legionella makes incursions into host cell by employed a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV), and translocates "effector" proteins into host cells for survival in the host cell. SidK is an effector from legionella, it directly bind the VatA subunit that is responsible for hydrolyzing ATP, leading to defects in LCV acidification and avoiding of lysosomal killing of LCV. The SidK protein was expressed in E.coli, the VatA protein was expressed in insect cell, and the interaction of SidK and VatA identified. Finally, the stable SidK-VatA complex was got. This is important for further study the structure of SidK-VatA complex.
Objective: To express Tat-MANF fusion protein and evaluate its biological activities. Methods: Tat-MANF fusion gene is constructed by PCR method to add the TAT sequence in the N-terminus and a His tag in the C-terminus of the MANF cDNA respectively. The Tat-MANF cDNA is inserted into the pET22b+ expression plasmid and prokaryotic expression system (BL21) is used to express the fusion protein. After purification, Tat-MANF protein is detected by SDS-PAGE and Western blot analyses. In order to evaluate biological activities of Tat-MANF, 6-Hydroxydopamine (6-OHDA) was used to induce apoptosis in human dopaminergic neuroblastoma cells. Tat-MANF is added to the cell-culture medium and the neuroprotection effect is detected by flow cytometry after 24h. B-endo3 cells were used as an in vitro blood-brain barrier model, in which B-endo3 cells are incubated with the FITC-labeled Tat-MANF and examined under microscopy after 4 hours. Results: Tat-MANF-His recombinant protein is successfully expressed in prokaryotic expression system. Western blot revealed that Tat-MANF-His recombinant protein can be detected by both anti-MANF and anti-His antibodies. Flow cytometry demonstrates that Tat-MANF significantly inhibit 6-OHDA-induced neuronal cell apoptosis similar to MANF. After 4 hours co-incubation with Tat-MANF-FITC, intracellular fluorescent signal is observed in the B-endo3 cells, indicating that Tat-MANF is able to cross cell membrane of brain vascular endothelial cells, a key component of BBB(blood-brain barrier). Conclusion: The recombinant Tat-MANF fusion protein is neuroprotective and may be able to cross blood-brain barrier, which may provide a novel therapy to Parkinson's Disease.
Stereoselectivity of 2-haloacid dehalogenase is one of the most important properties in biocatalysis, but the process of chiral recognition is not clear. Therefore resolving this problem becomes a research focus. The process of chiral recognition of dehDIV-R, a R-haloacid dehalogenase from Pseudomonas sp ZJU26 was studied. Firstly, by measuring the configuration of the product, the reaction catalyzed by dehDIV-R was defined as SN2 reaction. Through multiple sequence alignment and homology modeling of dehDIV-R, the key site of stereoselectivity of dehDIV-R was found to be Asn236. After molecular docking studies, the stereoselectivity of dehDIV-R was forecasted to be related to the steric hindrance for substrate to reach the specific reacting site. Asn236 of dehDIV-R was virtually mutated to amino acids residues with different steric hindrance, and the stereoselectivities of the mutant enzymes were predicted. According to the prediction results, the mutations of Asn236/Ala and Asn236/Ser were constructed by site-directing mutagenesis. After measuring their stereoselectivities, A1 with Asn236/Ala showed activity towards S- and R-substrate, and S1 with Asn236/Ser showed inverted activity towards S-substrate instead of R-substrate. These results were consistent with the prediction and the rationality of the model was proved.
