Engineered targeting fusion protein XE-TNFαm2 was prepared.In which,XE is the second excellular domain of CXCR4,a co-receptor of HIV/SIV. TNFαm2 is a mutation protein of TNFα.Its side effect was reduced 18 fold.It has been used in clinic for cancer therapy.In this study, Jurkat H2/9855, a standard cell line was donated toward by AIDS Reagent Program, NIH ,USA.In which, a deleted HIV mutant was inserted into chromosome of Jurkat cells to be 5’LTR-Tat-GFP-3’LTR. XE-TNFαm2 with different doses were added into 1x105/ml Jurkat H2/9855 cell suspensions respectively. After 24hrs,48 hrs and 72 hrs,the GFP in samples were determined by flow cytometry. The results showed that the increase of GFP expression level is along with the extended time of treatment with XE-TNFαm2,and it is depended on the dosage of XE-TNFαm2. This result indicated that XE-TNFαm2 is able to stimulate latent HIV to re-propagate in Jurkat H2/9855 cells.Thus,the host cell were became to be novel infected cells. On the other aspect, our previous study indicated that one of XE-TNFαm2 functions is to kill the cells infected by HIV/SIV. Accordingly, when the novel re-propagating HIVs start to pullulate, their gp120 must be appeared on the surface of host cells. Then,gp120 must be bound by XE in XE-TNFαm2, and latter’s killing signal must be transduced into cells.In that case, these host cells must be killed by XE-TNFαm2. Thus, the death of host cells leads to stop the propagation of un-maturated HIVs. Then, such kind of HIV without infection ability must be eliminated together with died host cells before that the re-propagated HIVs maturated to break host cells and to release out from cells.
The proteomics technique platform of Golgi apparatus isolated from gastric cancer cells was established successfully by using subcellular isolation with sucrose density gradient ultra-centrifugation, the separation of proteins of Golgi complex by 2-DE, the analysis of Electrophoresis patterns with ImageMaster 2D software, and the identification of proteins based on matrix-assisted laser desorption/ionization time of flight mass spectrometry. The results indicated that the well-resolved and reproducible 2-DE pattern of Golgi apparatus isolated from gastric cancer cells was obtained , and twelve proteins were identified, including proteins related to protein synthesis and folding, membrane fusion proteins, regulatory proteins, transportation proteins, proteins related to apoptosis, proteins related to cell proliferation. The first installment of the proteome technology of Golgi complex isolated from gastric cancer cells SGC7901 by 2-DE and MALDI-TOF MS will benefit for further investigation on the functions of Golgi complex.
Infections of rotaviruses are the primary cause of severe infantile gastroenteritis worldwide. VP4 is one of the major antigenic protein of the virus. VP4 has an essential role during the early interactions of the virus with the cell surface, including receptor binding and cell penetration. Cleavage of the VP4 protein prior to cell attachment by trypsin-like enzymes into two peptide fragments VP5* and VP8* that remain associated with the virus particle enhances its infectivity. In order to further investigate properties of this two proteins, open reading frame (ORF) sequences of VP5* and VP8* proteins derived from the VP4 of rotavirus strain TB-Chen were cloned and expressed and their immunological characteristics were analyzed. The results showed that VP5* and VP8* proteins could be highly expressed in E. coli cells; the expressed recombinant VP5* (rVP5*) and VP8* (rVP8*) could elicit specific antibodies in guinea pigs, these antibodies could recognize the recombinant VP4 derived from TB-Chen strain and the VP4 protein synthesized in Wa infected or SA11 infected MA104 cells. The results indicated that rVP5* and rVP8* have good immunogenicity and anti-rVP5* and anti-rVP8* antibodies have high specificity and cross reactivity.
Objective:To discuss the mechanism of p73 inducing tumor cells into apoptosis by constructing recombinant expression plasmid pBP73 and observing the expression and apoptosis-inducing effect of anti-p73 gene on colon cancer cell HepG2 Methods: Anti-p73 gene was cloned into the plasmid pBabe to form the recombinant plasmid pBP73, and then pBP73 was transformed into HepG2 cells and then agarose electrophoresis and flow cytometry were used to verify apoptosis of tumor cells. At last caspase-3 was detected by Western blotting. Results: Sequence analysis justified the recombination of plasmid pBP73. Virus could be collected from cultured supernatant of infected 293A cells with the titer of 5×107pfu. And 48h after infection, agarose electrophoresis of genomic DNA showed typical ladder-like pattern and flow cytometry analysis showed obvious apoptosis peaks with the highest percentage rate of apoptotic cells present and cellular caspase-3 expression could be seen in pBP73 transformed group. Conclusion: Anti-p73 gene could induce apoptosis in human colon cancer cells HepG2 in vivo by activating caspase-3 expression.
