Sodium Dodecyl Sulfate Polyacrylamide Gel Electropheresis Research Articles

Changes of different subtypes of adiponectin in epicardial fat in patients with coronary heart disease

Objective To study the difference of the secretion and synthesis of adiponectin in different parts of adipose tissue in patients with coronary heart disease (CHD). Methods The serum, epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT) in over 50-years old male patients with CHD or valvular heart disease as control group. The patients with CHD were excluded by coronary angiography in control group. Serum adiponectin concentrations were examined in CHD group and control group by enzyme linked immunosorbent assay (ELISA). Serum adiponectin types were classified in accordance with the molecular weight of high-molecular-weight (HMW), medium molecular weight (MMW) and low molecular weight (LMW) in CHD group and non-CHD group by gel filtration chromatography. Real-time PCR was used to detect the expression of adiponectin mRNA in epicardial adipose cells and subcutaneous adipose cells. The levels of HMW MMW LMW adiponectin in adipose cells were determined by non-hearting and non-reducing sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). Results As compared with non-CHD group, the concentration of serum HMW in CHD group was significantly reduced [(0.55±0.18) mg/L vs. (0.31±0.12) mg/L, t=3.472, P 0.05; LMW: (0.48±0.22) mg/L vs. (0.53±0.20) mg/L, t=1.712, P>0.05]. The expression of adiponectin mRNA in CAD epicardial group was significnatly downregulated when compared with autologous subcutaneous group (0.810±0.101 vs. 1.102±0.135, t=3.351, P<0.01) and control epicardial group (0.810±0.101 vs. 1.008±0.146, t=3.363, P<0.01). The proteomics revealed that the HMW content in CHD group decreased significantly as compared with non-CHD epicardial group and subcutaneous tissue group, and the rest two types of adiponectin showed no statistically significant differences. Conclusion The low expression of adiponectin in patients with CHD is located in the EAT and mainly decreased as the form of HMW adiponectin. Key words: Adiponectin; Epicardial adipose tissue; Coronary artery disease

Visible light driven CuBi2O4/Bi2MoO6 p-n heterojunction with enhanced photocatalytic inactivation of E. coli and mechanism insight

Here, a novel CuBi2O4/Bi2MoO6 (CBO/BMO) p-n heterojunction was fabricated and exhibited markedly improved photocatalytic inactivation capacity of E. coli cells under visible light excitation (λ > 420 nm) compared with pure CuBi2O4 and Bi2MoO6. The CBO/BMO-0.5 hybrid displayed the highest photoinactivation ability which could completely inactivate the E. coli cellswithin 4 h. The mechanism of photocatalytic disinfection towards E. coli of CBO/BMO heterojunctions was attributed to the disruption of cell-membrane, leakage and damage of cellular content including total protein and DNA as verified with SEM, fluorescence-base dead/live stain, sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and agarose gel electrophoresis (AGE). Additionally, the scavenge experiments showed that the reactive species h+, e− and •O2−play the predominant role in the photocatalytic system of CBO/BMO hybrids. The improved photocatalytic activity of CBO/BMO composites was mainly attributed to the promotion of spatial separation and migration rate of photoproduced electron-hole pairs, enhancement of visible light absorption and more generation of reactive species (•O2−) on the interface of catalyst and water which was demonstrated by nitroblue tetrazolium (NBT) and EPR. Our work indicated that construction of CuBi2O4/Bi2MoO6 p-n heterostructure photocatalyst is a promising environmental friendly alternative method to deal with the biohazards of pathogenic microorganisms.

