Table of Content

29 March 2014, Volume 30 Issue 3

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Papers

Advances in Molecular Breeding of Energy Plant Jatropha curcas

Wang Qiong, Liu Bobin, Kong Qianqian, Chen Jienan

2014, 30(3):  1-8. 

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Jatropha curcas as an important energy plant, has draw growing focus in conventional breeding, but it is still at the starting stage in molecular breeding. Advances in the research of Jatropha curcas gene cloning and tissue culture regeneration system were reviewed. Futhermore, the methods including Agrobacterium mediated genetic transformation, gene gun mediated transformation and nanocarriers transformation were summarized as keys. Meanwhile, the existing problems and prospects of the application of molecular breeding in Jatropha curcas were discussed.

Progress in Structure Function and Antifungal Mechanism of Plant Defensins

Zhu Licheng, Luo Hui, Ren Han

2014, 30(3):  9-14. 

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Plant defensins, a kind of cationic peptides with molecular weight ~5 kD, are the key components of plant innate immune system and play an important role in numerous plant physiological and biochemical activities. The three dimensional structure of plant defensins is so-called the Cys stabilized αβ motif(CSαβ)which three anti-parallel β-sheets and one α-helix that is stabilized by four disulfide bridges formed by eight conserved Cys residues. Plant defensins can inhibit a broad range of fungi and bacteria growth. The investigations that plant defensins were used as enzyme inhibitors, cancer cell proliferation inhibitors and ion channel blockers were done in a number of relevant literatures. The antifungal mechanism of plant defensins is more complex, but it should be first binding the complex lipids those from the outside of fungal plasma membrane, and cause a series of physiological and biochemical reaction including permeabilization of the fungal plasma membrane, increased intracellular ROS level, and fungal cell apoptosis etc. But it is not clear that plant defensin enter the fungal cell or not and its intracellular targets. The progress of the structure, function and antifungal mechanism of plant defensins in recent years were summarized.

Research of Hyaluronidases and Future Development

Su Kang, Ji Aiguo

2014, 30(3):  15-21. 

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Hyaluronidase is a kind of glycosidase which mainly degrade hyaluronic acid. Research on hyaluronidase- a negeleted enzyme for a long time increased in recent years. we mainly reviewed the current research on the several resources of hyaluronidase, including human hyaluronidase, bovine testicular hyaluronidase, animal venom hyaluronidase and bacterical hyaluronidase. Moreover, the enzyme activity assays and clinical application were reviewed. Finally the future research on hyaluronidase was prospected.

Casein Kinase 1α in Cell Signalling

Han Guocan, Jiang Shaojie, Zou Feiyan

2014, 30(3):  22-29. 

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Casein kinase 1α(CK1α)is a member of CK1 family(CK1α, β, γ1, γ2, γ3, δ, ε), which expressed in many eukaryotes ranging from yeast to humans, and the sequence is highly conserved. Mammalian CK1α involved in diverse cellular processes including membrane trafficking, cell cycle progression, chromosome segregation, apoptosis and cellular differentiation etc. In addition, CK1α is needed in the process of Wnt/β-Cat, Hh and NF-κB signalling pathway. The detail functions of CK1α in cell signalling, and provide reference for CK1α research was summarized.

Progress on the Secondary Metabolites and Applications of Streptomyces

Dai Fangping, Li Shiweng

2014, 30(3):  30-35. 

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Streptomyces widely distributed in the soil as a common microorganisms, researcher had focused much attention on them since they could produce some antibiotics applied in medical and other natural substances, so it has tremendous commercial and medical exploitation value. The research progresses on the kinds of secondary metabolites of Streptomyces isolated from terrestrial and marine habitats as well as extreme environment were reviewed. Meanwhile, The potential applications of the novel metabolites produced by Streptomyces in agriculture, medicine, environmental remediation and other fields in recent years were summarized.

