25 October 2006, Volume 26 Issue 10

    

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研究报告
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  Pilot-scale fermentation of rhNDPK-A producing E. coli in 50L culture

  China Biotechnology. 2006, 26(10): 1-6.

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  Production of recombinant human nucleoside diphosphate kinase A (rhNDPK-A) in pilot-scale. The primary seed culture was flask-shaked to 5.0~5.5 OD600, and then inoculated into 7L fermentor in ratio of 10%. Cultured to 9.6~10.5 OD600, the secondary seed culture was inoculated into 80L fermentor to carry out fed-batch culture. The obtained bacteria were homogenized under high-pressure and micro-filtered to remove the bacteria residue. Concentrated by superfilter, the crude rhNDPK-A was purified by ion-exchange and affinity chromatography. The results showed that the wet cell yield was 31.27g/L or 1560g/batch in 50L culture after 10h fermentation. The expression level of NDPK-A was 23.8%. In addition, the nutrition fed had significant effect on the density of culture. Compared with only carbon-material feeding, the density of culture was significantly enhanced in the condition of feeding carbon and nitrogen materials, but the expression level had not significant enhancement. In an optimum condition, the culture density was (38.30±0.28) OD600 U/mL; the wet cell yield was (2220.00±169.71)g/batch, i.e. (44.4±x3.4) g/L; the protein level was (22.00±0.42)%. Production of rhNDPK-A in pilot-scale provides materials for the basic research and new drug development of NDPK-A.
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  Cloning and functional analysis of a promoter of β-conglycinin subunit gene

  China Biotechnology. 2006, 26(10): 7-12.

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  A promoter fragment (7SαP ) of α subunit gene was isolated by PCR from genomic DNA in various soybean accessions, including cultivars Nannong99-10,N2899,Nannong 88-1, and wild soybeans Jiangpu -1 and ZYD4174.The sequences of 7SαP fragment from these five soybean accessions shared 99% homology, this indicated that the promoter regions of α subunit gene were conserved. Meanwhile , sequencing analysis showed that the 7SαP fragment contained several seed-specific motifs, such as RY motif, AGCCCA motif, ACGT motif and A/ T rich motif. The expression vector pBI121-7SαP was constructed with the 7SαP fragment (from Nannong99-10) and the GFP reporter gene for functional analysis. Arabidopsis plants were transformed by Agrobacterium mediated method. Southern blot results showed that the 7SαP had been integrated into the genome of Arabidopsis. Assay of GFP expression in the seeds of transgenic Arabidopsis was determined to identify the function of 7SαP promoter. The results showed that 7SαP was a seed-specific promoter.
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  Cloning and Expression of Vivrio Cholerae CTB Gene and the Recombinant CTB Protein Activation Assay

  China Biotechnology. 2006, 26(10): 13-17.

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  CTB protein possessed mucosal adjuvant immunoactivity. The CTB gene was amplified by PCR method from a strain V. cholerae. The nucleotide sequence of CTB gene was 375 bp and shared 96.0%~99.2% homology with other 6 CTB genes. The recombinant plasmid pTWIN1-CTB was transformed E. coli strain BL21(DE3) and was expressed with 0.8 mmol/L IPTG. The molecular weight of expression products was identical with expectative weight by SDS-PAGE electrophoresis. The CTB fusion proteins mainly assembled inclusion bodies and the outputs of proteins were approximately 20% of the total bacterial proteins. The CTB proteins possessed mucosal immunoactivity by GM1-ELISA assay.
技术与方法
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  Establishment of the Huntington's Disease in vitro Drug Screening Cell Model

  China Biotechnology. 2006, 26(10): 18-23.

