The results showed that a 280 bp fragment from the female sheep DNA（XX）was amplified, and two fragments（280 bp and 235 bp respectively）were got from the male sheep DNA（XY）. The accuracy rate of sex identification of 50 samples（25 females and 25 males）was 100%.

Establishment and Optimization of RAPD-PCR Reaction System and Primers Screening from Perca fluviatilis

Dong Yanyan Hu Wenge Lu Lipeng Chen Dengwen Yang Di Wang Yanping Han Jing

2013, 0(9):  114-118. 

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In order to get the best reaction system and suitable primerof Perca fluviatilis, using Perca fluviatilis genomic DNA as templates，by L17（5^{4）orthogonal experiment table, four major factors（Taq enzyme, Mg2+, dNTP and primer）that greatly affected the results of RAPD were chosen and design to seek optimal reaction condition.For a 25 μL reaction system contain, the optimal compositions included 30 ng DNA template 1.5 μL, 10 μmol/L primer0.7 μL, 10×buffer2.5 μL, 5 μmol/L dNTPs 0.4 μL, 5U Taq polymerase 0.2 μL, 2.5 mmol/L MgCl2 3.0 μL；useing the optimal system, 10 random primers were selected from 50 primers, and determine the optimal annealing temperature.Key words: Perca fluviatilis RAPD-PCR Condition optimization Suitable primers Annealing temperature}

Gene Cloning and Transfer into Tomato of Brucella Outer Membrane Protein OMP31

Wang Jingyan, Wang Jianying, Yu Hua, Zhao Hongyu, Zhao Liang, Xin Cuihua, Wang Bin

2013, 0(9):  119-123. 

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Transgenic tomato carrying Brucella suis membrane protein OMP31 gene was constructed for the development of edible vaccine. In the experiment, the Omp31 gene was inserted into a plant expression vector pJG045 using ligation-independent cloning or LIC；the recombinant plasmid was transferred into Agrobacterium tumefaciens AGL-0 by triparental cross；then the transformed Agrobacterium was used to infect tomato cotyledons discs, and transgenic seedlings were identified by antibiotics resistance screening and PCR；5 transgenic tomato plants were obtained.

Expression，Purification and Bioactivity of Fusion Protein GGH Analogue with N-terminal Extension

Feng Junshang, Dou Wenfang, Zhang Xiaomei, Xu Hongyu, Shi Jinsong, Xu Zhenghong

2013, 0(9):  124-129. 

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Fusion protein GGH Analogue Pichia pastoris Expression Purification Bioactivity

Effect of Extracts from Artemisia rupestris on P450 CYP6B6 Expression Profile from Cotton Bollworm（Helicoverpa armigera）

He Hai, Liu Ning, Zhu Yan, Liu Xiaoning

2013, 0(9):  130-135. 

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In order to investigate the cytochrome P450 CYP6B6 in midgut induced by flavones and terpenes extracts from Artemisia rupestris, we detected the CYP6B6 expression in mRNA level by Real-time quantitative PCR（qPCR）, and at protein level by Western blot.qPCR results showed that the P450 CYP6B6 mRNA levels in the midgut of the larvae were induced by these two extracts. For the time effect, when the larvae were exposed to 20 mg/100 g flavones extracts and 2 mg/100 g terpenes extracts respectively for 12 h the relative expression level of P450 CYP6B6 mRNA in the midgut was over-expressed, while at 24 h the expression was inhibited. For the concentration effect, when the larvae were treated with these two exyracts for 24 h, the relative expression level of P450 CYP6B6 mRNA was over-expressed by 20 mg/100 g flavones and 1 mg/100 g terpenes extracts, respectively, while the expression was inhibited by 40 mg/100 g flavones and 2 mg/100 g terpenes extrcts in the diet, respectively.

Studies on Fermentation Conditions for r-GOD Production by Pichia pastoris GS115

Wang Shuaikun Hao Jieqing Wang Zhenwei Shi Hui Meng Yanfa

2013, 0(9):  136-141. 

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Using methanol to induce the expression of Aspergillus niger glucose oxidase gene cloned in Pichia pastoris GS115 mut^{+ was deeply investigated for establishing a high-performance but low-cost method of recombinant glucose oxidase production. The medium, the pH, the time point of induction, the temperature and the methanol concentration for induction were optimized and studied in detail by shake flask experiment. The results of optimization were verified by enlarged fermentation in 50 L fermentor. Results suggested that the best medium culturing the Pichia pastoris were YEPD medium, the optimum time for induction was the mid-anaphase of logarithmic phase, the optimum inducing concentration of methanol was 1%, and the optimum pH and temperature conditions were pH6-6.5 and 25℃. According to the optimum conditions, the highest biomass and glucose oxidase expression level were obtained in 50 L fermentor with batch culture for 14 h and induced by 1% methanol for 48 h. The final cell density（OD600­­­）was 44, 105 kU enzyme activity and 1 000 mg protein were obtained in 1 L fermentation broth.}

Separation and Screening of Endophytic Bacteria in Xisha Wild Noni Morinda citrifolia L.）Seed

Cheng Chi, Liu Yang, Cao Yanhua, Li Hui, Li Jinxia, Yao Su, Tan Wangqiao

2013, 0(9):  142-145. 

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This is the first report that the endophytic bacteria in Xisha wild Noni seed were isolated, screened and identified by the microbial culture-dependent method combing with morphological observation, 16S rDNA amplification and phylogenetic analysis, indicated that the total 20 strains from Noni seed were clustered into 3 genus belonging to Proteobacteria, Enterobacter sp., Burkholderia sp. and Pantoea sp..