Research Advance of the Construction of Gene Engineering Strains for Glucosamine

Liu Dianlei, Li Piwu, Li Ruirui, Wang Sheng, Wang Ruiming

2014, 30(3):  36-41. 

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Glucosamine(GlcN), also called amino sugar, was a compound derived from the substitution of a hydroxyl group of glucose molecule with an amino group. GlcN found a wide-range of applications in health food and pharmaceutical industries. Glucosamine was currently produced by extraction and acid hydrolysis of chitin from shellfish waste, which was limited by the amount of raw material available and existed great risks of environment pollution. However, recombinant strain of gene engineering to overexpress the glucosamine could avoid those problems. the gene engineering strain, its metabolic pathway and fermentation of glucosamine were introduced. It is a great significance to ferment glucosamine and N-acetyglucosamine.

Progress of Pichia pastorisEngineering Bacteria on High-Density Fermentation

Min Zhaosheng, Guo Huiming, Yan Xu, Hong Housheng

2014, 30(3):  42-49. 

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High-density fermentation has been a key technique tache to improve the expression level of the protein, and fermentation process is an important factor in high-density fermentation. The Pichia pastoris protein expression on its genetic characteristics, host strains, expression vectors and the expression of heterologous proteins in the areas of basic researches were reviewed. In addition, we briefly discussed high-density fermentation of Pichia pastoris in the selection of engineering bacteria, designation and optimization of culture medium, control of the key biochemical parameters of fermentation process, and methanol induction. In the end, the problems existing in industry high-density fermentation progress were put forward, and the development trend was prospected.

The Mechanisms of Immune Stress and the Impact on Animal Growth

Lin Binbin, Zhang Bin, Li Fengna, Li Yinghui, Fan Juexin

2014, 30(3):  50-54. 

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Immune stress not only affects the growth and performance of the animal, and may result in death, even vaccinated to prevent and control the disease on the body will produce some damage, then a serious impact is taken by immune stress on livestock production, bringing huge economic losses. The paper described the concept of immune stress, giving a summary of the animals and research expectations for the immune stress mechanism of action and providing reference materials for livestock production and scientific research, so that make certain animal health can be protected and maximize their performance, and will not result in waster feed, thus improving the breeding efficiency.

Research Progress on miR-155, EMT and Tumour Invasion/Metastasis

Wu Liting, Cui Lei, Wang Yanlin, Huang Liming

2014, 30(3):  55-59. 

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EMT(epithelial-mesenchymal transition)is one of the key steps for tumor cells to obtain their ability of invasion and metastasis. miR-155 is an important regulator for EMT and effects EMT by targeting the key proteins involved in this process and inhibiting their expression. The function of miR-155 is tissue-and cell-specific, which can promote or inhibit tumor invasion/metastasis in different tumor tissues and cell types depending on the biological characteristics of the targeted protein moleculars. It reviewed the relationship among miR-155, EMT and tumor invasion/metastasis.

Development of a Real-time Turbidimeter-based LAMP Method for Detection of NPTⅡ Gene in the Genetically Modified Plant

Kuang Xiaoshan, Hu Songnan, Wang Xiaoyu, Tang Shiming, Cheng Xiaowei, Feng Jiawang

2014, 30(3):  60-64. 

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According to NPTⅡ gene(HE582394.1), the common selective marker gene of genetically modified plant(GMP), six specific primers were designed. A loop-mediated isothermal amplification(LAMP)method for screening GMP was established. Real-time monitoring of the LAMP reaction was achieved by a turbidimeter. Specificity, sensitivity, stability and repeatability of this method were tested. The results showed that the method can specifically detect NPTⅡ gene, and the detection sensitivity of the method was 0.5%. With the genetically modified maize MON863(NPTⅡ positive)samples of 10%, 1%, 0.5%, 0%(W/W)as templates, stability and repeatability testing was conducted, and false negative rate was 0. The results showed that the LAMP method is suitable for specifically detecting NPTⅡ gene in GMP.

