The pcD-awte candidate malaria DNA vaccine could induce protective immune-responses through animal test, so it is necessary to establish a suitable preparative protocol with which the vaccine can be applied in clinical test as soon as possible. Here we reported an efficient preparative method, with which plasmid DNA vaccine could be produced in high yield and purity. Escherichia coli DH5α containing recombinant plasmid pcD-awte was grown through fed-batch fermentation in 5L fermentor with basic culture media of improved TB. After fermentation, the crude plasmid DNA was obtained by alkaline lysis and the plasmid DNA was further purified with a three-step efficient chromatographic procedure, including Sepharose 6FF size exclusion chromatography , Plasmidselect thiophilic aromatic chromatography and Source 30Q anion exchange chromatography. The purified plasmid DNA was identified, which showed that the yield of final product reached 43.9 mg/L and that the quality met the standards of pharmaceutical-grade plasmid DNA.
To generate recombinant adenovirus expression vector of human Sema4C gene and observe its expression in mouse myoblasts cell line C2C12 for ensuring easy access to investigate the role of Sema4C gene during myogenesis. The recombinant plasmid was packaged and amplified after being transfected in HEK293 cells through Lipofectamine. After infecting C2C12 myoblasts with recombinant adenovirus vector, the adenoviral infection efficiency was determined by confocal microscope which showed that the expression of green fluorescence could be detected at 12h and then reached peak at 24h after recombinant adenovirus infection. The infection efficiency was almost 100% confirmed by FACS examination. Detection of WB indicated that the expression of Sema4C in C2C12 of recombinant adenoviral infection group was significantly higher than that of the control group (P<0.01). To further investigate the role of Sema4C gene during the proliferation and differentiation of C2C12 myoblasts, proliferation index of C2C12 cells were detected by FACS and the myogenic differentiation was also observed after adenovirus infection. Our data firstly showed that overexpression of exogenous human Sema4C gene could not only increase the percentage of G0/G1 cell cycle and decrease the proliferation index but also promote myotube formation of C2C12 cells.
E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria. In this report, we described a high expression and efficient purification scheme and kinetic assay on interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 induced with IPTG. SDS-PAGE revealed that the expected protein with a molecular weight 20.6kD was soluble and amounted to about 30％ of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90％. The resulting SSB protein was a correctly folded tetramer with an apparent binding to ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 M as determined by gel filtration and surface plasmon resonance.
In the present study, we inserted the core-streptavidin cDNA into downstream of multi-cloning site of plasmid pOPE101-8E5 by DNA gene recombination technology. And then, the variable fragments of heavy and light chain of the scFv-8E5 were replaced by the scFv-C4 variable fragments to construct the expression vector pOPE101-C4::core-streptavidin. After transformed the vector pOPE101-C4::core-streptavidin into E.coil, the fusion protein C4::core -streptavidin-His-tag can be expressed by inducing with IPTG, and the expression level and activity of the expressed fusion protein analyzed by SDS-PAGE and Western blot. The results show that a scFv-C4::core-streptavidin fusion protein of 45kDa was obtained, which can bind proteins of 60kDa & 45kDa from the KG1a cells lysate simultaneously. The binding function can be detected by the binding of core-streptavidin and biotin directly.
Chinese oak silkworm, Antheraea pernyi is an economic insect rearing outdoors. It is mainly cultured in the area of Northeast of China. A. pernyi diapause by pupa stage for hibernation. The pupa is big, easy fix, keep time long, need not the feeding, easy conveyance etc. advantage. A. pernyi pupae as biological reactor product foreign protein may be large-scale mechanization production, reduce operational and trivial with labor force. In this paper, A. pernyi nucleopolyhedrovirus (AnpeNPV) as gene expression vector successfully expressed β–galatosidase gene (LacZ) in A. pernyi culture cells (AnPe cell) and diapause pupae, moreover purified recombinant AnpeNPV expressing LacZ (AnpeLacZ) obtained using AnPe cell. For β–galatosidase yield of the AnpeLacZ, their peak activity was 40.9 units/ml at 12 days post-infection (p. i.) in AnPe cell cultured using TC-100 medium (10 % FBS) and is 59.9 units/ml at 18 days p. i. in AnPe cell cultured using SF-900Ⅱ medium, respectively. The latter was slightly high but slow at time. The β–galatosidase activity of AnpeLacZ in A. pernyi pupae who store 7 months at 5 ℃ reached peak at 15 days p. i., female was 14.3 units/g and male was 11.7 units/g, respectively. The female was better than male. Results demonstrate that AnpeNPV and A. pernyi pupae could be developed and used as a novel baculovirus gene expression system which may be mechanization and large-scale production.