744 bp of N- terminal of mcm7 gene was amplified by PCR and was cloned into prokaryotic expressing vector pGEX-5x-3 to construct pGEX-5x-3/MCM7N. A batch of E.coli BL-21 were transformed with pGEX-5x-3 and pGEX-5x-3/MCM7N, respectively. GST protein and GST-MCM7N fusion protein were obtained from the recombinant E. coli BL-21 after IPTG induction and were purified with Glutathione Sepharose 4B. The fusion proteins with 54 kDa molecular weight were specifically recognized by both anti-GST antibody and anti-MCM7 antibody in Western blotting analyses. GST pull-down analysis showed that GST-MCM7N fusion proteins interacted directly with androgen receptor (AR) protein after GST-MCM7N incubated with prostate cancer LNCaP cell lysate and His-AR fusion protein, respectively. The result demonstrated that the N- terminal of MCM7 protein can interact directly with AR in vitro.
Mouse MDFIC (MyoD family inhibitor domain containing) protein is a newly found transcriptional factor, which shows high homology with HIC (Human I-mfa domain containing protein) and may play important roles in myogenic differentiation. In this article, cDNA encoding full-length of MDFIC was amplified by PCR and inserted it into prokaryotic expression vector pGEX-4T-3. The recombinant plasmid pGEX-4T-3- MDFIC was then transformed into E. coli BL21 and induced. The expression product was indentified by SDS-PAGE and Western blotting analysis. The fusion protein was purified by affinity chromatography with glutathione sepharose-4B column and then was used to immunize rabbits to generate polyclonal antibody. Indirect ELISA analysis shows that the titer of the antibody is higher than 1:640 000. Besides, Western blotting analysis indicates that the antibody can detect both exogenous and endogenous MDFIC protein. The polyclonal antibody of MDFIC provides an important tool to study the function of MDFIC and further to explore the mechanisms of myogenesis.
To express and purify the fusion protein of GST-tagged DNA fragmentation factor 45(DFF45) in prokaryocytes and prepare anti-DFF45 polyclonal antibody. The expression plasmid pGEX-5X-1/DFF45 was constructed and transformed into E.coli BL21, and the expression of GST-DFF45 protein was induced by IPTG. After purification, the fusion protein GST-DFF45 was used to immunize Oryctolagus cuniculus to obtain the antibody serum. Then the anti-DFF45 serum was purified by CNBr-activated Sepharose? 4B. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by Western blotting and immunofluorescence. Furthermore, we detected the expression of DFF45 protein in several human cell lines, and observed the cellular localization of DFF45 protein. The fusion protein was successfully expressed and purified, and the specific polyclonal antibody was prepared. The titer of the polyclonal antibody was up to 1:20 000 detected by ELISA, and the high specificity of the antibody was confirmed by Western blotting and immunofluorescence. Moreover, we found that DFF45 protein showed differential expression in several human cell lines. We also found that DFF45 protein was located in both cytoplasm and nucleus. The successful preparation of DFF45 polyclonal antibody and the confirmation of its different expression levels in human cells provides a tool for the further study of the relation between DFF45 and tumors.
A full-length VP73 gene sequence was synthesized, the its higher antigen area is predicted by using DNAStar software, and one pair of specific primers for this area are designed by using Primer Premier 5.0. Amplified 243bp fragment of the VP73 gene (vp73l) by PCR was digested and cloned into the prokaryotic expression vector pET-32a. The positive clone of inserting VP73L gene with correct reading frame was confirmed by sequencing and colony PCR. After induction by IPTG, the fusion protein was highly expressed in Escherichia coli BL21 (DE3) in soluble form. The recombinant protein was purified with His-Bind affinity chromatography. Western blotting analysis revealed that the recombinant protein could react with rabbit anti-African swine fever virus VP73 polyclonal antibody. Fusion expression of African swine fever virus VP73L is helpful to to prepare ASFVserological diagnostic reagent in next work.
The radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) and its mutant (mRsPHGPx) fusion proteins expressed in Escherichia coli BL21(DE3) with high efficiency were purified with Glutathione Sepharose 4B affinity chromatography and cleaved on-column with PreScission protease, and both the recombinant proteins without tags were obtained at a purity more than 90%. The enzymatic measurement confirmed that the activity of mRsPHGPx was far less than that of RsPHGPx. Effects of RsPHGPx and mRsPHGPx on restoration of B16 melanoma cells irradiated with ultroviolet were investigated. The results showed that the active RsPHGPx could significantly increase the cell viability and reduce the lipid peroxidation of cell membranes, while mRsPHGPx with very low activity had no effects, suggesting RsPHGPx could inhibit the ultroviolet-mediated injury in cell membranes mainly by rapidly scavenging various lipid peroxides.