Molecular Cytogenetic Identification of Perennial Wheat Hybrid at the Fifth Generation

Jin Song Zhou Xuan Li Zhenghong Li Jilin Zhang Yanming

2014, 30(3):  65-72. 

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The morphology and molecular cytogenetic testing of 15 materials were conducted in F5 generation of perennial wheat hybrids obtained from octoploid Trititrigia and Thinopyrumintermedium hybridization. The results showed that most of the materials were contained in E and St chromosomes or chromosome fragments. Among them, eight intermediate types(Tritelytrigia type)had the parents’ traits, such as developed root system, lush plants, more tillers, and powerful stress resistance, and so on. But the number of chromosomes was remained instability, ranging from 42-56, and there were six materials with renewable trait. In addition, seven common wheat types that the number of chromosomes was between 41-43, they were no regeneration, but contained Thinopyrumintermediate chromosome or chromosome fragment. Those common wheat types had a large and multiflorous spike, disease resistance and other characteristics, which may be E or St chromosome substitution or translocation. These results indicated that the genes of perennial wheat that determined renewable, cold resistance and perennial characteristic were primarily in the part of E and St chromosomes.

Cloning and Expression Pattern Analysis of Actin Gene Fragment from Elytrigia elongata

Guo Qiang, Meng Lin, Mao Peichun, Tian Xiaoxia, Li shanshan, Zhang Lin

2014, 30(3):  73-78. 

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A pair of degenerate primers was designed based on the conserved sequences of the actin(ACT)genes from other plants. Total RNA was extracted from the roots of Elytrigia elongata to obtain an ACT gene fragment by reverse transcription polymerase chain reaction(RT-PCR). Results showed that the length of EeACT gene fragment was 598 bp encoding a 198 amino acids. The deduced amino acid sequence of EeACT shared over 90% amino acid homology compared with other plants ACT. No significant differences in expression level of EeACT were found between shoots and roots among low temperature, salt and drought treatments for 12 h, indicating that expression of EeACT is stable.

Cloning and Protein Sequence Analysis of CHS Gene Family in Fusarium oxysporumf.sp. cubense Race 4

Mao Chao, Yang Laying, Dai Qingdong, Huang Junsheng

2014, 30(3):  79-85. 

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In order to know the characteristics of chitin synthase genes in Fusarium oxysporum f. sp. cubenes race 4 and lay an information foundation for further study of their functions, the degenerate primers were designed according to the sequence of several filamentous fungi’s CHS genes. The FOC4 CHS genes were amplified by PCR and RT-PCR methods. Their cDNAs were sequenced and protein sequences were analyzed. The results showed eight CHS genes were identified in FOC4. Through phylogenetic analysis, the FOC4 CHS proteins can be classified into two families. They also can be classified into seven classes through characteristics of the amino acid sequences analysis. The FOC4 CHS amino acid sequences showed high identity with other Fusarium species by homology camparison analysis.

Study on Genetic Transformation of LfMADS1 Gene into Lilium ** formosanum' Raizen No.1'

Li Yunhua, Liu Qing, Liu Qinglin

2014, 30(3):  86-93. 

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In order to create pollen-free double flowers, the in vitro bulblet scales of Lilium × formosanum ‘Raizen No. 1’were taken as explants, the effects of Kan and Carb on regeneration from bulblet scales were studied at first. Then a orthogonal test were conducted to screening such factors as pre-culture time, bacterial concentration, infection time and co-culture time, and in this way the transformation system was optimized. Through this system, LfMADS1 gene was transferred into bulblet scales mediated by Agrobacterium, and the transgenic plants were molecularly identified. The results indicated that Kan 100 mg/L was optimal to screen adventitious buds of Kan-resistant；Carb 500 mg/L was able to control growth of Agrobacterium. There are some key points include pre-culture for 1d, immersion of explants into bacterial suspension(OD 600 0.8)for 10 min, and co-cultivate for 5 d in the optimized transformation system. Four transgenic lines were obtained through this system identified by PCR, in which the exogenous gene had successfully integrated into genome of one line confirmed by PCR-Southern. These transformed lilies can be a new germplasm for the breeding of pollen-free or double flower lilies.

