Myeloid differentiation factor MyD88 is a critical adaptor molecule that integrates and transduces intracellular signals in inducing the differentiation of dendritic cells (DCs). We searched the domain regions within MyD88 which could potentially affect the function of dendritic cells and found that MyD88 aa155-171 motif not only regulate the activity of transcription factor NF-κB but also control the production of cytokines and expression of costimulatory molecules. Indeed, aa155-171 motif deleted type MyD88 (MyD88?155-171) transfected RAW264.7 exhibited the reduced NF-內B and AP-1 activity and interrupted the expression of CD86 and B7H1. Meanwhile, lower level expression of cytokines such as IL-12,IFN-r were observed by means of cytokine array techniques in MyD88-/- DC trasfected with MyD88?155-171 as compared to the MyD88 transfected cells. Thus, aa155-171 motif inside MyD88 could affect the expression of costimulatory molecules, cytokines secretion and interrupt Toll like receptor signal pathway, suggesting that this motif may play an important role in regulating responses of innate immune system.
ABSTRACT：Aim: To approach the relationship between cellular immune function and cerebrovascular disease(CVD) injury by estab1ish two-dimensiona1 electrophoresis(2-DE)maps of Splenolymphocyte in rat with intracerebral hemorrhage(ICH) and cerebral ischemia ,and identify the differentially expressed proteins．Methods: SD rats were randomly divided into normal group, intracerebral hemorrhage group (intracerebral hemorrhage induced by VII-collagenase and cerebral infarction group (a thread of a middle cerebral artery occlusion model). The Splenolymphocyte of rats were isolated and the total proteins were extracted and separated by 2-DE .The differentially expressed proteins were analyzed using PDQUEST analysis software，and then identified by peptide mass fingerprint(PMF)based on matrix-assisted laser desorption／ionization time of flight mass spectrometry (MALDI—TOF—MS). Resuits：Glia maturation factor gamma 、Rho, GDP dissociation inhibitor (GDI) beta 、Peroxiredoxin-II 、Hypothetical protein 、Beta-actin 、NADH dehydrogenase [ubiquinone] flavoprotein 2、60S acidic ribosomal protein P0、40S ribosomal protein SA were significantly increased, annexin III was significantly reduced in in rat with intracerebral hemorrhage and cerebral ischemia. Conclusions：The well-resolved and reproducible 2-DE pattern of Splenolymphocyte were established in rat with cerebral hemorrhage and cerebral ischemia and ten differentially expressed proteins were characterized.This approach may lay a foundation for the further investigation of the relationship between cellular immune function and cerebrovascular disease(CVD) injury.
To investigate the effect of Hemangiopoietin (HAPO) on the hematopoiesis reconstitution in lethally irradiated Balb/c mice. Methods: In the live animal experiment, Balb/c mice underwent total body irradiation at 700 cGy 137Cs γ radiation and were treated after irradiation with HAPO or recombinant human granulocyte colony stimulating factor (rhG-CSF). Survival rate of mice after radiation, the number of bone marrow cells, blood routine, spleen node forming and colony forming units were detected. In vitro experiment, cells from bone marrow of Balb/c mice were washed and resuspended, then cultured in 96- well plates. HAPO or rhG-CSF were added in the culture system before or after irradiated for 24 hours or 72 hours. MTT assay was used to assess the viability of cells. Cells from bone marrow of Balb/c mice cultured in 24-well plates were irradiated, then cultured for another 3 weeks with HAPO in culture medium to assess ability of in vitro hematopoiesis reconstitution. Result: rhG-CSF and HAPO treated mice both showed increased survival rate and increased colony forming units. The changes of the peripheral WBC level after radiation were remarkable. The recovery of peripheral WBC number in HAPO group was quickest compared with that in rhG-CSF group and PBS control group. The number of bone marrow cells at day 14 of rhG-CSF group was higher than that in HAPO group, but the number of bone marrow cells at day 32 of rhG-CSF was lower than that in HAPO group. The number of bone marrow cells at day 42 of rhG-CSF was below normal. The number of bone marrow cells at day 42 of HAPO group was nearly normal. The number of CFU-GEMM in HAPO group was most compared with that in rhG-CSF group and PBS control group at day 7, 14 and 21 after radiation. The survival rate of cells after radiation in HAPO group was markedly higher than that in PBS control group, but the survival rate of cells after radiation in rhG-CSF group was no notable difference compared with that in PBS control group. In MTT assay, both HAPO and rhG-CSF incubation stimulated proliferation of bone marrow cell at 72 hours after radiation. Bone marrow cells formed Hematopoietic islands in HAPO group after radiation and were positive for sca-1 and CD31. CD31 positive endothelial cells increased around the Hematopoietic islands. There was no Hematopoietic islands formation, few CD31 positive endothelial cells and no sca-1 positive cells in PBS control group. Conclusion: we confirmed HAPO promoted hematopoiesis reconstitution in lethally irradiated Balb/c mice in vitro and in vivo study, increased survival rate of mice and stimulated hematopoietic stem cells proliferation.