Fh antagonist to Botrytis cinerea was preserved in laboratory. Through a series of morphological and biochemical characteristics, Fh belongs to Bacillus. In order to enhance the inhibitory effect of this strain, a 6.4 kb DNA fragment containing chitinase gene from pUC1965 was inserted into vector pBE2 to construct recombination plasmids called pBE2-chib . A new strain named Fh-chib was formed by transferring the recombination plasmids into Bacillus Fh. The PCR identification of chitinase gene and chitin plate culture confirmed that Chitinase gene was transferred successfully into the wild strain Fh. The original chitinase activity of Fh-chib using the vector pBE2 was 4.06U/ml. Fh-chib had an increased inhibitory effect against Botrytis cinerea on tomato by 23.2% through chitin plate culture.
Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) has been extensively used for many years to control H.armigera in China. HaSNPV chitinase is considered responsible for the liquefaction process of cadavers, and it may be a safer pesticide to control H.armigera.The HaSNPV chitinase gene was constructed into pET28a and transferred into E.coli strain Rosetta, which contain colons rarely used in E.coli. The chitinase was successfully expressed as (His) 6-chiA. It was expressed as inclusion body and was purified by Ni-NTA Column Affinity Chromatography under denaturing conditions. The inclusion body was then refolded by two different ways, both of which can produce activated enzyme.
An antagonistic endophytic bacterium, designated as strain SD-6，was isolated from healthy tomato leaves using dual culture method. Based on phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as Paenibacillus polymyxa. In vitro antifungal spectrum assay vindicate that it was active against a broad range of phytopathogens and inhibited the growth of thirteen fungal pathogens. In order to further evaluate the biocontrol potential against rice bakanae disease caused by Fusarium moniliforme, crude protein was extracted from fermentation broth using ammonium sulphate precipitation. The crude protein inhibited the mycelial growth and spore germenation of F.moniliforme, and caused distortion, tumescence of a portion of the germinated spores. This is the first report about isolating an antagonistic endophytic P.polymyxa from tomato leaves, which produced antifungal protein and possessed the inhibitory activity against a wide range of fungal pathogens. The protective properties of selected P.polymyxa strain make it as potential tool to reduce deleterious impact of fungal diseases. The strain SD-6 and associated proteins are potentially useful in controlling rice bakanae disease.
Hyaluronic acid (HA), which was mainly produced by fermentation in industry, had many significant applications in the fields of health products,cosmetics, and clinical treatments etc. The rapid and precise measurement of HA concentration in fermentation broth played an important role in microbial production of HA. Quantitative analysis of HA concentration in fermentation broth was modified on basis of original cetyltrimethylammonium bromide (CTAB) turbidity method. The water bath incubation of HA and acetate buffer was eliminated, the optimal CTAB buffer concentration was reduced to 2.5 g/L and the HA-CTAB complex reaction time was restricted at 5 minutes. The interference of fermentation broth to CTAB measurement was further observed. It is revealed that Mg2+ concentration exhibits a significant impact on CTAB results, but glucose, arabinose and D-glucuronic acid not. The ethanol precipitation coupled with the modified CTAB measurement was then proposed to implement the effective quantification of HA in fermentation broth.
By combining the extraction method of plasmid from E.coil and the method of plasmid from lactic acid bacteria reported before to improve the common methods used in plasmid extraction kit,a fast,safe and high efficient method for extraction of plasmid DNA from lactic acid bacteria was developed.The optimal concentration of lysozyme was proved to be 20mg/ml,the optimal processing time was 30min,and the usage toxic ethidium bromide was avoided during the whole process of extraction. Experimental results showed that,the developed method showed an increased extraction efficiency,as well as good reproducibility,and thus provided a good foundation for the following molecular improvement of lactic acid bacteria.
DNA microarray bears the advantages of fast, high-throughput, parallel-oriented, and has a broad application prospect in the detection of bacterial pathogens. One of the key step is how to select the appropriate target gene of bacteria. The bacterial ribosomal gene has been utilized as universal target gene; however, it has the shortage of non-specific hybridization. With more and more genome information and gene functions available, many more genes reveal better genus or species-specificity, such as virulence genes and resistance genes, which provide more specific targets for the detection of pathogenic bacteria using microarray. Therefore, microarray technology will be more sensitive, accurate, and will play a greater role in the study of pathogenic bacteria.