The expression and purification of a new complement inhibitor complement receptor of the immunoglobulin superfamily

Objective To construct the prokaryotic expression plasmid pSmartI-SUMO-complement receptor of the immunoglobulin superfamily (CRIg) of the CRIg, next express and purify it by E. coli expression system, finally detect the activity of the expressed product. Methods Total RNA was extracted from human liver cell HHMa, and the gene sequence encoding the extracellular domain of human CRIg was cloned onto the pSmart-I vector by reverse transcriptase-polymerase chain reaction (RT-PCR) and Seamless cloning. Positive colonies were selected for plasmid extraction and sequencing, and the expressed products were detected by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. Finally, the activity was detected by alternative pathway hemolysis assay. The experiment was repeated three times and averaged to plot the hemolysis curve. Results The PCR product amplified from human CRIg was 819 bp in length. The sequencing result was aligned with the extracellular segment of human CRIg. After the plasmid was transformed into Rosetta strain, it was found by IPTG that CRIg could be stably expressed in the supernatant. Hemolysis experiments demonstrated that a 25 μm protein concentration could almost completely inhibit hemolysis. Conclusion CRIg can achieve soluble expression in prokaryotic expression system. Key words: Complement receptor of the immunoglobulin superfamily gene; Complement; Immune inhibitor; Seamless cloning; Prokaryotic expression plasmid

Highly efficient visible light driven photocatalytic inactivation of E. coli with Ag QDs decorated Z-scheme Bi2S3/SnIn4S8 composite

Here, a novel Ag QDs modified Bi2S3/SnIn4S8 composite with direct Z-scheme charge carriers transfer model was synthesized through combining with chemical reduction and solvothermal methods for high-efficient photodisinfection toward E.coli under visible light stimulation (λ>420 nm). The 200 Ag QDs/Bi2S3/SnIn4S8 hybrid exhibited the strongest photocatalytic inactivation activity of E.coli which could completely inactivate the bacteria with 4 h visible light excitation. The photoinactivation mechanism can be ascribed to the wreck of cell membrane, leakage and damage of intracellular biomolecules including protein and DNA as demonstrated by fluorescence-based live/dead stain, sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and agarose gel electrophoresis (AGE). Moreover, the trapping and verification experiments of free radical revealed that the reactive species h+, e− and O2- play the chief role in photodisinfection reaction. The photoelectrochemical techniques disclosed that the enhanced photocatalytic activity could mainly be assigned to the synergistic effect of Ag QDs, Bi2S3 and SnIn4S8, which could improve the visible light absorption ability, separation and transfer efficiency of photoinduced charge carriers, enlarge specific surface area through the surface plasma resonance (SPR) effect and the electron trapped and injected of Ag QDs. Additionally, the Ag QDs/Bi2S3/SnIn4S8 composite displayed a potential application in organic contaminant removal. Our work firmly exhibited a promising environmental friendly substitutable strategy for wasterwater purification caused by pathogenic bacteria and organic pollutant.

Structure Changes in Relation to Digestibility and IgE-Binding of Glycinin Induced by pH-Shifting Combined with Microbial Transglutaminase-Mediated Modification

This study aimed to evaluate the effects of pH-shifting treatment combined with microbial transglutaminase (MTG)-mediated modification on the structure, digestibility, and IgE-binding of glycinin. Glycinin was incubated in acidic (pH 1.0) or alkaline (pH 13.0) solutions to induce protein structure to unfolding followed by refolding for 1 h at pH 7.0. Afterwards, glycinin was incubated with MTG under appropriate conditions. Sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE), circular dichroism, UV absorption spectra, and surface hydrophobicity were considered to measure the changes in the structure of glycinin. The digestibility and IgE-binding of glycinin were determined by Tricine-SDS-PAGE and ELISA, respectively. The results showed that pH 1.0 shifting caused the unfolding of the spatial structure of glycinin and the formation of some polymers via disulfide bond. After glycinin was incubated with MTG, the protein preferentially underwent embedding and folding. The acidic compound-modified glycinin was stable for digestion. Under pH 13.0 shifting treatment, glycinin was partially hydrolyzed, and the MTG-modified alkaline-treated glycinin was slightly affected with a good digestibility. Compound modification could reduce the IgE-binding of glycinin, especially under alkaline conditions. Our findings suggested that alkaline pH shifting combined with MTG cross-linking can be an efficient approach to reduce the IgE-binding of glycinin with a labile digestion.