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  To develop a Huntington's disease(HD) cell model in vitro to screen drugs targeting the aggregation of polyQ，different length of CAG repeat fragments were amplified by random primer PCR, identified by DNA sequencing and were fused to the N-terminus of CAT in the pCAR system respectively which had been constructed and identified before. Recombinant plasmids were transformed into and induced to express in the host E.coli. SDS-PAGE and chloramphenicol resistance test were done to determine the solubility of the polyQ and chloramphenicol resistance levels of the fusions. With different length of CAG repeat fragments cloned and expressed in the CAT-fusion protein reporting system, we found that when the length of the fragments increased over 40, their encoding polyQ expressed as insoluble protein and chloramphenicol resistance levels are lower, while under 40, the polyQ expressed as soluble ones and chloramphenicol resistance levels are higher. A in vitro HD model that could minimize the pathological process of the HD thus has been developed. With which by measure the recombinant bacteria's resistance to chloramphenicol we can determine the polyQ' solubility and folding state in vitro by quality and quantity. Thus this model can be used to screen drugs or bioactivity materials that can inhibit aggregation of the polyQ, which thereby shedding new light on the prevent, diagnosis and therapy of HD.
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  Study on A GFP Expression System for Detecting Estrogenic Compounds

  China Biotechnology. 2006, 26(10): 24-29.

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  Based on transcriptional regulation of estrogenic compounds, a recombinant 3×ERE EGFP-N1 reporter vector was constructed by inserting 3×ERE TATA sequence and transfected into MCF-7 breast cancer cells. Stable transfectants were selected and their GFP fluorescence intensity was measured using flow cytometry after adding E2 and other chemicals. 17β-estradiol(E2) induced a dose-dependent increase in GFP intensity in the cells, reaching maximum response at 1×10－10 mol/L and EC50 value was 1.5×10－11 mol/L, so done by phytoestrogen daidzein and polydatin , their EC50 values were 2.4×10－7 mol/L and 6.2×10－6 mol/L respectively, but grape seed polyphenols (GSP) had no significant estrogenic activity. Estrogen antagonist tamoxifen blocked GFP expression induced by E2 in a dose-dependent way and had a maximum inhibition response at 1×10－7 mol/L. The cell model can be used to detect ligands of estrogen receptor by testing GFP intensity induced by chemicals
研究简报
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  Prokaryotic expression and preparation of polyclonal antibody for IBDV non- structure protein VP5 gene

  China Biotechnology. 2006, 26(10): 30-34.

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  With a pair of primers designed according to the sequence of Infectious bursal disease virus(IBDV), the VP5 gene of IBDV was amplified from vvIBDV-Gx, IBDV-Gt, respectively. Then IBDV VP5 was cloned into expressing vector pET30a and pET28a. The recombinant plasmid was identified by restrictive digestion, PCR and sequence analysis, then named pET30a-GxVP5, pET28a-GtVP5, respectively. The recombinant plasmid was transformed into E.coli BL21(DE3) and induced by IPTG. SDS-PAGE results indicated that Gx-VP5, Gt-VP5 were about 24kDa, 23kDa, respectively. These recombinant fusion proteins mainly existed as inclusion bodies. High titer anti-VP5 serum was also prepared in BALB/c mice immunized with purified Gx-VP5 fusion protein inclusion. ELISA results showed that titer was above 1:25600 and Western blot analized that the expressed protein Gx-VP5 reacted with polyclonal antibody and 6×His monoclonal antibody. It explained that the expressed protein Gx-VP5 possess specific satisfactory immunological reaction.
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  Construction of TK Gene-deleted PRV SH Strain Containing a Single LoxP Site

  Pu-Yan Chen

  China Biotechnology. 2006, 26(10): 35-39.

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  Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and Economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. In this study, we constructed a recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID50 assay indicated that rPRV2 grows well on RK-13 cells. The LD50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.
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  Effect of Saponin upon the Activation of Different Chemokine Receptors

  China Biotechnology. 2006, 26(10): 40-43.