Variations in the amino acid sequence of Infectious canine hepatitis virus (ICHV) structural proteins are the molecular basis for the antigen diversity of the virus.Majority of antigenic sites for the virus neutraliation are present on Loop1 and Loop2 of hexon.ICHV (the isolated strain) DNA was isolated and purified from the cultured MDCK cells.The Loop1 and Loop2 fragments were amplified by polymerase chain reaction(PCR) method,and then was connected by ligase T4.The target fragment was then connected with vector pET28a.The nucleotide sequence ecoding Loop1 and Loop2 was determined.The nucleotide sequence identity of Loop1 region between the isolated strain and CLL, RI261 and Toronto A26/61 strains is 100%, 100% and 83.8%, and the nucleotide sequence identity of Loop2 region between the isolated strain and CLL, RI261 and Toronto A26/61 strains is 88.1% , 88.1% and 99.3%, and amino acid identity is 93.6 , 93.6% and 98.6%.The recombinent Loop protein was expressed in E. coli and was approximately 36kDa in size,and then was purified.
The effect of exogenous cell factors as mLIF, hSCF, bFGF and hIL-11 on the stage of the 19th and 28th EPGCs proliferation and differentiation were elucidated by means of MTT test when combined added in the culture medium. The regulated mechanism of those cell factors was discussed as well. When the cell factors as mLIF, hSCF, bFGF and hIL-11 combined in the medium, it showed evident effect on proliferation of stage 19th EPGCs compared to the control group when cultured for 72h (p<0.05). The proliferation of EPGCs was obviously inhibited when the concentration of mLIF and hSCF beyond 20ng/ml, the suitable concentration of mLIF was 10~20ng/ml, hSCF was 15~20ng/ml. The combined of hSCF and bFGF was more efficient than single cell factor added. The suitable concentration of bFGF was 10~20ng/ml. Similar to hIL-11, when combined with the other cell factors, the number of viable cells increased distinctly（P<0.05）, the suitable concentration of hIL-11 was 0.10~0.20ng/ml. The results would promote and facilitate development of chicken EPGCs line and also highlight a general role in stem cell proliferation mechanism.
Eukaryotic DNA element called Matrix Attachment Regions (MARs) can function on regulating the structure and activity of chromosome. In order to study in vivo chromatin accessibility and transcription regulation involved MAR, here, we show that alpha 1-antitrypsin MAR (α1-AT MAR) is cloned from human blood and incorporated in pEGFP-C1 vector. non-MAR-containing and MAR-containing plasmids were transfected into HEK-293 cells with LipofectamineTM 2000, respectively. Pools of clones were assayed after 20 days of selection of G418. Semiquantitative RT-PCR and fluorescence microscope analysis show that this MAR has a positive effect on modulate nearby gene expression. Then, chromatin immunoprecipitation (ChIP) where it co-localizes with newly CMV promoter and RNA polymeraseⅡ(RNAPⅡ) is detected by PCR, The result demonstrates that more RNAPⅡwas recruited to the CMV promoter in presence of MAR. ChIP can be used to confirm the MAR-mediated transcriptional activation and provide more dynamic information than RT-PCR in real time. The technology is also providing a platform for our research in gene expression regulation.
Bacillus cereus B-04 was isolated in zibo soil and proved as a biocontrol agent of Botrytis cinerea. The aim of this study was to enhance the biocontrol effect of this strain by introduction of a β-1,3-glucanase gene. A 4.1kb DNA fragment containing β-1,3-glucanase gene was inserted into vector pBE2 and pHY300PLK to construct a new plasmid, PBE2-glu and pHY300PLK-glu,which was introduced into Bacillus cereus B-04, resulting in a new strain named B-04-glu.Restriction digestion and β-1,3-glucanase plate culture and PCR experiment confirmed that B-04-glu contained a functional β-1,3-glucanase gene. Compared with wild type B-04 in pot experiment, B-04-glu had increased effect against Botrytis cinerea caused by tomato.