Objective: To clone the genes encoding human anti-DNA-PKcs antibodies from large phage antibody library for the use of cancer therapy or diagnosis in future. Method: Human DNA-PKcs protein fragments with high antigenicity and low homologous to other proteins were defined through antigenicity analysis and BLAST searching. After prokaryotic expression and purification, the protein segments were coated onto immuno-tube. Panning of large scale phage library against antigen was conducted to select specific antibodies against DNA-PKcs. The binding activity and specificity were tested by ELISA method. Soluble scFv was prepared through transfecting HB2151 with the selected phage antibody genes. Result: 250 AA of DPK3 and 257 AA of DPK4 segments were defined as high antigenicity and low homology to other protein through bioinformatic analysis. After 4 rounds of panning, 26 clones were identified with the specific binding ability with DPK3, and 31 clones with DPK4. DNA fingerprinting revealed 5 individual positive clones to DPK3 and 21 individual positive clones to DPK4. Sequencing analysis showed that variable regions of these ScFvs belong to different subgroups. Conclusion: Human DNA-PKcs antibody genes were successfully obtained from large scale phage antibody library.
Abstract Objective: To express the EMCV 3AB gene by prokaryotic expression systerm , and prepare monoclonal antibodies against it. Method: NSP 3AB gene of Encephalomyocarditis virus (EMCV) was amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and a recombinant protein 3AB with high antigenicity was expressed in E. coli. Balb / c mice were immunized by purified recombinant 3AB protein of inclusion-body, and the splenocytes of the immunized mice were fused with murine myeloma cells to produce hybridoma cell line. Results: After subcloning by 3 times, one strain of hybridoma cell line steadily secreting antibodies of 3AB protein was obtained, named 2D12. The McAb belongs to IgG1 /κ. The McAb and was confirmed by indirect immunofluorescent assay (IFA) and Western-blotting. Conclusion: These results can provide a potential value for structural and functional studies of EMCV-3AB and early diagnosis of Encephalomyocarditis virus infection.
Objective: Using the method of bioinformatics to predict the mutual epitope of Plasmid-mediated AmpC β-lactamases (pAmpCs) and to realize prokaryotic expression, purifica- tion and identification of AmpCs-Trx fusion protein. Method: Using the multi-sequence compa- rison tool to find various types of pAmpCs's relative conservative region, and using molecular biology software Biosun to predict the parameters of the accessibility, the antigenicity, the epitope, the flexibility and the hopp-woods, etc . Through comprehensive analyzing, find out the area of mutual epitope pAmpCs1. Based on the preference of the E. coli codon, obtain the nucleotide sequence pampcs1. Then the pampcs1 fragment was synthesized using overlapping PCR and the expression vector which is called pET32a (+) / pampcs1 was constructed. Adopting sequence verified, to make induced expression for the E.coli BL21(DE3) which contains the recombinnant plasmid. After the expressed product was analyzed by SDS-PAGE, to purify pAmpCs1-Trx fusion protein by using Ni-NTA affinity chromatography. And using the method of Western blotting to identify the antigenicity of pAmpCs1. Result: According to the result of prediction, the area of mutual epitope have been found out, and successfully established the recombinant expression vector pET32a(+)/pampcs1. BL21(DE3) strain was induced by IPTG, a specific expression band with a relative molecular mass 23×103 was detected by SDS-PAGE,and the purified fusion protein can be detected by multiclonal antibody against AmpC β-lactamases. Conclusion: Having been successfully established AmpCs1-Trx fusion protein prokaryotic expression vector and having been obtained the highly purified AmpCs1-Trx fusion protein. Initially prove the antigenicity of the AmpCs1-Trx fusion protein , settle a good foundation for preparing specific antibodies against pAmpCs1 and detecting pAmpCs.
