Human cytomegalovirus (HCMV) is extremely species specific and does not replicate in experimental animal tissues.To overcome the problem and establish suitable animal models for studying antiviral strategies, the expression of HCMV UL49 gene was explored in mice. UL49-GFP gene was subcloned into the adenovirus shuttle plasmid pDC316, the products(pDC316-UL49-GFP)were co-transfected with helper plasmid-pBHGloxΔE1,3Cre into HEK293 cell lines by liposome reagent, recombinant adenovirus(Ad-UL49-GFP) was generated and confirmed by PCR and Western blot. Ad-UL49-GFP was propagated in 293 cells and purified. The titer of viral stocks was determined by end-point dilution assay. The purified adenoviruses were delivered into mice via the tail vein injection. Fluorescence quantitative PCR and Western blot experiments were used to examine the tissue distribution and duration of UL49 gene expression. The results showed that the recombinant adenovirus were present in vivo. The expression level in tissues arranged in descending order was liver, spleen, kidney, heart and lung . 3 days after injection, the liver, spleen, kidney, heart and lung expressed protein UL49 in high lever and then declined gradually. 14 days after injection, UL49 protein expression was disappear in some organs except liver and spleen. In conclusion, transgene animal model carrying UL49 gene was successfully established. Therefore, the system may be suitable for selecting anti-HCMV drugs targeting UL49 gene.
AIM: To construct a recombinant adenovirus carrying Fhit gene, a tumor suppressor in many types of cancer, and to observe its biological function on the proliferation of colon cancer cells. METHODS: Fhit gene was cloned from the fetal liver cDNA library using the PCR method. The PCR product was inserted into the T vector to construct the plasmid pMD18T-Fhit. The Fhit fragment from the pMD18T-Fhit was inserted into the vector ptrack-CMV to construct a shuttle plasmid ptrack-CMV-Fhit. After PmeI digested and linearized process, ptrack-CMV-Fhit was co-transformed into Escherichia coli strain BJ5183 together with the adenovirus backbone vector pAdEasy-1 to generate a recombinant adenovirus plasmid by homologous recombination. The adenovirus plasmid was identified by PacI digestion and transfected into 293A cells to package a recombinant adenovirus which expressed the Fhit protein. Furthermore, the adenovirus rAd-Fhit was infected into colon cancer cells，and the expression of the ectogenic protein was detected by Western blotting. Finally, the proliferation of colon cancer cells was observed in adenovirus-infected cells by the MTT assay. RESULTS: We constructed the recombinant adenovirus encoding Fhit gene and expressed it in colon cancer cells successfully. We detected that the proliferation of colon cancer cells was inhibited obviously in rAd-Fhit-infected cells with comparison to the control groups. CONCLUSION: Fhit may function as a tumor suppressor in colon cancer cells, and the adenovirus-mediated Fhit can be a novel strategy for the colon cancer therapeutics.
Objective:To clone, express and characterize the HA（hemagglutinin） and NA（neuramidinase，NA） protein of avian influenza virus H5N1. Methods:On the basis of successful clone the full length HA and NA gene and sequence analysis of avian influenza virus H5N1,Ligated part of the gene into pET32a（＋）.full of the gene into pGEX4T-1.An expression vector pET32a（＋）/HA(49~1587bp),pET32a（＋）/NA(121~1141bp),pGEX4T-1/HA、pGEX4T-1/NA were constructed and expressed in E.coli BL21/rosetta induced by IPTG. recombinant protein was purified through Ni2＋ and GSTrap 4B affinity chromatography column. Western blotting and ELISA were used to determine the antigenic of the recombinant protein. Results:The recombinant capsid gene can be overexpressed in E.coli. SDSPAGE showed that the gene could express product as same as I might expect. The purity of the protein is greater than 90%.ELISA and Western blotting showed that the recombinant protein has good antigenic. Conclusion:The HA and NA protein of avian influenza virus H5N1 has been successful cloned and expressed, which could be useful for developing diagnose reagents or vaccine of H5N1.