The crude polysaccharide PNM was extracted from the submerged culture of Phellinus nigiricans by the method of ethanol precipitation. PNW is a heteropolysaccharide and GC analysis indicated that PNM consisted of xylise, mannose, galactose and glucose in the quality radio 0.05：0.63：0.31：1.00. PNM was purified by freeze thawing, methods of Sevag and preparation on Sepharose CL-6B column chromatography, it was PNMⅠ. HPLC analysis manifested that it was uniform fraction and the moleculer weight was about 29 throusand. Initially investigated the effects on the lymphocyte proliferation in vitro. The results were that both PNM and PNMⅠhad the most significant effects on the proliferation of mouse spleen cell in vitro at 400μg/ml.
By using Brine Shrimp as primary screening assay and silkworm as second screening assay, the strain of GX-29 with high insecticidal activity substances was selected from the soil. It was identified as Streptomyces fulvissimus based on analysis of 16S rDNA gene sequence. The insecticidal stability was also studied by means of Brine Shrimp tracking activity. The result shows that temperature, ultraviolet are not greatly affected the insecticidal activity, and its bioactivity kept steady in neutral and slight alkaline condition.
Response surface methodology was implied to optimize the culture components for lipopeptide production of Bacillus natto TK-1. In the first step, two level factorial design of Plackett-Burman was used to evaluate the influence of related factors. It showed that there were three factors play the important role in the medium, they were peptone, yeast extract powder, CaCl2. The path of steepest ascent was used to approach the optimal region of the fermentation conditions subsequently. In the third step, the concentrations of these three main factors were further optimized using Box-Behnken and response surface analysis. Under the optimized fermentation conditions, the diameter of haemolysis of fermentation culture increased 29.3 % than before. HPLC analysis was also used to estimated the precise production of lipopeptide. By using the optimal culture, its production was 30.2% higher than preliminary culture. It showed that the HPLC method was in accordance with the blood plate method. Furthermore, at three batches cultivation, the experiment value under the optimal conditions agreed with the predictive value. It showed that Response Surface Methodology was proper and a good choice for optimization.
Batch fermentation of antibiotic substance CF66I by Burkholderia cepacia CF-66 at various temperatures ranging between 23℃～33℃ were investigated and the dynamics characteristics during fermentation process were also analyzed. Based on the results, a two-stage temperature-shift strategy in which temperature at 30℃ was controlled at first 20 h, and then changed to 25℃ till the end of fermentation was developed. By applying this temperature-shift strategy in CF66I fermentation, the CF66I activity reached 3.783 u/ml and increased 26.1%, compared to single temperature at 23℃, 25℃, 28℃, 30℃ and 33℃.
Abstract: The main collagens in skin are type I and III. In this research, total collagens were separated from rat skin by enzymatic digest and acetum methods. The collagens were denatured at 60℃ and digested with trypsin. Characteristic peptides typical for collagen type I and III were identified with high performance liquid chromatography/mass spectrometry. The relative abundance of collagen type I/III was determined according to characteristic peptides. The result indicated that the content of collagen type III in rat skin decreased with growth stage, and content of collagen type I increased slightly with growth stage, the ratio of collagen type I/III keep stable after 8 weeks.
Macrophages are involved in many important biological processes and membrane proteins are the key effector molecules for their functions. However, membrane proteins are difficult to analyze by 2-DE based method because of their intrinsic tendency to self-aggregate during the first dimension separation (IEF). To circumvent the obstacle hampering membrane protein analysis, we combined one-dimensional SDS-PAGE with capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using this technique, we identified 458 GO annotated membrane proteins with extremely high confidence, including most known markers of peritoneal macrophages (e.g., CD11b, F4/80, CD14, CD18, CD86, CD44, CD16 and Toll-like receptor). Thirteen other CD antigens (CD243, CD98, CD107a, CD107b, CD36, CD97, CD205, CD206, CD180, CD191, CD300, CD45and CD29), and 18 Ras-related small GTPase were also identified. In addition to those known macrophage membrane proteins, a significant number of novel proteins have also been identified. This research provides a valuable data set of macrophage membrane proteins, thus allowing for more comprehensive study of membrane proteins and a better understanding of the function mechanisms of macrophages in many biological processes.