Selection, Identification and Characteristics of a Cucumber Growth-Promotion Strain of Rhizobacteria

Ge Chunhui, Meng Ajing, Ma Yanru, Yang Xinhua, Wang Xinyong, Sun Jiusheng

2014, 30(3):  94-99. 

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Salkowski colorimetric was used to evaluate IAA metabolism of different strains, the use of plates and pot trials were conducted to detect that strains improve plant viability；strain was identified by the method of combination strain’ Physiological -biochemical character and 16S rRNA sequence, and further the influence of different carbon sources and nitrogen source in medium on metabolism ability of IAA was explored. Results showed that eight strains that could excrete IAA were isolated from cucumber roots. Strain SGM7 was the highest in IAA excreting capability(23. 59 mg/L in 48 h of cultivation). Based on its morphological feature, Some of its physiological and biochemical characteristics and its 16S rRNA sequence analysis, it was identified as Bacillus Pumilus. The optimum fermentation medium contained 1% of protein, 2% of maize powder, 0. 25% of wheat bran, 0. 05% of ammonium sulfate, and 0. 05% of potassium nitrate. Strain SGM7 had the highest in IAA excreting capability(35. 87 mg/L in 24 h of cultivation).

Identification and Antagonistic Activity of Endophytic Bacterial Strain NG14 Isolated from the Fruits of Paracel Islands Noni(Morinda citrifolia L.)

Liu Yang, Li Jingxia, Yao Su, Zhang Mingjuan, Chen Jianguo, Cheng Chi

2014, 30(3):  100-105. 

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Endophytes related with healthy and efficacy component production of plants. In this study，the bacterial strain NG14 isolated from Paracel Islands Noni(Morinda citrifolia L.)fruit. NG14 was identified as Paenibacillus polymyxa by polyphasic taxonomy basing on morphologicial and physiological methods，and 16S rRNA gene phylogenetic analysis. Through the antagonism test，results showed that the strain NG14 had a comprehensive and fine antagonistic activity against pathogens chosen for this research.

Isolation and Identification of Cellulose-Decomposing Microorganisms in Deep-litter System

Liao Qing, Jiang Zhepu, Xingying, Wei Guangpo, Huang Dongliang, Li Yangrui

2014, 30(3):  106-110. 

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43 cellulose-decomposing microorganisms were isolated from deep-litter systems. Five of them, with bigger transparent circles and disintegration effect of filter paper piece were screened using Congo red differential medium and filter paper method. Then 2 strains with higher capacity of decomposing cellulose were obtained by determinations of CMC enzyme activity, FPA enzyme activity and natural cellulase activity, and named as F5 and F21, respectively. The 2 strains were identifined as Bacillus subtilis and Streptomyces sp. basing on 16S rDNA phylogenetic analysis.

Screening of Solid Media and Optimization of the Fermentation Conditions for Trichoderma reesei FS10- C

Luo Yang, Teng Ying, Liu Fang, Ma Wenting, Li Zhengao

2014, 30(3):  111-116. 

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In order to provide a foundation for the application of the Trichoderma reesei FS10-C on remediation of heavy metals-contaminated soils, the solid fermentation substrate and the fermentation conditions were studied. The fermentation substrate was screened through a single factor, then confirmed the appropriate fermentation conditions using a method of orthogonal combination and different factors. In addition, an enlarge experiment was also carried out in a 50 L solid fermentation tank. The result showed that the mixture of orange peel and wheat bran were deemed the best fermentation material for Trichodermaressei FS10-C. The optimal conditions in plate as following:the proportion of orange peel to bran and solid material to water were 1∶1 and 1∶1.2 respectively, inoculum 5%, the temperature of fermentation was 28℃. Under this optimal fermentation conditions, the spore number were up to 2.43×10 ¹⁰ CFU/g after 14 days, and the effect was satisfactory when it had been applied to solid fermentation tank, the spore number were 1.44×10¹⁰ CFU/g. Through the screening of soild media and optimization of the fermentation conditions, the cost of production was reduced, and FS10-C biomass through the optimization of fermentation conditions was increased. The solid fermentation of FS10-C had the potential of mass production.