Objective:To find out genes that are responsibe for the protection of astrocyte against the neurotoxicity of rotenone. Methods: To address this question, MN9D cells was used as a cell model of dopaminergic neurons and rotenone a toxin to initiate mitochondrial deficiency and wonder whether ACM could protect MN9D cells against rotenone toxicity and its possible protective mechanism. Cells treated with ACM or not were exposed to rotenone before assay of cell viability，signal transduction pathway for apoptosis and so on. Results: The results showed that rotenone decreased viability of MN9D cells in a dose-dependent manner and ACM treatment significantly attenuated rotenone toxicity at each concentration. Results of genechip found 104 differentially expressed genes, among which there were 62 up-regulated genes and 42 down-regulated ones after ACM treatment. These genes involved many functions such as signal transduction, transcription regulation, metabolism, cell cycle, etc. According to previous literature and the present study, 3 genes, namely Nrf3, GCL and NQO1 were likely to be associated with ACM protection and were confirmed by real-time PCR. Conclusions:The findings suggest that astrocytes can protect MN9D cells from oxidative stress and mitochondrial dysfunction caused by rotenone. Glutathione and neurotrophic factors may partially account for the protection. Some meaningful genes related to ACM protection were selected by genechip. Further research into these genes may provide clues to the detailed mechanism of ACM protection and shed light on accurate role of astrocytes in Parkinson’s disease.
In eukaryotic cells, heat shock factor 1 is the main specific transcription factor mediating the enhanced expression of heat shock proteins when cells experience stresses. It is kept in a latent state by inhibitory complexes under unstressed conditions. It is only transiently activated in response to diverse forms stresses. Dominant-positive heat shock factor 1 has been developed through deletion-mutation. It can activate the expression of endogenous heat shock proteins in the absence of stresses. Environmental neurotoxins play an important role in the pathogenesis of Parkinson’s disease. The neurotoxins induce cell death of dopamine neurons through oxidative damage. The results of western blot and dual-luciferase analysis indicate that the expression of HSP70 was greatly up-regulated in dopaminergic SH-SY5Y cells transfected with dominant-positive heat shock factor 1. To investigate the effect of dominant-positive heat shock factor 1 on 6-OHDA–induced apoptosis in dopaminergic SH-SY5Y cells, the release of lactate dehydrogenase was detected. The result argues that dominant-positive heat shock factor 1 significantly inhibits neurotoxin 6-OHDA–induced cell death in SH-SY5Y cells. The cyto-protective role may be attributed to HSP70 activated by dominant-positive heat shock factor 1. Taken together, it is possible that dominant-positive heat shock factor 1 can be used to prevent or cure Parkinson’s disease.
Angiopoietin-2(Ang-2) was a specific vasular growth stimulating factor. It could be highly expressed in malignant tumor cell only, and had a close affinity with the amount of tumor vessel. So it could be used to make prognosis as serum marker. Serum Ang-2 quantitative detection may be helpful for malignant tumor diagnosis and pathogenic mechanism research. A Ang-2 chemiluminesscence quantitative assay kit was developed based on the recombinant Ang-2 antigen expressed in E.coli and the Ang-2 monoclonal antibodies selected from Ang-2 antigen immunizing mice. Several characteristics of the Ang-2 quantitative assay kit were analyzed. The recombinant Ang-2 protein purity was ≥92%. The sensibility of the Ang-2 quantitative assay kit is 0.1ng/ml, the linearity range of it is 0.5-25ng/ml. No coss-reactions to lipoprotein, GLB, α1-acid glycoprotein, HGB, mucoprotein, hyaluronic acid or CEA were observed. The addition accuracy was between 97.6%-102.7%. CV (within batch) ≤4.1%, CV (batch to batch) ≤8.6%. After be kept in 37℃ for 4 days, the technical datas were almost same as be kept in 4℃. At last， the Ang-2 quantitative assay kit was use to detecte the Ang-2 in 307 gynecology malignant tumor patients’ serum and 1000 healthy people’s serum. 73.2% cervical carcinoma patients, 83.3% oophoroma patients, 76.7% endometrial carcinoma patients and 70.4% other gynecology malignant tumor patients can be detected. All the results showed that the Ang-2 quantitative assay kit we developed can be used for gynecology malignant tumor diagnosis and pathogenic mechanism research.
Code gene being cloned from the human genome by the primer5′extension, as the same time that code had been optimized partly to gene in the cloned procession. It was integrated into the gene secreted Pichia pastoris expression vector pPIC9K and translated yeast Pichia pastoris. PCR, SDS-PAGE and Western blotting confirmed hGM-CSF gene was integrated into the yeast genome and expression was successful. The expressed hGM-CSF was secreted into medium at the level of 389mg/L. Experiments in vivo showed that activity of hGM-CSF was normal. SDS-PAGE electrophoresis revealed that the hGM-CSF expression had occurred in glycosylation, N-glycosidase F to glycosylation also confirmed this point.