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  The effect of Saponin upon the activation of various chemokine receptors was studied by [35S]GTPγS binding assay on cell lines of CHO/CXCR1, CHO/CXCR2, CHO/CXCR3, CHO/CXCR4 and CHO/CCR5 which stably expressed chemokine receptor CXCR1, CXCR2, CXCR3, CXCR4, and CCR5, respectively. The results indicated that: ① On CHO/CXCR1 and CHO/CXCR4, the addition of 10 μg/ml of Saponin to the assay system could facilitate the activation of the receptor and the specific binding of [35S]GTPγS to receptor G-protein complexes, enlarge the stimulation ratio (CPMAgonist/CPMBasal) of [35S]GTPγS binding assay from 125% and 184% to 481% and 415%, respectively; ② On CHO/CCR5, 10 μg/ml of Saponin had no effect on the activation of the receptor, the specific binding of [35S]GTPγS to receptor G-protein and the stimulation ratio; ③ On CHO/CXCR2 and CHO/CXCR3, 10 μg/ml of Saponin inhibited the activation of the receptor and reduced the stimulation ratio from 171% and 168% to 130% and 114% respectively. It was revealed that Saponin had something to do with receptors but not G-protein. And it was concluded that the effect of Saponin upon the activation of various chemokine receptors were different.
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  Platforms of Oligonucleotide Array Comparative Genomic Hybridization and Its Application

  China Biotechnology. 2006, 26(10): 44-49.

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  The array CGH technique (Array Comparative Genome Hybridization) has been developed to detect chromosomal copy number changes on a genome-wide and/or high-resolution scale. It is used in human genetics and oncology. Generally PCR amplified bacterial artificial chromosomes (BACs) or cDNAs have been spotted as elements on the array. Recently Oligonucleotides as spotted elements on the arrays have been successfully explored. Compared with BAC array CGH, oligonucleotide array CGH(oaCGH) can save considerable time and efforts, be designed with more flexibility and provide much higher resolution with high sensitivity . We expect a gradual transition from BAC array CGH to oligonucleotide array CGH in the coming years. The combination of oaCGH and other high-through put analysis can lead to discovery of a host of novel oncogenes , tumor suppressors as well as tumor drug resistance genes. This review compares the different platforms of oaCGH and its development in recent years.
综述
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  Progress in Construction Methods of Diverse Mutated Gene Libraries for Protein

  China Biotechnology. 2006, 26(10): 50-56.

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  Directed evolution has been proven to be an effective method for proteins engineering. In the past few years, many new protocols for directed evolution were developed on the basis of oligonucleotide -directed random mutation, error-prone PCR and DNA shuffling. In this article, these new methods and their characteristics were summarized to provide a reference for choosing an approach to modify specific protein properties. The recent researches indicated that the combined directed evolution, coupling the directed evolution with rational design and computational design approaches respectively, is becoming increasingly important in protein engineering and is becoming the new direction for directed evolution.
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  Progress in the Study of Chemokine CXCL9/Mig

  China Biotechnology. 2006, 26(10): 57-61.

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  Chemokine CXCL9/mig (monokine induced by IFN-γ) belongs to the subfamily of chemotactic cytokines known as CXC-chemokines. In vivo CXCL9 is mainly induced by IFN-γ in macrophages and primary glial cells. In vitro, CXCL9 can be secreted by cells such as macrophages, microvascular endothelial cells and neutrophils, in response to the synergy of IFN-γ and TLR(toll-like receptor) ligands. CXCL9 is a chemoattractant for activated T lymphocytes, tumor-infiltrating T-lymphocytes, but not for neutrophils or monocytes. The receptor specific for CXCL9 is CXCR3, a G protein-coupled protein which has seven transmembrane domain. This review focuses on the structure and the chemical characterization of CXCL9, as well as its effects on autoimmune deseases, allograft rejection, cancer therapy, and so on.
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  Progress in vaccine development of hepatitis C virus infection

  China Biotechnology. 2006, 26(10): 62-68.

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  Hepatitis C virus (HCV) accounts for the majority of cases of transfusion acquired hepatitis and may cause chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine against HCV and treatment is expensive and not always effective. In this review, we describe the adaptive host immune response in viral clearance of HCV infection and summarize the recent progress in vaccine development of HCV infection, focusing on the fields of DNA vaccine candidates, recombinant viral vector vaccine candidates and combined (prime-boost) vaccine candidates.
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  Advances in Herpesviruses-encoded MicroRNAs

  China Biotechnology. 2006, 26(10): 69-74.