Construction of a Versatile Escherichia coli-Bacillus subtilis Shuttle Vector and Its Application in Protease Gene Expression

Yang Jian, You Zhiqing , Mai Zhimao, Li Jie, Tian Xinpeng, Zhang Si

2013, 0(9):  146-150. 

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A versatile Escherichia coli-Bacillus subtilis shuttle vector pY980coli was constructed by genetic recombination. Resistance gene and replicon of pUC18, a cloning vector for Escherichia coli, were integtated into the Bacillus subtilis expression plasmid pWB980. Protease gene hspa from a marine bacterium was then inserted under P43 promoter of pY980coli, and the recombinant vector was transformed into Bacillus subtilis WB600. Protease activity was detected in the fermentation broth.

Exploring and Function Characteristics of Exo-1，4-β-D-glucanase CelB Gene of Bacillus licheniformis

Pang Hao, Chen Yan, Wu Qianqian, Liu Chunyu, Guo Yuan, Lin Lihua, Huang Ribo

2013, 0(9):  151-157. 

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Screening of DNA sequence similar with exo-1, 4-β-D-glucanase from the genome data of Bacillus licheniformis with bioinformatic techniques, one DNA fragment CelB showed high similarity was found. This DNA was cloned, expressed, and the result protein was purified. The CelB showed activity to cellulose substrate. Based on the conserved activity sites of glycoside hydrolase family 48 protein, amino acid sites of CelB were choosed for mutation and the mutant of CelB lost the activity, thus it showed that CelB contain the typical active sites of family 48. In this work, a exo-1, 4-β-D-glucanase gene was successfully found and cloned from B. licheniformis, it establishes the foundation for the further study of the cellulase system and their working mechanism of B. licheniformis.

Comparative of Avermectin Related Genes in Two Strains with Deferent Avermectin Production

Yang Han^{1，3 Zhong Juan, Sun Fenghui1，3 Shu Dan, Luo Di, Tan Hong}

2013, 0(9):  158-164. 

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In this research, we cloned and sequenced 2 genes[SAV-940（aveC）and SAV-4584（wblA）]which may closely relate to Avermectin production, in both Avermectin overproduction strain Streptomyces avermitilis AV-326 and the initial strain Streptomyces avermitilis AV-95 which the overproduction strain resulted from. In addition, Real-time quantitative PCR was applied to detect the relative expression levels of the 2 genes, at 4 time points（36 h, 48 h, 120 h and 240 h, respectively）in the fermentation process. Results showed that amino acids sequencees respectively coded by the 2 genes were exactly the same in both two strains；but expression levels of gene aveC were obviously higher in the high performance strain than in the initial one at all four tested time points, while those of gene wblA were remarkably higher in the initial strain during Avermectin synthesis period.

Expression and Immune Characteristic Analysis of Multi-epitope Antigen of Infectious Bursal Disease Virus

Hu Wei, Yu Nina, Zhang Haihong, Liu Wen

2013, 0(9):  165-169. 

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To express multi-epitope antigen of infectious bursal disease virus（IBDV）and analyze its immune characteristics, the multi-epitope recombinant gene resulting from VP2 and VP3 sequences was designed and amplified by PCR synthetic technology, and was cloned into pBVIL1 plasmid. The multi-epitope antigen was expressed by temperature induction at 42℃ with fusion and its immune reactivity was detected by IBDV standard antiserum, and the immunogenicity was evaluated using the titre of antiserum from mice. The results showed that multi-epitope fusion antigen was expressed with 31 kD molecular weight, its immune reactivity was confirmed using standard antisera through western blot assay and the antiserum titer was 1∶6 400 with ELISA method after mice immunization. It was concluded that the prokaryotic expression vector was constructed successfully and the multi-epitope antigen possessed the IBDV antigenic properties, which could be provided for pathogen diagnosis and genetic engineering vaccine research.

Transient Expression of Human EGFL6 in HEK293 Cells

Kang Zhongkui Zhang Jinxia Jiang Haowu Wang Hong Song Qifang Huang Jianfang Deng Ning

2013, 0(9):  170-176. 

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To construct eukaryotic expression vector of EGFL6 and transient transform HEK293T cell to investigate EGFL6 effects on tumor cells with EGFL6 conditioned medium. EGFL6 gene fragments and enhanced green fluorescence protein（EGFP）gene fragments were sub-cloned by PCR and combined to construct EGFP-EGFL6 fusion protein gene fragment by overlapping PCR. The sequencing identified EGFP gene fragments and EGFP-EGFL6 gene fragments were inserted to eukaryotic expression vector pAAV to construct the expression vectors. Purified by endotoxin-free kit, the expression vector plasmids were transiently transformed into HEK293T cells. The expression of EGFL6 fusion protein was identified by fluorescence localization and Westernblot analysis. The effects of EGFL6 on tumor cell proliferation of A375 and SKOV3 were assayed by CCK-8 reagent using EGFL6 expression conditioned medium. The restriction enzyme assay showed that the expression vectors of pAAV-EGFP and pAAV-EGFP-EGFL6 were constructed successfully. EGFP fluorescence localization and western blot assay results showed that EGFP-EGFL6 fusion protein was expressed successfully in HEK293T cells. The cell proliferation assay showed that EGFL6 enhanced tumor cell proliferation of A375 and SKOV3 and the cell proliferation rates reached about（37.79±14.05）% and（30.53±6.31）%. EGFL6 was expressed in HEK293T cells and the EGFL6 expression conditioned medium could promote tumor cell proliferation.