Phenol Degradation by Brevibacillus borstelensis and Kinetic Analysis

Ge Qilong, Wang Guoying, Yue Xiuping

2014, 30(3):  117-122. 

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An effective phenol-degrading strain was isolated from activated sludge, and was identified as Brevibacillus borstelensis based on morphological, biochemical, physiological experiments and 16S rDNA gene sequence analysis. The strain could utilize phenol as sole carbon and energy source and the optimum degradation condition were temperature of 30℃, initial pH7.0 and shaking speed of 160 r/min. The biodegradation experiment indicated that the strain could completely degrade 1 600 mg/L phenol in 72 h. Also, the results showed that the higher the phenol concentration was, the stronger the substrate inhibition imposed. The process of its growth was also investigated using Haldane kinetic model. The parameters were:μmax(maximum specific growth rate)=0.334 h ^-1, Ks(half-satruration constant)=14.07 mg/L, Ki(inhibition constant)=196.89 mg/L. The phenol degradation kinetics of the strain followed a similar trend to that of its growth. Metabolic pathway research showed that the strain could be induced to synthesize extracellular catechol-1, 2-dioxygenase.

Effect of the lipA Inactivation on the Cell-bound Lipase Yield of Burkholderia sp. ZYB002

Shi Shaolei, Lin Hong, Shu Zhengyu, Liu Yanru, Li Xin, Wu Hailong, Huang Jianzhong

2014, 30(3):  123-129. 

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Burkholderia sp. ZYB002 strain can produce large amount of extracellular lipase and cell-bound lipase. The type of the cell-bound lipase from Burkholderia sp. ZYB002 was qualitatively analyzed, which will help to develop the whole cell lipase in the organic synthesis. Series of potential cell-bound lipase genes were presumed from the known genomic DNA information of Burkholderia cepacia J2315, which was highly homologous to that of Burkholderia sp. ZYB002. The lipA gene from Burkholderia sp. ZYB002 was selected and then inactivated by homologous recombination. The mutant with lipA inactivation was screened and the cell-bound lipase activity from Burkholderia sp. ZYB002-Δ lipA was tested. The cell-bound lipase activity from Burkholderia sp. ZYB002-Δ lipA decreased by 42%.

Isolation and Identification of Pathogenic Aeromonas veronii Biovar Sobria from Monopterus albus

Chen Honglian, Jiang He, Hu Wang, Ling Jun, Duan Guoqing

2014, 30(3):  130-136. 

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Large colonies with the same morphology and color were isolated from liver, spleen and kidney of diseased mud eel, Monopterus albus. 5 isolates randomly selected were proved to be the same bacteria by 16S rRNA sequence analysis. Further research of one strain named HS120920 among them was developed. According to morphology, physiological and biochemical characteristics, analysis of their 16S rRNA and gyrB sequences, the strain was identified as Aeromonas veronii biovar sobria, the LD50 was 5.79×10⁷ CFU/mL. The susceptibility test showed that the strain was highly sensitive to 17 drugs such as seven quinolones, five aminoglycosides, but showed low sensitivity or resistance to other 3 drugs.

Cloning and Expression Analysis of Na⁺-K⁺-ATPase α in Red Tilapia(Oreochromis sp.)at Different Water Salinities

Wang Zhongliang, Zhang Jiandong, Huang Jiansheng, Tang Baogui, Zhou Hui, Shi Gang, Pan Chuanhao, Wu Zaohe, Chen Gang

2014, 30(3):  137-145. 