Objective: To investigate the immunogenicity of SARS-CoV N DNA vaccine and the feasibility of live-attenuated Salmonella typhimurium as the carrier to deliver the N DNA vaccine. Method: The recombinant attenuated salmonella strain CS022 harboring the pcDNA-N DNA vaccine was constructed. And mouse was immunized with the recombinant strain via intranasal and oral routes. Cellular and humoral immune responses were assessed by ELISA, lymphocyte proliferation assays, ELISPOT and FACS. Result: The oral immunization with the transformed salmonellae elicited strong immune responses mainly including high level of N-specific antibody, a dramatic activation of IFN-γ-secreting cells, a high level of lymphocyte proliferation, and a high level of activated CD8+ T cells. Conclusion: Live-attenuated Salmonella typhimurium could effectively deliver the SARS-CoV N DNA vaccine in vivo. These encouraging pre-clinical data provide a rational basis for undertaking a new immune style to investigate SARS vaccine.
The cytolytic effect of perforin is a mechanism of anti-virus, killing microbial-infected cells and tumor cells. Perforin is a very important non-specific immune factors in fish. In order to understand the function of perforin,the cDNA of grass carp perforin C-terminal peptide was amplified from grass carp liver and kidney cDNA library. It contains a protein kinase C conserved region 2(C2). The cDNA was connected with pET32a, and transformed to expression bacteria DE3. PFP-C was expressed by a prokaryotic expression system and then purified by affinity chromatography. It showed a significant haemolytic activity when tested with rabbit red cells, the optimal pH for haemolytic activity was 7.5, and its haemolytic function dependents on Ca2+ apparently.
According to the sequence of SmLEA gene, a DNA fragment of 1 038 bp upstream of the coding sequence of SmLEA gene was amplified by DNA walking with the genomic DNA of Salvia miltiorrhiz as the template. Sequence analysis showed that the fragment contained some putative ciselements relating to abiotic stress, ABA, seed specific expression.So the S.miltiorrhiz seedings was treated with 100μmol/L ABA，200mmol/L NaCl，4℃, and subjected to dehydration. The real-time PCR showed that expression levels of SmLEA was increased obviously, which was in accordance with the sequence analysis..
OsRacD belonging to rice Rho family of the small GTP binding proteins, is a pivotal gene involved in rice photoperiod fertility conversion of photoperiod sensitive genic male sterile rice, which influences rice fertility via controlling the pollen tube growth.T20N site-directed mutation was introduced into its highly conserved G1 motif by PCR-mediated method to mimic its GDP-binding state based on the sequences alignment and conserved domains analysis. The prokaryotic expression vector of OsRacD and T20N-OsRacD were constructed , and the His6 tag fused proteins were expressed and purified from E.coli. After identified by Western blot,the GTP hydrolysis activities were detected. The results showed that the GTPase activities of T20N-OsRacD were significantly reduced comparing with that of OsRacD, suggested that OsRacD and T20N-OsRacD have different biochemical characteristics.
In order to achieve the high production of cutinase by recombinant Bacillus subtilis WSHB06-07, the effect of various initial sucrose concentration on enzyme production was investigated in 3 L fermentor. It was found that sucrose concentration of 38 g/L is suitable for cell growth and cutinase production. Based on this sucrose concentration, fedbatch as well as constant-fed fermentation were investigated. The results showed that, using constant-fed fermentation from 16 h to 31 h and sucrose feed rate of 4 g/(L·h), the cutinase activity in the culture media reached 545.87U/ml, which was increased by 67.8% compared to that of batch fermentation. By this way, high cutinase productivity was gained and it is beneficial for industrial production.