The cDNAs encoding mouse TAp63γ and its two deletion mutants were inserted into the pGEX-2TK expression vector and transformed into competent cell of E.coli BL21(DE3), respectively. The three recombinant plasmids expressed effectively soluble GST fusion proteins in E.coli BL21(DE3) when induced by IPTG under the most optimal expression conditions. The three soluble expressed crude protein lysates were achieved by harvesting cell pellet through centrifugation, breaking the cell wall through sonication and dissolving with Triton X-100. The three near-homogeneous purified GST fusion proteins judged by SDS-PAGE were obtained by Glutathione-Sepharose affinity chromatography. Electrophoretic mobility shift assay (EMSA) indicated only wild type GST-TAp63γ protein can bind to the p63 DNA-binding consensus motif, which is also the p53 consensus sequence. Furthermore, sequence alignment and homology modeling accounted for to some content the conservation of important residues and the high similarity of three dimensional structure of the intact DNA binding domain are necessary for the DNA binding activity of mouse TAp63γ protein.
Objective: To prepare recombinant chimeric toxins containing luffinS2, Ⅰ type of GnRH and domain Ⅱ of Pseudomonas exotin, in which luffinS2 is toxic moiety, GnRH is target moiety and PEⅡ is translocation moiety, and analyze its lethal effect to some tumour cells in vitro. Methods: The gene of GnRH-PEⅡ-luffinS was amplified by overlapping PCR techniques and cloned into pET32a vector, and then the recombinant plasmid was transformed into E. coli BL21(DE3). Positive strains were induced and expressed recombinant proteins were purified by Ni-NTA affinity column. Purified protein which was cleaved to remove the Trx fusion protein by rEK and then its cytotoxicity to Hela, A549, HepG-2, SP2/0 and CEF were analyzed by XTT method. Results: We constructed the recombinant expression plasmid which succeeded to express in E. coli and be purified with purity of 94％. The IC50 of GnRH-PEⅡ-luffinS were 13.50μg/ml, 13.74μg/ml, 16.79μg/ml and 26.07μg/ml respectively and the recombinant protein had shown no toxic effect to CEF. Conclusion: GnRH-PEⅡ-luffinS have cytotoxicity to cancer cell lines.
Thermostable L-arabinose isomerase (L-AI) is the most potential enzyme for the biological production of D-tagatose from D-galactose, a novel functional factor. Gene araA encoding the L-arabinose isomerase from Bacillus stearothermophilis IAM 11001 was cloned and expressed in Escherichia coli. The araA gene of 1491 bp has 95% identity with L-AI from Thermus sp. IM6501. The GenBank accession number for the nucleotide sequence of this araA gene determined in this work is EU394214.The bacterium was induced by IPTG and analyzed by SDS - PAGE, approximately 59 kDa exogenous protein was observed on the SDS - PAGE. The recombinant L-AI was purified to electrophoretical homogeneity with affinity chromatography, and the activity of recombinant L-AI was also studied. The bioconversion rate of D-galactose to D-tagatose reached 39.4% after 24 h whole cell reaction.
The gene encoding the phosphatidylserine synthase in Escherichia coli K12 Sgal- (ExPASy P23830) was amplified by PCR. After DNA sequence analysis, it was inserted into the inducible expressive shuttle vector pBES of Bacillus subtilis, which was constructed in this lab, and the recombinant plasmid pBES-pss was then transformed into competent cells of the Bacillus subtilis strain DB104. The positive transformant DB104 (pBES-pss) was grown on Bacillus subtilis common fermentation medium, which contained 30μg/mL kanamycin. After 2 hours cultivation, sucrose was added and increased to the final concentration of 2% for induction and this phosphatidylserine synthase was secreted into the medium. The result of SDS-PAGE showed that the molecular weight of the protein was 52kDa and the result of enzyme coupling colorimetric method showed that the enzyme activity was 1.50U/mL. The recombinant Bacillus subtilis has increased the yield of phosphatidylserine synthase which will be used for industrial biosynthesis of phosphatidylserine.