PentraSorb C-Reactive Protein: Characterization of the Selective C-Reactive Protein Adsorber Resin.

C-reactive protein (CRP) is well known as a general marker of inflammation. It furthermore represents a reliable risk factor for cardiac events and mediates tissue damage in acute myocardial infarction (AMI). It has been demonstrated that selective CRP depletion by extracorporeal apheresis in a porcine AMI model had beneficial effects on the infarcted area and the cardiac output. We therefore developed a novel adsorber for CRP apheresis from human plasma (PentraSorb CRP). It is intended for use in the clinic as therapy for patients suffering from AMI or other acute inflammatory diseases with elevated CRP plasma levels. The PentraSorb resin specifically bound CRP from human blood plasma and almost no other proteins as determined via Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). The resin further efficiently and selectively depleted CRP from plasma with low as well as high CRP concentrations (10-100 mg/L) at different flow rates, ranging from 17 to 40 mL/min. The resin was regenerable for up to 200 times without losing its CRP binding capacity or affecting biocompatibility. The depletion of CRP from plasma was comparable between the utilized small-scale column (0.5 mL resin) and the PentraSorb CRP adsorber (20 mL resin volume). The established features can therefore be applied to the clinical setting. In summary, PentraSorb CRP provides a novel, specific, and efficient CRP-binding resin that could be used in apheresis therapy for patients suffering from inflammatory diseases such as AMI, stroke, acute pancreatitis, and Crohn's disease.

Construction, Expression, and Characterization of rSEA-EGF and In Vitro Evaluation of its Antitumor Activity Against Nasopharyngeal Cancer.

Staphylococcal enterotoxin A is well known as a superantigen and able to be used for cancer immunotherapy. In this study, recombinant Staphylococcal enterotoxin A was genetically conjugated to epidermal growth factor to produce a chimeric protein recombinant Staphylococcal enterotoxin A–epidermal growth factor expressed in Escherichia coli. The recombinant Staphylococcal enterotoxin A–epidermal growth factor protein was purified using Strep-Tactin affinity chromatography and Endotoxin Removal Resin and identified by sodium dodecyl sulfate-polyacrylamide gel electropheresis and liquid chromatography-tandem mass spectrometry analysis. Furthermore, in vitro experiments showed purified recombinant Staphylococcal enterotoxin A–epidermal growth factor could successfully bind to the human nasopharyngeal carcinoma cell line CNE2, significantly promote the proliferation of human peripheral blood mononuclear cells, and enhance the secretion of several cytokines that have broad antitumor activities, such as interferon-γ, tumor necrosis factor-α, and interleukin-2 . Importantly, recombinant Staphylococcal enterotoxin A–epidermal growth factor significantly inhibited proliferation of CNE2 cells and promoted apoptosis in CNE2 cells when cocultured with peripheral blood mononuclear cells. Finally, both the binding of recombinant Staphylococcal enterotoxin A–epidermal growth factor and the toxicity of recombinant Staphylococcal enterotoxin A–epidermal growth factor-activated peripheral blood mononuclear cells were demonstrated as specific and only effective on high epidermal growth factor receptor-expressing cell lines. In all, our work suggests that recombinant Staphylococcal enterotoxin A–epidermal growth factor serves as a promising novel immunotherapeutic agent. More in vivo and in vitro studies are needed to verify its antitumor potency, as well as investigate the underlying mechanisms in cancer immunotherapy.

Effect of heating temperature and duration on the texture and protein composition of Bighead Carp (Aristichthys nobilis) muscle

ABSTRACTThe effect of heating temperature and duration on the texture and protein composition of bighead carp (Aristichthys nobilis) muscle was investigated. Samples were heated at 90°C, 100°C, 110°C, and 120°C for different times. The results showed that cooking loss increased significantly with increasing temperature and heating time, and a fractional conversion model was used to describe the increase. Hardness, chewiness, and gumminess exhibited a similar four-phase change with two peaks at 90°C, 100°C, and 110°C, and the trend was not observed at 120°C. Analysis of sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) revealed that contents of myosin heavy chain decreased entirely to disappear and contents of actin significantly decreased. Increasing the intensity of the heavy molecular weight bands indicated the aggregation of myosin. Content of water-soluble proteins presented an increasing trend, which reflected the protein degradation and the release of low-molecular-weight compounds.