In this paper, the way of “extracting－salting－chromatography” was used to purify the phycoerythrin and phycocyanin from Porphyra yezoensis in process scale-up. First, by comprehensive comparison of efficiency, the Sephadex G-25 was selected from four resins (Sephadex G-25、G-100、S-300 and CL-6B) as the best choice used in crude extract desalting of phycobiliprotein. Then the preparation process of phycobiliprotein was scaled-up with raw material(Porphyra yezoensis) increased from 1g to 20g,and finally to 400g. The results indicated that the yields of purified phycoerythrin and phycocyanin (absorption spectra purity above 3.2) increased during according to process scale-up, with 0.323% phycoerythrin and 0.148% phycocyanin obtained from 400g frozen Porphyra yezoensis blades respectively. It is no doubt that the process involved in the experiment is a potential way for large scale preparation of phycobiliproteins of high purity.
Numerous clinical trials (67) are using adeno-associated vectors (rAAV) as a gene delivery system so far. It becomes more and more alluring by its safety, efficiency, stability and long expression profiles. Recently, exciting outcomes have been obtained by rAAV clinical application in a retinal degeneration disease, Leber’s congenital amaurosis. Clinical trails provide better information for us to understand about the rAAV based gene drugs and bring more challenges as well, immunogenicity and drug safety, etc, in particular.
The cancer stem cell hypothesis posits that cancer stem cells, which are rare among the tumor cell population and share many common properties with normal stem cells, and have been positively identified and successfully isolated from some cancers, are responsible for tumorigenesis and implicated in multistage cancer progression, particularly with respect to metastasis. A better understanding may provide insights into the development of cancer stem celltargeting therapeutic strategies. The examines what is known regarding cancer stem cells’ concept, comparison with normal stem cell, potential origins, assays for isolation and their roles in metastasis.
As the major commercial source of natural rubber, Hevea brasiliensis attracts much attention. However, the heterozygous nature, long breeding cycle are strong limitations for conventional breeding. While genetic engineering, which can be used to widen the germplasm base and produce desirable agronomic traits quickly and efficiently, offers a viable alternative approach to complement traditional breeding. Comprehensive analysis indicates that in the past two decades, with calli derived from immature anther or integumental tissues of immature fruit as receptors, both biolistic and Agrobacterium-mediated transformation methods were employed for developping rubber genotypes with improved latex yield, tolerance to tapping panel dryness syndrome, producing high-value recombinant proteins, etc. Being recalcitrant to tissue culture, the transformation efficiency of Hevea is comparatively low, and the procedures are still needed to optimize. Finally, breeding objectives and strategies to improve transformation efficiency were also proposed in the review.
Strain breeding is very important in fermentation industry. Compared with other breeding methods, microbial protoplast fusion technology(mPFT) has advantages such as high recombination frequency, little limitation by genus or sex, and complete transfer of genetic material. Now mPFT has became one of the most staple methods in micro-organisms breeding. Here the procedures of mPFT in combination with the relational study in recent years was reviewed, including the factors that affected the formation, regeneration, fusion of protoplasts, and the ways of selecting fusants. Furthermore, the latest application of mPFT in micro-organisms breeding and the prospect of mPFT were discussed.
In recent years, more and more of the enzyme proteins have been carried out using recombinant microorganism bioreactor for large scale production. For reasons of improved the catalytic capability and environmental suitability, or enhanced expression level of the protein, a variety of genetic engineering technology according to protein molecule modification have been applied extensively.Major strategies and achievements of molecular modification for microbial enzyme, such as site-directed mutagenesis, error-prone PCR, DNA shuffling and optimum codon design were reviewed.
Microcapsule technology recently has been applicated and developed in pharmaceutical，chemical, food and so on. The achievments have been obtained on the novel technics of microcapsulation, analytical methods of microcapsule characteristics, and methods of characterization for the appearance and porous structures of microcapsules.The novel developments on the structure and property of microcapsules were reviewed.
Biomass resources are strategically vital for the sustainable development of a country. The United States has recently made a variety of plans or roadmaps and taken significant actions in order to promote the R&D of biomass resources.These plans and actions for the key issues in the development of biomass resources were analysed.
United States is a big biofuels market and a dominant country in the research and development of advanced biofuels. It has set up an ambitious development goal and strong supportive policy measures for advanced biofuels, and implemented the Biomass Program , currently focusing on the research, development and demonstration of cellulosic biofuels, and also embarking upon the research of third generation biofuels. US government attaches great importance to related planning, analyses and inter-agency cooperation. On the basis of the progresses on the frontiers of basic and applied research, it has been vigorously advancing the demonstration and commercialization of biofuels technology, in an effort to accelerate the transition to advanced biofuels.