cDNA encoding goose IL-2 (goIL-2) was cloned from Con A-stimulated goose SMC using oligonucleotide primers based on the conserved sequence of duck IL-2 (duIL-2), chicken IL2 (chIL-2) and turkey IL-2 (TuIL-2). Sequencing results showed that the goIL-2 nucleotide sequence was 768 bp in length, encoding a polypeptide of 141 amino acid residues. The predicted nucleic acid and amino acid sequences of goIL-2 were of 90.1% and 83.6% identical to duIL-2, 69.7%-75% and 61.0%-63.1% to chIL-2, tuIL-2 and quail IL-2, 25%-30% and 14%-17% with mammalian IL-2s, respectively. Amino acid sequence analysis showed that the predicted protein had a leader sequence composed of 21 amino acids, and four conserved cysteines allowing the formation of two intrachain disulfide bonds. GoIL-2 mRNA was detectable using RT-PCR assay in goose splenic mononuclear cells from 2 h to 24 h after stimulation. Three-dimensional structure prediction indicated that the mature goose IL-2 protein contained A, B, C and D four α-helix bundle structures and two β-sheets. Phylogenetic analysis showed that the goIL-2 had close relationship with duIL-2 and had distant relationship with tuIL-2.
The spermatozoa from mature guinea pigs were incubated in modified TALP under 5% CO2 in air at 37 ℃. The capacitation condition was assessed by chlortetracycline (CTC) staining, the localization and level of tyrosine phosphoryalted proteins during the capacitation were detected by indirect immunofluorescence or Western blot. The results showed that the number of sperm tyrosine protein phosphorylated increased from 31% to 92% during capacitation period. The site of phosphorylated tyrosine-specific fluorescence was expanded from the head of spermatozoa to the whole head and principal and mid-piece areas of the flagellum. 40kDa protein in sperm extracts taken at 0 to 0.5h after incubation was detected by anti-phosphotyrosine monoclonal antibody. 1h after incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. 3h after incubation, three proteins with relative molecular weight of 40, 45, 80kDa were detected.
A new kind of curcin (curcin 2), induced by several kinds of stresses from Jatropha curcas leaves, under the control of the 35S CaMV (cauliflower mosaic virus) promoter, was introduced into tobacco genome by Agrobacterium tumefaciens-mediated transformation method. Curcin 2 protein was only detected in the transgenic tobacco plants transformed with the cur2p fragment (coding premature curcin 2 protein), but not in the plants transformed with cur2m fragment (coding mature curcin 2 protein). The transgenic lines expressing curcin 2 showed increased tolerance to tobacco mosaic virus (TMV).
PCR is one of the common techniques in molecular biology, which can amplify nucleic acids through the cycle of denaturation, annealing and extension. Based on the principle of common PCR, rapid PCR is to realize the amplification of nucleic acids in less time without affecting the specificity, sensitivity and fidelity of the reaction. A lot of research work in this field has been going on in recent years. And this article will make a review of the development of rapid PCR with emphases on the improvement of DNA polymerase, the choice of additives and the improvement of thermocyclers.
Human papillomavirus (HPV) infection causes almost all cases of cervical cancer, the second most common cause of death from cancer among women worldwide. HPV can also cause many other kinds of cancers and diseases including skin warts and anogenital mucosal condylomata et al. Usually HPV vaccines can be divided into two categories: prophylactic vaccines and therapeutic vaccines. Progress of the two kinds of vaccines were reviewed to give a reference to novel vaccine research.
As a group of important transcription factors, NF-κB are composed of members of Rel/NF-κB family. For most kinds of cells, NF-κB with IκB (inhibition protein) binding normally is existed in inactived form in cytoplasm. With efficient stimulus, NF-κB can be activated through signal transduction pathways, resulting in the escape form from IκB binding, and should be transported into nuclear where is the proper locale for it to exert its transcriptional regulation function on a great deal of genes. However, in abnormal cases, there will be mistaken expression and activation of NF-κB, which has been testified to be close relationship with many disease genesis, including inflammation and cancer, and so on. However, in the other cases, activation of NF-κB could repress disease development. To inhibit the abnormal activity and function of NF-κB has been shown to be an effective strategy for therapy of relative diseases. With relative research development, NF-κB should have bright prospect as a target for novel drug design and relative disease therapy.