Objective: To express Mycobacterium tuberculosis protein CFP10-ESAT-6-MPB64 in baculovirus insect cell expression system, and identify its immunogenicity. Methods: The target gene CFP10-ESAT-6-MPB64 was connected to pFastBac vector, then the pFastBac-CFP10-ESAT-6-MPB64 plasmid which was harvested would transformed to DH10Bac competent, and the target gene was transposition into Bacmid by Tn7 transposase fragment, therefore Bacmid-CFP10-ESAT-6-MPB64 Shuttle vector was obtained. The shuttle vector was packaged by liposomes and transfected Sf9 cells to harvest P1-generation virus, then high titers of P4 generation virus was harvested by repeat transfected Sf9 cells three times. The target protein CFP10-ESAT-6-MPB64 was purified from the supernatant by Co affine chromatography, which were used to immunize Balb/c mice. Antibody changes in serum would be detected, and the proliferation of immunized mice spleen cells would be detected by MTT,detected the IFN-γ secretion by CFP10-ESAT-6-MPB64 stimulated spleen cells by ELISA method. Result: CFP10-ESAT-6-MPB64 successfully expressed in insect cells. The purity of target protein is over 90% and yield up to 42mg/L after purification. Purified protein can effectively stimulate Balb/c mice to produce antibodies, increase the content of IFN-γ medium in mice spleen cells, and significantly promoting proliferation in spleen cells between 1~50μg/ml. Conclusion: CFP10-ESAT-6-MPB64 which has immunogenicity was successfully expressed in baculovirus insect cell expression system, that open a new avenue for tuberculosis vaccine production.
The previous study has indicated that the targeted inactivation of ompH gene decreased the lethality of serotype A Pasteurella multocida C48-3 in mice after intraperitoneal injection. The ompH gene was cloned from P. multocida C48-3 and expressed in Escherichia coli M15, and immune serum was generated by immunizing rabbit with the purified recombinant protein OmpH. Differences of adhesion ability, serum resistance and antiphagocytic activity between the wild-type strain C48-3, mutant strain ΔompH and its complemented strain C48-3C were compared by biological experiments. The adhesion of wild-type strain and complemented strain to CEF cells was inhibited significantly by treatment with immune sera as compared to the treatment with normal serum. The mutant strain was sensitive to the bactericidal action of chicken serum, whereas the wild-type strain and complemented strain were both resistant. The mutant strain was readily taken up by mouse peritoneal macrophages in the normal rabbit serum, whereas the wild-type strain and complemented strain were both resistant. In addition, the rabbit antiserum against OmpH promoted the phagocytosis of C48-3 and C48-3C by mouse peritoneal macrophages, but mutant strain was not affected. These results demonstrate that the OmpH is a major virulence factor of P. multocida.
Mature map30(signal peptide sequence excluded) was amplified from the genome of bitter melon and inserted on the expression vector pET28a. The final expression vector pET28a-map30 was transformed into E. coli Rostta (DE3) for MAP30 expression. The recombinant MAP30 was identified by SDS-PAGE, Western blotting and LC-MS. After purification by nickel-affinity chromatography, The DNA-cleaving activity was analyzed by electrophoresis after supercoiled plasmid pUC19 as substrate treated with MAP30 of various concentrations. Meanwhile MAP30 was used to treat MCF-7 human breast tumor cells, MTT, DNA Ladder, AO/PI double-stained is used to detect the anti-tumor activity. Results show that map30 gene was successfully expressed in E. coli Rostta (DE3)and been identified by LC-MS and Western blot using His-tag mAb. First discovered through Escherichia coli heterologous expression of recombinant MAP30 like natural MAP30 protein also exert the activity conversion supercoiled plasmid pUC19 to relaxed or linear forms. The cytotoxicity assay of recombinant MAP30 showed that it exhibited dose-dependent and time-dependent inhibition to MCF-7, AO/PI double-stained displayed typical of apoptosis. It through genetic engineering technology preparation MAP30 protein to further study of its biological activities in vitro and laid important foundation for the future clinical application.
An extracellular polysaccharide secretion related gene exoD was cloned from genome of S.fredii WGF03 and the influence of genes on extracellular polysaccharide synthesis as well as nodulation,nitrogen fixation with host plant was investigated. ΔexoD mutant was constructed through homologous double-crossover using suicide plasmid pk18mobsacB as a vector. Compared with the wile type strain, the mutant strain produced less exopolysaccharides (EPS) on YMA medium plate and the motility was decreased.Nothing changes in the growth situation on the medium containing less than 350 mmol/L of NaCl. The plant test showed that the number of nodules was more, but small, varying shapes, and nitrogen-fixing enzyme activity was also significantly decreased after inoculation mutant strain. This demonstrated that exoD gene affect EPS synthesis in S.fredii WGF03, and involve in nodulation and nitrogenase activity.