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The full-length cDNA of red tilapia(Oreochromis sp.)Na ⁺-K ⁺-ATPase α was cloned using homological cloning and rapid amplification of cDNA ends(RACE)methods. Results showed that the full-length cDNA was 3 379 bp, containing a 5'untranslated region(5'-UTR)of 143 bp, a 3'-UTR of 164 bp and open reading frame of 3 072 bp bp encoding 1 023 amino acids with an estimated molecular weight of 112.5 kD and an estimated isoelectric point of 5.26. BLAST analysis revealed that the Na ⁺-K ⁺-ATPase α of red tilapia shared a high homology of 97%-99% with the corresponding proteins of other know species, respectively. Phylogenetic analysis showed that Na ⁺-K ⁺-ATPase α of red tilapia shared closest relationship with the corresponding proteins of Oreochromis mossambicus and Sarotherodon melanotheron, respectively. The expression pattern of Na ⁺-K ⁺-ATPase α at different water salinity was analyzed by fluorescent quantitative real-time PCR technology, and results showed that the expression of Na ⁺-K ⁺-ATPase α reached the highest level at 25, and fell to a relatively lower level at 32. In the same salinities, the expression of Na ⁺-K ⁺-ATPase α mRNA increased significantly(P<0.05)during the adaptation phase(12 h), and in regulatory phase(after 24 h)the amounts of Na ⁺-K ⁺-ATPase α mRNA fell to a relatively lower level, while the expression levels were still higher than that of fish in control group(P<0.05).

Detection of Quorum Sensing in Vibrio parahaemolyticus Isolated from Macrobrachium rosenbergii

Wang Ying, Zhu Suqin, Zhang Caili, Zeng Mingyong

2014, 30(3):  146-150. 

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Vibrio parahaemolyticus VIB461 and VIB800, which were isolated from Macrobrachium rosenbergii, were found to produce three kinds of quorum sensing signals using  three different quorum sensing reporter strains V.harveyi JMH612,   V.harveyi JMH597 and  V.harveyi JAF375, and LuxS gene of the two strains were also amplified by PCR. The results showed that both strains produced three kinds of singnal molecules:HAI-1, AI-2 and CAI-1. The level of three signal molecules all ascended with an increase in bacterial population density, reached high levels during the late exponential phase or early stationary phase, and decreased afterwards. The PCR amplification sequences both shared more than 95% similarity with the reported LuxS gene of V. Parahaemolyticus, which indicated that both strains harbored LuxS gene.

Purification Effect of Denitrifying Bacteria and Photosynthetic Bacteria on Aquaculture Seawater

Xing Guowei, Li Yanqin, Li Fengchao, Guan Yueqiang, Kang Xianjiang

2014, 0(3):  151-154. 

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To determine the optimum proportion(R/ H ratio)of two bacterial strains, the Rhodovulum sulfidophilum and the Halomonas alimentaria were immobilized with sodium alginate and added to purifying bio-ceramic units in different proportions. The purification effects was tested in the recirculation aquaculture seawater system by observing the removal rate of nitrate, nitrite and ammonia as the purification index. The results showed that a highest removal rate of nitrate and ammonia at a R/ H ratio of 75%, even in the water conditions with lower ammonia concentration. At the R/ H ratio, the removal rates of ammonia, nitrate and nitrite were 79.72%, 90.61% and 77.06%, respectively.

Prokaryotic Expression and Polyclonal Antibody Preparation of the Conserved Region of Yak Prdm9 Gene

Zeng Qin, Huang Lin, Lin Yaqiu, Jin Suyu, Zheng Yucai

2014, 0(3):  155-158. 