The application of phage display technology for the generation of monoclonal antibodies to haptens represents a very exciting alternative to traditional mammalian based hybridoma technology,which combines production ,selection and affinity maturation in numerous imaginative ways.However it is still has some technical and logistical problems,none the less,it holds great promise in hapten-specific antibodies.
Plant could produce different biological agents in tuppenny costing as a bioreactor.It was a atrractive expression system and has unique advantages for the production of recombinant protein for high level expression of forein protein.It reviews the characteristic and current progress of three different plant expression systems including stable transformation system,transient expression system,and chloroplast transformation,also,discussed the existing problems and perspectives of the plant bioreactor.
Scientists have been searching an effective tool for solving the safety issue of GM (genetically modified) crops for many years. The group led by Chinese American scientist Yi Li, a professor at University of Connecticut, made a great breakthrough in this field after 6 years’ intensive research: his group published the “Gene-deletor” technology in 2007. The technology was developed from two recombination systems Cre/LoxP and FLP/FRT, and FLP/Cre was driven by Organ/Tissue-specific promoter, all transgenic foreign genes will be thoroughly removed from pollen, fruit and seed after they have accomplished function. It could effectively prevent GM gene flow into non-biotech crops or weeds, and may help alleviate public concerns on ecological risks and food safety caused by GM plants. Here, we introduce the concept, principle of “gene-deletor” technology and discuss its applications in genetic engineering research.
With the development of genetic engineering, more and more transgenic plants and their products released to people's lives and greatly changed our lives. The controversy over potential ecological risks and the impact of transgenic plants on human health are becoming increasingly acute. Therefore, the techniques for detecting transgenic plants must be established and perfected.The advances in detection techniques of transgenic plants were summarized. The progresses of PCR, ELISA as well as gene chip in transgenic plants detection were emphasized,and their characteristics were compared. Furthermore, detection on specific metabolites in transgenic plants was also introduced. Based on detection of the differential protein, one project of two-dimensional electrophoresis technology for traceability of genetically modified organisms and products was put forward finally.
S-nitrosylation, the covalent attachment of an Nitric oxide (NO) moiety to Cys residues of proteins, resulting in the formation of S-nitrosothiols(SNO), is a prevalent posttranslational protein modification involved in redox-based cellular signaling. S-nitrosylation has been shown to regulate the function of many proteins which are involved in a wide array of cellular activities. The growing body of evidence now suggested that S-nitrosylation may also have a centrol function in plant disease resistance.The basic concepts of S-nitrosylation, the detection methods, functional studies and the recent progress of S-nitrosylation in plant disease resistance were summarized.
As a good biorenewable energy, microalgae biodiesel may play an strategic role in resolving the energy crisis and global warming.The development of microalgae biodiesel was summarised from the following aspects：the technology process for producing microalgae biodiesel, the oilrich microalgae species,the key bottle-neck of microalgae biodiesel（inconsistency between growth rate and oil content）, the methods promoting oilaccumulation in microalgae, the large-scale culture(mass culture), the harvest of microalgae and the acquisition methods of microalgae biodiesel, etc. The future core research direction and prospect of microalgae biodiesel are also mentioned.
Xylan is the major component of hemicellulose, which is the second largest recyclable natural resource after cellulose. And xylanase is a very important enzyme system which can break down xylan into xylooligosaccharides and xylose, thus it has a high potential for use in feed, paper making, food industry and biotransformation. There are many researches on microbial express xylanase, that involved bacteria and fungi.The process of ferment, yield and characteristic of xylanases were reviewed. The prospect for development of xylanase production was also discussed here.
Glyphosate-tolerant crop currently takes the biggest share of GM cultivation area all around the world. Thus, the cloning, expression and function of EPSPS become main focuses of modern bio-molecular breeding, and putting the function genes and transformation techniques into their own exclusive property rights by IP protection, such as patent, becomes the general developmental strategy of developed countries and bio-technology companies. The current situation of IP protection, core patent distribution and industrialization of EPSPS gene throughout the world were analyzed by retrieving data of global EPSPS gene patents and transformation events. The result shows that applications of EPSPS patents soared up in recent years and the applicants were mainly from the US, France and China etc., but most of the key technologies and industrialization were owned by international companies like Monsanto, Bayer, Pioneer Hi-bred, and Syngenta, and the initiative of certain industrial development was controlled consequently.