Objective: To prepare and analyze the rabbit polyclonal stress associated phosphorylation antibodies specific for the T103 (Thr103) site of Tudor-SN protein. Methods: The phosphorylated T103 (pT103) site-containing polypeptide was synthesized; Antiserums were collected from the New Zealand white rabbits immunized with the polypeptide four times and purified through AKTA protein purification system. The Western blotting analysis,immunofluorescence experiment and In-cell Western assay were performed using the purified anti-pT103 antibodies. Results: (1) The phosphorylated peptide "TIENKpTPQGRC" was synthesized and about 75 ml rabbit antiserums were collected (2.08 mg/ml); (2) The phosphorylation signal was detected and increased under oxidative stress conditions in Western blotting analysis; the granule signal was also observed in the cytoplasm of HeLa cells and co-localized with the endogenous Tudor-SN-labeled stress granule during stress. (3) The phosphorylation level of Tudor-SN at T103 site fluctuates during the oxidative stress and after the removal of stress. Conclusion: The rabbit polyclonal anti-pT103 antibodies specific for the T103 site of Tudor-SN were prepared successfully. It will help to study the role of Tudor-SN phosphorylation modification in cellular stress responses.
There are three kinds of aspartate kinase in the metabolic pathway of Escherichia coli, including LysC, MetL and ThrA. Lysine, methionine and threonine are synthesized after aspartic acid phosphorylation pathway caused by aspartate kinase so that aspartic acid is not able to be accumulated to a high concentration in E. coli. Aspartic acid phosphorylation could partly inhibit by gene knock-out. Single gene mutants which lack LysC, ThrA and MetL respectively, are constructed from W3110.After chloroamphenicol resistance removed by pCP20, double gene mutants which lack LysC-ThrA and LysC-MetL respectively are constructed. All mutants are finally confirmed by check primer. These constructions are based on Red recombination system. Concentration of aspartic acid is determined by high performance liquid chromatography. The results show that all mutants except MetL single gene mutants are able to accumulate more L-aspartic acid than wild type. This will lay a foundation for strain improvement by metabolic engineering and contribute to production of aspartic acid by fermentation.
Objective: The purpose was to improve the efficiency of scarless gene knockout in E. coli genome by the optimized method. Methods: The efficacy of scarless gene knockout in E.coli genome by two steps Red homologous recombinantion system and endonuclease I-SceI screening was investigated via optimization of the homologous DNA length with target sequence and the inducer concentration for production of I-SceI for the selection of positive clones. The scarless knockout of nanKETA clusters in the strain CLM37 was taken as a model. The new strain growth behaviour with the scarless knockout of nanKETA was investigated via the comparing the growth curves of wildtype E. coli CLM37. Results: The nanKETA clusters were knockouted scarless successfully in E. coli CLM37 genome, and the efficacy of scarless processing was increased up to 90% via extending the length of the homologous DNA with the genome, from the 80 base pairs normally used up to 684 base pairs, and increasing the concentration of the inducer tetracycline for producing I-SceI, from 500 μg/ml upto 1000 μg/ml. It is shown that the knockout of nanKETA clusters in E. coli CLM37 did not impact the growth. Conclusion: The efficacy of gene scarless knockout in E.coli can be significantly improved via extending the length of the homology DNA and increasing the concentration of inducer tetracycline.
Consolidated bioprocessing (CBP) is a promising technology for lignocellulosic ethanol production, and the key to CBP is the engineering of a microorganism that can efficiently utilize cellulose. Saccharomyces cerevisiae is a traditional ethanol producing strain, and it has many advantages as the CBP host strains, so the expression of cellulase in S. cerevisiae is causing great interest. The factors that influence the expression of cellulase genes in S. cerevisiae, including gene expression cassette expression elements (promoter, signal peptide and terminator, etc.), cellulase gene copy number and existing forms, the sources of cellulase gene and so on were reviewed. In addition, research progress about one or more cellulase genes expressed in S. cerevisiae and the construction of CBP strains were briefly introduced.