ABSTRACT: AIM A cell based reporter gene assay had been established and optimized for high throughput screening to find agonists for human histamine 3 receptor (H3R) from Chinese herb components. METHODS The H3 receptor plasmid (H3R/pCDNA3.1-hygro) and reporter gene plasmid (3XCRE-LUC) were cotransfected HEK293 and a stable cell line was selected with antibiotics for H3 receptor agonist screening. Upon agonist binding, H3 receptor was activated and lead to a decrease of the high expression of luciferase gene induced by forskolin. RESULT To identify and optimize the assay condition, the effects of some factors were examined, such as compound solvent selection, final concentration of forskolin, agonist and forskolin incubation time, final concentration of DMSO. A steady cell line and a reliable method were established for H3R agonist screening, the Z’factor value was above 0.5, the signal did not change using HEK/H3R/CRE cell line passage 5 to passage 20. By using this assay system, two herb components were identified and regarded to contain agonists for H3 receptor. CONCLUSION This drug screening cell model has been successfully used for histamine 3 receptor target high throughput drug screening.
It has been reported that a gene can be knocked out by homologous recombination technology in EL350 genetically engineered bacteria strain. However, the study about the mutation and genetic targeting of Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT) gene by this system has not been reported. In this paper, rabbit full length HPRT gene BAC clone LBNL1-304M19 is used as the template. A 47kb rabbit HPRT gene fragment, which does not have promoter and exon1, is cloned into pBACLinkSp plasmid to form pBACLinkSp-rHPRT recombinant plasmid via Gap-Repair by Red recombination system. Then, different homologous arms are designed to delete different coding region of HPRT gene on the basis of the pBACLinkSp-rHPRT plasmid. Three different HPRT gene targeting vectors have been constructed. Meanwhile, the efficiency of deleting different sizes of DNA fragment by homologous recombination technology has also been studied. These three different HPRT gene targeting vectors form the basis for exploring the gene targeting in rabbit fibroblast cells and embryonic stem cells, and making rabbit HPRT gene knockout models in the future.
The plasmid pRSET-A, a common prokaryotic expression vector, encodes a N-terminal 34aa precursor peptide with a histidine tag sequence. Anti-histidine-tag antibody is usually used for identification of expression of the recombinant protein expressed using pRSET-A. A pair of primers that contained flanking hydropathic amino acid codons were synthesized, to amply the sequence coding for 10~34 aa in the precursor sequence. The amplified fragment was inserted into pRSET-A to generate the first double repeat of the precursor gene. By utilizing a pair of isocaudamer BamH I and Bgl II sites, and another downstream Hind III site of plasmid pRSET-A, following a series of simple double digestions and ligation of the resulted products, a series of repeat (3, 4 and 6) precursor peptide fragment genes were derived. The 6 repeat polymer of the precursor peptide was successfully expressed, and used to immunize goats for preparation of anti-precursor peptide antibodies. The antibody produced effectively identified the recombinant protein with the completed precursor peptide, but not the recombinant protein only had the His-tag sequence and lack the 10~34 aa sequence. These results demonstrated the repeated short-peptides polymer can be quickly and efficiently constructed by utilizing a pair isocaudamer sites in production of subunit-vaccine or immune-regulating antigens.
Objective: To establish a method of constructing antibody microarray based on sandwich immunoassay. Methods: Modified glass slides were robotically printed with capture antibodies against MCP-1, then dilutions of the cytokine were applied to the arrays and the protein was detected with biotin-labeled antibody coupled with Cy3- conjugated streptavidin. A laser confocal scanner was used to obtain the images and the signal intensity was analyzed subsequently. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, blocking buffers, reproducibility and quantitative ability of the system, two-cytokine analysis and shelf life of the post-printing slides. Results: The signal intensities increased with increasing capture antibody concentration; 2%BSA and 5% casein were proper as the blocking buffer for this system; The results revealed high reproducibility with regard to intra-array (1.3%) and the inter-array (8.7%) variation at the capture antibody concentration of 125ug/ml and good qualitative ability with a correlation coefficient of 0.9995 between the antigen concentration and the signal intensity; Besides, an antibody microarray for the parallel analysis of two cytokines was established and the printed arrays could be stored for at least two months without any apparent change of the performance parameters. Conclusion: A method for constructing antibody microarray based on sandwich immunoassay was established which might lay a foundation for fabrication of antibody microarray with multiplexing and quantitative capacity.