The impact of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3 gene on the proliferation of prostate cancer cells

Objective To investigate the expression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3 (PFKFB3) gene in the prostate cancer tissues and its impact on the proliferation of prostate cancer cells PC3 and DU145. Methods Immunohistochemical staining was used to detect the expression of PFKFB3 in the prostate cancer tissues and the adjacent tissues (antibody concentration 1∶50). Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR, 40 cycles, 188 bp) and Western blotting [30 μl, 10% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)] were applied to detect the mRNA and protein expression levels of PFKFB3 expression in the prostate cancer cells and prostate epithelial cells. After knocking out PFKFB3 gene, plate cloning (40 μmol/L H2O2) formation assay and cell growth assay [20 μl methyl thiazol tetrazolium (MTT)] were applied to measure the cell proliferation. Results The expression of PFKFB3 in the prostate cancer tissues (2.42±0.16) was significantly higher than that in the adjacent tissues (1.38±0.10, P=0.031). The mRNA and protein expression levels of PFKFB3 in prostate cancer cells were significantly higher than those in the prostate epithelial cells (P=0.015), 2.0-2.6 times and 2.5-4.5 times respectively. When knocking out the PFKFB3 gene, cloning formation assay showed that the cloning formation rate decreased as 22%-35% compared to the control (P=0.018). Cell growth assay showed that the proliferation of prostate cancer cells was inhibited as 28%-30% at day 4 (P=0.026). Conclusion PFKFB3 gene is highly expressed in prostate cancer cells and prostate cancer tissues. PFKFB3 plays a promotional role in the prostate cancer cells. Key words: 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3; Prostate cancer; Proliferation

Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L-1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The Km and Vmax values of the purified protease for casein were calculated to be 4.89 mg mL-1 and 1111.11 U mL-1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry.

A study of outer membrane protein and molecular epidemiology of carbapenem-resistant Acinetobacter baumannii

To study the characteristics of the distribution and drug resistance of Acinetobacter baumannii, and the epidemiology of the main strains among wards and hospitals, and to investigate the role of outer membrane protein in producing resistance against carbapenems. 145 Acinetobacter baumannii strains were collected from July 2013 to July 2014 from Huangdao Hospital Affiliated to Qingdao University and 401st Army Hospital. Antimicrobial susceptibility test was carried out with K-B disk diffusion method. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) was used for DNA typing and test of homology. The carO gene of outer membrane protein was amplified by PCR, and the outer membrane proteins were extracted by ultrasonication and ultracentrifuge method from 30 randomly selected carbapenem-resistant Acinetobacter baumannii and 17 carbapenem-sensitive strains. Sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) was used to analyze the expressions of outer membrane proteins. 145 Acinetobacter baumannii strains were generally resistant to 16 common antimicrobial agents, with the highest susceptibility rate of 79.3% for minocycline, followed by susceptibility rate of 40.7% for amikacin. There were 128 carbapenem-resistant strains (resistance rate of 88.3%), 137 multidrug-resistant strains and 126 extensively drug-resistant strains. The detection rates of carO gene were 97.7% (125/128) and 17.6% (3/17) in carbapenem-resistant and sensitive strains respectively. Around position of relative molecular mass 47,000, 16 of 17 sensitive isolates were expressed this protein, while 20 of 30 resistant ones were detected nothing or fade; 13 of 17 sensitive isolates were expressed around position of relative molecular mass 37,000 and 29,000 while 25 were detected nothing or fade around position of relative molecular mass 37,000 and 23 were detected nothing or fade around position of relative molecular mass 29,000 in 30 resistant ones. 145 Acinetobacter baumannii were classified into 8 types based on ERIC-PCR electrophoresis patterns, and the major prevalence types were type A (71 strains) and type E (37 strains). Drug resistance of clinically isolated Acinetobacter baumannii is a serious problem in two hospitals; drug-resistant strains are spread and epidemic among wards and hospitals. The carO gene of outer membrane protein is widespread in carbapenem-resistant Acinetobacter baumannii. The loss or fading of outer membrane protein may play an important role in Acinetobacter baumannii resistance to carbapenems drugs.