Abstract：Diabetes mellitus with the increasing prevalence has become one of the diseases which threaten the heath Diabetes mellitus has become one of the diseases which threaten the heath of human being in the 21st century．A goal of research in diabetes is to find a way to increase the number of functional insulin-producing cells. Islet transplantation has been considered to be the most effective approach to cure type Ⅰ and part of type Ⅱ diabetes mellitus．This approach, however, is severely limited by an inadequate supply of donor islets available for transplantation．Moreover, recent progress of stem cells research has shown that stem cells may act as a new source of islet transplantation in diabetes mellitus treatment. Recent evidence indicates that Glucagon-Like PeptideⅠ(GLP-1) plays a very important role in targeted differentiation of stem cells into Insulin-Producing Cells and pancreatic development. GLP-1 is an intestine-derived insulinotropic hormone that stimulates glucose dependent insulin production and secretion. GLP-1 can induce differentiation of stem cells into insulin-producing cells, which is achieved by up regulation of PDX-1 expression.PDX-1 is a transcription factor critical for pancreatic development and endocrine cell neogenesis and a marker for pancreatic stem cells. These new findings suggest an approach to create Insulin-Producing cells in vitro by expanding stem/progenitor cells and then to convert them into Insulin-Producing cells by treatment with GLP-1. Thus GLP-1 may be a means by which to create Insulin-Producing cells ex vivo for transplantation into patients with insulinopenic type Ⅰ diabetes and severe forms of type Ⅱ diabetes. This article reviews recent progress about GLP-1 and targeted differentiation of stem cells induced by GLP-1.
Compared with complex glycoproteins in humans, therapeutic proteins produced in yeast expression systems typically carry high-mannose sugar chains which can lead to rapid clearance, enhanced immunogenicity and poor pharmacokinetic behaviour in clinical application. After the recreation of the N-glycosylation pathways by eliminating endogenous glycosylation reactions, introducing mammalian glycosyltransferases and expressing some sugar transporters, human-like complex glycoproteins with terminal sialic acids could be produced. This review summarizes yeast N-linked glycosylation processes, the heterogeneity of glycosylation, the genetic engineering of yeast glycosylation pathways to produce fully humanized glycoproteins and the application of endo-β-N-acetylglucosaminidase for in vitro synthesis of complex glycoproteins in recent years. Moreover, existing problems and future development directions are discussed.
Bacterial cellulose (BC) is a natural polymer that has bioactivity, biodegradability and biocompatibility. It displays unique physical, chemical and mechanical properties including high crystallinity, high water holding capacity, nanofibre-network structure, high tensile strength and elastic modulus. Due to its unusual material properties, BC has recently become a kind of attractive biomedical material in the international research. This paper describes BC’s properties, study history and its applications as biomedical materials, especially gives emphasis to introduce the applications of BC on scaffold tissue engineering, artificial blood vessels, artificial skin and the treatment of skin wound, as well as the present study status.
Incorporation of a selectable marker gene during chloroplast genetic transformation is essential to obtain transformed chloroplasts. Many selective marker genes have been used in chloroplast transformation. Progress on selective marker genes of chloroplast genetic transformation is reviewed; especially the antibiotic-free selective marker—betaine aldehyde dehydrogenase (BADH) and the marker elimination system are discussed.
Abstract：This article introduced the new advbance in production of polyunsaturated fatty acid by microbial fermentation.The screening of high yield mutant strain and the important factors that work during the process in the production of PUFA are all discussed .The separation，purification of PUFA are also briefly investigated. At last the development prospects of PUFA production by Microbial Fermentation were briefly outlined.
Green algae are invaluable plant for the development of biological energy research and industrial application. In this article, first, we briefly summarized the known pathway of hydrogen production by green algae; second, we mainly studied [Fe]-hydrogenase, the key control parameters of hydrogen production; lastly, we forecast the future of developing biological energy of green algae.
Systems Biology including Systems Biomedicine is emerging internationally and providing opportunities for revolutions of Traditional Chinese Medicine (TCM) and conventional western drugs (WD), while the latter has been taking more advantages because WD has much stronger research basis at molecular levels than TCM. Thorough understanding of molecular network including gene network would automatically induces design of WD into formula western drugs equipped with TCM philosophy, but such molecular formula drug is eventually one of the key aims of modernization of TCM. So TCM is facing big threats it never met. This situation requires a nationwide and international strategy with intelligence and resolution.