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  MicroRNAs (miRNAs) are small (approximately 22 nt) RNAs that exhibit a diversity in sequence，structure，abudance，and expression profile. Bioinformatic approaches and direct cloning methods have identified >3500 miRNAs from various species. MiRNAs play a pivotal role in the regulation of genes involved in diverse processes such as development，differentiation，and cellular growth control. Recently，many viral-encoded miRNAs have been discovered, most from the herpesviruses viral family. Virus-encoded miRNAs seem to evolve rapidly and regulate both the viral life cycle and the interaction between viruses and their hosts. The detailed study on the virus-encoded microRNAs and the role they play in the progress of viral infection，replication and expression will be beneficial to better understand viral molecular biology，and also provide new strategies for the prevention and treatment of virus.
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  The Study on Somatic Cell Cloned Cattle in China

  China Biotechnology. 2006, 26(10): 75-80.

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  Because of the important role of cloning technique in husbandry production, therapy of the disease, study of the basic theory and the conservation of animal resource, the efficiency of the cloning technique was becoming the present focus of the study. The factors related to the cloning technique were reviewed in the paper. The effect of different cell lines on the efficiency of somatic cell cloned cattle were different, the blastocyst rate, pregnant rate and calving rate of fetal cells and the cumulus cells were higher than ones of adult fibroblast. The study about refrigerant conservation of cloned embryos and oocyte indicated that the vitrification refrigeration method could be used to freeze the oocyte and the cloned cattle embryos. The new progress of the study on mammalian cell clone and the efficiency of somatic cell clone were also discussed in the review, and the urgent problem in somatic cell clone was presented in the article.
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  Advances in isobaric Tags for Relative and Absolute Quantitation Techniques Research

  China Biotechnology. 2006, 26(10): 81-85.

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  Quantitative proteomics is a novel subject of proteomics research. There are several new techniques employed for the protein quantitative study. Isobaric tags for relative and absolute quantitation （iTRAQ） technology for protein quantitation using mass spectrometry is a recent powerful means of determining relative and absolute protein levels in up to four samples simultaneously. The iTRAQ reagent produced high quality, reproducible result in enriched complexes, organelles, and whole cell lysates. This review, recommend the status of the recent promising techniques and on their possible future evolution.
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  Advances of Bio-hydrogen Production in Chlamydomonas reinhardtii

  China Biotechnology. 2006, 26(10): 86-90.

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  This article has a review on the research advances of hydrogen production in Chlamydomonas reinhardtii as a renewable energy source. The mechanism of hydrogen metabolism, culture conditions, characteristics of hydrogenases and approaches to improve hydrogen productivity in C. reinhardtii using molecular biological methods, bioinformatics and bioengineering technology were addressed. The approaches includes the modification of hydrogenase's tolerance to O2, heterologous expression of hydrogenase genes, screening of enzymes essentially for H2 production, using S-deprived medium, cell immobilization or 2-stage culture method to improve H2 production. The possibility, economics and trends of bio-hydrogen production using C. reinhardtii are also analyzed.
产业发展
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  Strategy analysis of Chinese antibody industrialization

  China Biotechnology. 2006, 26(10): 91-95.

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  Recombinant antibodies have become the major growth trends in biotech industry following their success on therapeutic application and good revenue. But the low level of mammalian expression and laggard fermentation process constrained the development of antibody industry in China. In this paper, we mainly reviewed the global advances of antibody industry, compared the respective advantage between dihydrofolate reductase and glutamine synthetase expression system, continuous perfusion and fed-batch processes. Finally, based on our knowledge and experience of antibody expression and fermentation, we believed that the suitable strategy of antibody industrialization, e.g. the fermentation model and scale, should depend on the comprehensive consideration of entrepreneur for the productivity, manufacturing capacity and market revenue. It may be a wise choice to use glutamine synthetase expression system and continuous perfusion process for the need of Chinese antibody industrialization.