Application of label-free quantitative proteomics in the screening for differentially expressed serum proteins in patients with atopic dermatitis

Objective To screen differentially expressed serum proteins by label-free quantitative proteomics in patients with atopic dermatitis,in order to discover serum protein markers for atopic dermatitis.Methods Serum samples were collected from 6 patients with atopic dermatitis and 6 healthy human controls.Then,the samples from the patients were pooled together,so with those from the controls.Proteins were extracted from the two joint serum samples followed by separation with one-dimensional sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE).Peptides were obtained by enzymolysis in protein gels,which were subsequently identified by high performance liquid chromatography tandem mass spectrometry.The Mascot software was used for database searching,and the Scaffold software for relatively quantitative analysis of proteins.Results Totally,76 proteins with a difference of greater than 1.5 folds were identified between the serum samples of patients with atopic dermatitis and controls.There were 50 up-regulated and 26 down-regulated serum proteins in the patients compared with the healthy controls.Among these differentially expressed proteins,26 were only detected in the sera of the patients,and 14 proteins in those of the controls.Conlusion Differences exist in the expression of serum proteins between atopic dermatitis patients and healthy people. Key words: Dermatitis, atopic; Proteomics

Sulfated modification of the polysaccharide from Cordyceps_gunnii mycelia and its biological activities

A chemically new sulfated polysaccharide (SPS50) was prepared from the water soluble polysaccharide (PS50), isolated from Cordyceps_gunnii mycelia, by concentrated sulfuric acid method. The yield of crude SPS50 was 62.34% and its specific rotation was [α]D20=−36.75°. The structural characteristics of this chemically sulfated polysaccharide were determined based on the infrared analysis (IR), high performance liquid chromatography (HPLC) and sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). Its biological properties including anti-oxidant and anti-tumor activities were also investigated. The results showed that the anti-oxidant capacity of SPS50 was not as good as SP50 and the anti-tumor activity of SPS50 was much better than PS50. SPS50 showed evident growth inhibition on K562 cells. The tumor inhibition ratio of SPS50 against K562 cells was 69.92%.

Study on proteomic profile of normal ependyma and ependymoma of spinal cord by using three-diamensional Shot-gun and MS/MS technology

Objective To research focused on proteonic profile of normal ependyma and ependymoma of spinal cord by using three-diamensional Shot-gun and MS/MS technology.Methods Eight spinal ependymoma and four normal ependyma tissues were obtained through operations.The samples were progressed total proteins extraction,sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)electrophoresis,gels cutting,protein enzymolysis and MS/MS.The samples were progressed total proteins extraction.The identified proteins were analysis by bioinformatics. Results A normal spinal ependyma proteome consist of 2758 proteins while the ependymoma proteome consist of 2721.1631 proteins were detected both in tumor group and in control group,1090 proteins were only detected in tumor group while 1127 proteins only in control group.Conclusion All the proteomic profile studies by using three-diamensional SDS-PAGE and Shot-gun and MS/MS technology provide a foundation for the further tumor-genetical research of spinal ependymoma. Key words: Proteomics; Spinal ependymoma; Bioinformaties; Mass spectrometry

Expression, purification and immunocompetence analysis of a recombinant membrane protein, Tp0136, of Treponema pallidum