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The conserved region of Prdm9 cDNA was obtained from yak testis by RT-PCR and cloned into pMD19-T vector．Following sequence confirmation, the cDNA was ligated with pET-32a(+)vector and then transformed into E. coli BL21(DE3)to construct expression strains．The recombinant PRDM9 protein was efficiently expressed in the form of fusion protein through induction with IPTG．After purification by Ni-NTA, the recombinant protein was injected into Japan White rabbits, and polyclonal antibody against the conserved region of PRDM9 of yak was obtained and the antiserum was collected after the titter reach 1∶4, the antibody can be used in the future research．

Analysis of Structure and B Cell Epitope Predicition of Mycoplasmaovipneumoniae DnaK Protein

Wang Hui, Luo Chengbo, He Dao, Kong Lingping, Zhou Bijun, Wen Ming, Cheng Zhentao, Wang Kaigong

2014, 0(3):  159-164. 

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The study is based on the amino acid sequence of heat shock protein Hsp70(DnaK protein)of Mycoplasma ovipneumoniae(Mo)separatated in guizhou province, using bioinformatics methods—DNA Rtar software and online server bioinformatics analysis tools, through the Jameson-Wolf method, Kyte-Doolittle method, Emini method, Welling method and Karplus-Schulz method respectively forecast the parameters, such as antigenic index, hydrophilic, flexibility and surface polarity, and comprehensive analysis, forecasts the B-cell epitopes of the protein, at the same time protein secondary structure prediction and simulation of 3D structure. Results showed that Hsp70 protein of Mo has a low homology with other mycoplasma which can make the sheep disease, explain the protein has good specificity. Mo Hsp 70 protein is an good antigenicity, presents the rules of spatial structure, while C-Terminal(394-598 aa)is more stable and largest region, might be advantage B-cell epitope regions.

Complete Gene Sequencing and Genetic Analysis of PCV2 Isolates from Guizhou Province

Li Tao, Wang Kaigong, Cheng Zhentao, Zhou Bijun, Wen Ming , Cui Yalan

2014, 0(3):  165-170. 

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It was to get the molecular biology data of the PCV2 strains in Guizhou province, 3 isolations of PCV2(GZ-LZ1strain, GZ-KY1strain and GZ-PX1 strain)were cloned and sequenced. The sequences were then multiple-aligned with representative sequences published in GenBank, and phylogenetic analysis was performed, both using bioinfromatics software. Sequence analysis showed that complete gene of all the 3 PCV2 isolates were 1 767 bp, the accession numbers were, KC788503, KC788504and KC788505. In pairwise comparisons, they were closely relate to each other and to the strains of vaccine producted in China with high nucleotide sequence identity from 97.0% to 98.3% and 95.5%-98.2%, respectively. However, they were more divergent from those of other countries or orther regions of China with lower level of similarities from 77.5% to 99.9%. GZ-LZ1 and GZ-PX1 were closely relate to the the strainYM-SH of vaccine, the strain of GZ-KY1 Phylogenetic analysis revealed that all of the 3 isolates were in the branch of PCV2d, strains of was closely relate to the SC-10 strain, ZJ-38 strain and HLJ-10.

Expression and Antiviral Activity of Chicken Interferon Alpha in Bombyx mori

Li Tiantian, Yang Ling, Yi Yongzhu, Shen Guifang, Zhang Zhifang, Li Yinü

2014, 0(3):  171-176. 

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Chicken interferon alpha plays a significant role in infection prevention and treatment of chicken virus diseases. The purpose of this study is to acquire efficient expression and production of biologically active chicken interferon alpha by the baculovirus expression vector system. The full length of chicken interferon alpha(ChIFN-α)gene was optimized and synthesized. The optimized ChIFNα was cloned into the baculovirus transfer vector pVL1393. Thus, the recombinant plasmid pVL-ChIFNα was constructed. The pVL-ChIFNα was co-transfected with the BmBacmid DNA. The hemolymph of silkworm larvae was collected after infected with the recombinant virus. Western blot analysis showed that chicken interferon-alpha protein with the molecular weight of 19 kD was expressed. The activity of the expressed chicken interferon-alpha was detected by inhibiting cytopathic effect in the Vero-VSV*GFP test system. The antiviral activity was about 3. 2×10 ⁵ U/mL. The chicken interferon alpha protein was successfully expressed in the baculovirus expression vector system, which would be valuable in developing efficient and cost effective chicken interferon biological product.