During the process of bioethanol production, Saccharomyces cerevisiae cells are often stressed by the accumulated ethanol, which can lead to inhibition of S. cerevisiae growth and low bioethanol yield. To maintain the survival, S. cerevisiae cells have evolved a set of stress responses to environmental stimuli including ethanol stress. Fully understanding the mechanism of S. cerevisiae responses to ethanol will facilitate the development of strategies to improve the ethanol tolerance of S. cerevisiae and contribute to the construction of industrial feasible strains with high bioethanol yield. Under the stress of accumulated ethanol, some protectants, such as trehalose, heat shock proteins (HSPs), and proline can improve the ethanol tolerance of S.cerevisiae cells. As an important carbon source and energy storage material, trehalose can not only stabilize the cell membranes, proteins and nucleic acids, but also enhance the ethanol tolerance of S. cerevisiae. Furthermore, the up-regulation of HSPs can also improve the ethanol tolerance of S. cerevisiae cells. The progresses of protective roles of trehalose and HSPs for enhancing the ethanol tolerance of S. cerevisiae were focused on.
Biobutanol is regarded as a promising biofuel amid the energy crisis and global problems brought by excess use of fossil energy. In recent years, microorganisms including Escherichia coli are of interests in bioengineering studies for bioproduction of butanol, mostly done by heterologous expression of clostridia derived synthetic butanol pathway. Currently, the maximal yield of butanol production reported in Escherichia coli has been close to that in natural strains; however, there still existed many problems daunting the attempts to further improve the biobutanol level by using this microorganism. The engineered metabolic pathways for formation of n-butanol in E. coli were briefly summarized, the limiting factors existed in bioengineering studies were analyzed, and the possible solutions that might help in further improvement of biobutanol productivity using E. coli as host strain were shared.
Escherichia coli is one of the most commonly used hosts for recombinant protein production. Extracellular protein expression in E. coli, which is favored by its improved correct folding of target protein, reduced formation of inclusion bodies and simplified purification process, is attracting increasing attention. Among all the secretion systems of E. coli, the type I secretion system has become one of the most popular secretion pathways due to its fast secretion speed, high expression ability and harmlessness to cell physiology. The components and mechanism of type I pathway of E. coli and efficient strategies for enhancing secretion efficiency of recombinant protein are summarized, which provides the theoretical basis for production of recombinant protein.
A lot of PEGylated proteins have been approved. Therefore, the naming scheme of Chinese Approved Drug Name relating to this kind of products must be revised to adapting their development.The progress of PEGylation technology and INN for these drugs were introduced. The nomenclature for PEGylated recombinant protein in China was discussed.
In the past years, the planting area of the transgenic cotton has a rapid growth in many countries. For further study on technology systems and intellectual property protection of transgenic insect-resistant cotton, the intellectual property strategy of transnational biotechnology Corporation has been studied——taking cotton event MON531(Genuity Bloogard TM) for example. The results showed that in the field of the transgenic cotton MON531, there are three valid patents in multiple countries, which made an effective and overall protection of the main functional gene, regulatory elements and the commercial varieties. And for the plant variety rights, nineteen plant varieties have been authorized in America, while only one related application has been examined in China. The strategy research of intellectual property protection related the cotton event MON531 can be used for reference for making our development of genetically modified crops and intellectual property protection.
The scaffold material that have three-dimensional structure is another main content of tissue engineering. Current tissue engineering scaffolds are divided into three kinds including natural biomaterial, synthesized organic material and inorganic material. Scaffold material is a hot research topic in recent years, which is more perfectly studied and developed in the field of artificial skin that is the earliest tissue engineering product and made much investigation and exploration in other fields such as artificial bone, cartilage, nerve, blood vessel, skin, liver, spleen, kidney and bladder, etc. Unlike traditional tissue engineering scaffolds that need to be previously fabricated and shaped up in vitro, the injectable scaffolds that have grown up in bone and cartilage tissue engineering practices have many advantages, which is one of the important development direction of future tissue engineering scaffolds.