The culture conditions in 250 ml flask for the production of CoQ10 by Rhodopseudomonas palutris J001 were investigated. The results showed that the optimal initial pH, temperature, rpm of rotary shaker, medium volume in the flask and inoculum’s size were 6.5-7.5, 28-31℃, 100 rev min-1, 200 ml and 10%, respectively. The suitable carbon source was NaAc and suitable nitrogen sources were (NH4)2SO4 and NH4Cl. But the effects of phosphorus source on the cell growth and CoQ10 production were insignificant. With the aid of response surface method, the optimum concentrations of NaAc and (NH4)2SO4 were 5.39（g l-1）and 0.385（g l-1）for the growth of biomass, respectively, which the ratio of C/N was 14/1; and the optimum concentrations of NaAc and (NH4)2SO4 were 5.70（g l-1）and 0.365（g l-1）for the production of CoQ10, respectively, which the ratio of C/N was 15.6/1.
In this study, a sequence-specific M1GS ribozyme (M1GS-HCV/C20) has been successfully constructed by covalently linking an oligonucleotide (guide sequence, GS) to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The engineered ribozyme is targeted to the most conservative sequence (5'UTR) of HCV genome, and can effectively cleave the substrate RNA segment in vitro. Undoubtly, the M1GS-HCV/C20 we got would be a useful experimental material to futher study its cleavage activity in vivo, and can be even used for evaluating its anti-viral effect in the animal model. It was believed that this study would markedly facilitate the research of a general gene targeting agent for anti-HCV applications, and layed the foundation for developing a new nucleic acid drug and a novel strategy of anti-HCV therapy.
In this study the second intron, pPpMADS4, of an AGMOUS (AG) homologous gene PpMADS4 was cloned from peach (Prunus persica) and its function studied in transgenic Arabidopsis thaliana. Sequence analysis revealed the presence of several putative cis elements in the second intron of PpMADS4, and all the second introns cloned from seven different peach cultivars possess highly conserved cis elements although SNP-rich regions were identified. Furthermore, the pPpMADS4∷minimal35S∷GUS-0029 vector were constructed and transformed into wild-type A. thaliana. β-glucoronidase staining showed that it was expressed specifically in carpel and stamen at late floral developmental stage.
One alkaline protease from Actinomucor elegans AS3.2778 was purified 22.7 fold with a total yield of 16.1% and a final specific activity of 6094 u/mg protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is pH 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at < 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.
The expression of genes are subject to meiotic sex-chromosome inactivation(MSCI) during spermatogenesis, but lots of gene transcripts are reactivated in postmeiotic. The studies on transcripts show that the transcription of X and Y chromosome specific genes occur in different mammalian species spermatozoa in some extent. The study techniques of the transcripts had made great progress, such as DNA microarray, Serial Analysis of Gene Expression, Suppression Subtractive Hybridization and so on. With further development of these technologies, the transcription and regulation mechanism of sex-determining genes in spermatozoa will be more and more clear, and greater progress will be made on the research of sex specific protein.
As one of the most widely used technology in cell fusion, cell electrofusion is a rapidly developing cell engineering method, which utilizes dielectrophoretic force to induce cell alignment. Aligned cells are electroporated and subsequently electrofused within high electric field strength. These fused cells obtain different genetic materials from individual parent cells, which can show new genetic characteristics. At present, cell electrofusion is of important significance in biomedical and agricultural research. In the paper, the theoretical fundamental of electrofusion and up-to-date development are reviewed. Some relative research in our laboratory is also described.
It's commonly accepted that the infection of HPV with high-risk is the important factor in the occurrence of cervix cancer. Recombinate technologies are used to develop HPV vaccines, which is the only effective method to prevent cervix cancer so far. From the both aspects of expression vectors and codon optimization, this article reviewed the widely used expression vectors in recent years and their characteristics ；as well as the research results on obviously enhancement of capsid protein by optimizing the virus capsid gene according to codon preference, which provide basis for the further manufacturing and optimization of HPV vaccines.
Plant bioreactor called mocular farming has enormous potential to produce recombinant protein infinitely. Products expressed in plants has nature physico-chemical property and bioactivity. Plant bioreactor could be an safe, economic and convenient production system which has been widely applied in industries and agriculture, especially in the life science and pharmaceutical industry. The application of recombinant transgenic plant in the production of vaccines, antibodies and pharmaceutical proteins has become a hot point in the plant genetic engineering both at home and broad. However, there are some limiting factors of application such as yield, downstream processing and so on. This review discussed the advantages and research progress for the mocular farming of pharmaceutical proteins recent three years, focusing on the existing problems and new strategies in this area.