Expression and Antibody Preparation of Glypican3

Cheng Hua, Fang Xiangdong, Wu Meng, Shi Lei, Cui Shuo

2014, 0(3):  177-181. 

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Human glypican3 protein is a potential cancer therapeutic targets and diagnostic markers. In order to obtain anti-GPC3 monoclonal antibody, GPC3 gene was cloned into an expression vector pGEX-2T, which was transformed into E. coli BL21(DE3). Induced by IPTG, a molecular weight of 58 kD protein was obtained. After immunizing BALB/c mice, cell fusion and screening, one monoclonal antibody was generated. Western blot analysis revealed that the antibody has a strong specificity and high titer can be used for GPC3 protein function and applied research.

Expression and Purification of the Diphtheria Toxin Variant CRM197 in Corynebacterium diphtheriae

Zhu Tao, Deng Ligong, Duan Lei, Chang Yunsong, Shao Zhongqi, Mao Huihua, Yu Xuefeng

2014, 0(3):  182-186. 

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The CRM197 protein is a variant of the diphtheria toxin(DTx, 58 kD)characterised by a single mutation(i.e. a glycine-glutamic acid substitution in position 52)that reduces its toxicity. The protein nonetheless retains the same inflammatory and immunostimulant properties as thediphtheria toxin, and it is widely used as a safe carrier in conjugatedvaccines. A new and alternative procedure for the production of full-length CRM197 in Corynebacterium diphtheriae was proposed. The gene of CRM197 was synthesized and then expressed in Corynebacterium diphtheriae. Expression vector pCKM5.1 was first electrotransformed into Escherichia coli-s-17, reisolated, and subsequently transferred into the C. diphtheriae recipient strain by conjugation. The procedure involved the use of a modified non-deferrated growth medium that allowed for fast growth of bacteria and enhanced toxin production as a result of concomitant iron depletion. The CRM197 products are secreted into the culture medium as soluble body, accounting for around 70% of the total cellular protein, then recovered by filtering or precipitation, and subsequently purified using anion-exchange chromatographic method. The purity of the final preparation reached 95% and its biological properties is maintained. Consequently, the procedure could be suitable for a large-scale industrial production.

The Expression and Anti-apoptotic Function of Herpes Simplex Virus Type 2 Latency Associated Transcript-RL1

Gao Ruidi, Yang Huilan, Zhong Feifei, Lü Fangbiao

2014, 0(3):  187-192. 

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It was to study the expression of herpes simplex virus type 2 latency-associated transcript(LAT)RL1 and its anti-apoptosis function induced by actinomycin D in vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, expression was determined by green fluorescent protein and RT-PCR .The changes of Vero cells morphology induced by Actinomycin D were observed by Hochest33342 fluorescence staining, cells apoptosis rate was detected by flow cytometry . Membrane potential JC-1 fluorescence was observed, and Caspase 3 detected of apoptosis protein activity. Results showed that the green fluorescent protein has a high expressed efficiency in Vero cells, and target gene was detected by RT-PCR. Hochest33342 staining reavealed that Vero cells transfected with pEGFP-RL1 and induced apoptosis by actinomycin D had no changes in morphology. Flow cytometry assay showed that the cells apoptosis rate were no significant difference between pEGFP-RL1group and the normal group, but the cells apoptosis rate of pEGFP-RL1 was remarkable lower than the pEGFP-C2 group. Transfected with the recombinant plasmid pEGPF-RL1 cells by JC-1 staining, the proportion is much larger than red cells green cells transfected with empty vector pEGFP-C2. Caspase-3 results indicate that induction of apoptosis by transfected with empty vector pEGFP-C2 Vero cells, and the activity was significantly higher than those transfected with pEGFP-RL1 by actinomycin D-induced apoptosis of Vero cells and normal cells. HSV-2 LAT RL1 gene can effectively expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.