Objective To clone and express the Tp0136 gene of Treponema pallidum,to purify the recombinant protein and to evaluate its immunocompetence.Methods The full-length Tp0136 gene was synthesized,then subcloned into the expression vector pET28a to construct a recombinant plasmid,pET28a-Tp0136,which was subsequently transfected into E.coli Rosetta for protein expression.The recombinant protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography,and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot.New Zealand rabbits were immunized with the recombinant Tp0136 (rTp0136)protein,and anti-Tp0136 polyclonal antibodies in sera of the rabbits were examined by indirect enzyme linked immunosorbent assay (ELISA) with rTp0136 protein as the coating antigen.Also,positive sera were obtained from patients with syphilis and examined by Western blot to identify the immunoreactivity of the rTp0136 protein.Results The E.coli expression vector pET28a-Tp0136 was constructed successfully,and rTp0136 protein was also successfully expressed with a molecular weight of about 50 kD and a purity above 95％.High titres of anti-rTp0136 antibodies were detected in sera of rabbits immunized with the rTp0136 protein,and Western blot showed that the rTp0136 could specifically react with the sera from syphilitic patients,which proved the high immunogenicity and immunoreactivity of the recombinant protein.Conclusions The full-length Tp0136 membrane protein is successfully expressed with a high immunocompetence,and Tp0136 membrane protein may play an important role in the pathogenic mechanisms of Treponema pallidum. Key words: Treponema pallidum; Tp0136; Gene expression; Membrane proteins; Immunocompetence

Construction and expression of the recombinant plasmid pET32α-Sj26GST of Schistosoma japonicum in Escherichia coli BL21(DE3)

Objective To construct and express the recombinant plasmid pET28α-Sj26GST-Sj32 of Schistosoma japonicum(Sj) in Escherichia coli BL21 (DE3). Methods Total RNA was extracted from Sj adult worms by ultrasound-breaking, Sj26GST and Sj32 antigen gene was respectively amplified by RT-PCR from the total RNA; Sj26GST-Sj32 fusion gene obtained with gene splicing by overlap extension(SOEing) was cloned into prokaryotic expression plasmid pET28α and transformed into Escherichia coli BL2 (DE3) to construct pET28α-Sj26GST-Sj32;BL21 (pET28α-Sj26GST-Sj32) was induced with isopropyl-β-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blotting. Results The 1991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pET28α by restriction analysis and PCR identification, the recombinant plasmid pET28α-Sj26GST-Sj32 was successfully constructed; the relative molecular mass of the expressed recombinant protein was approximately 69 × 103 by SDS-PAGE, and the amount of the expressed protein was 25% of the total bacterial proteins; the fusion protein could be recognized by sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant plasmid pET28α-Sj26GST-Sj32 is successfully constructed and highly expressed in Escherichia coli in fused form with His-tag, and the expressed fusion protein shows specific antigenicity. Key words: Schistosoma japonicum; Recombinant plasmid pET28α-Sj26GST-Sj32; Escherichia coli; Vaccines, synthetic

Cloning and expression of Chlamydia trachomatis heat shock protein 60 gene.

Objective To clone and express Chlamydia trachomatis (Ct) heat shock protein 60 (hsp60) gene. Methods The hsp60 gene fragment was amplified from Ct chromosomal DNA by PCR. After purification and digestion with enzymes Sal I and Not I , the hsp60 gene fragment was inserted into the compatible site of prokaryotic expression vector pET-28a. The constructed recombinant plasmid was identified by PCR, restriction enzyme cleavage and sequencing, then, it was transfected into an expression strain Escherichia coli BL21 (DE3). The expression of fusion protein was induced by isopropy-β-D- thiogalactoside (IPTG) in the host bacteria, and the expressed product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blot. Results PCR and restriction enzymes cleavage analysis confirmed that the hsp60 gene was successfully cloned into the recombinant plasmid. DNA sequencing showed that the sequence of cloned gene was fully consistent with the published sequence in Genebank. As revealed by SDS-PAGE, the size of expressed fusion protein approximated 60 kilodaltons, and Western-blot confirmed the expressed product to be the expected protein. The final concentration of fusion protein was 17.85 mg/L with a purity of more than 90%. Conclusions A recombinant expression plasmid pET-28a-hsp60 is successfully constructed in this study, and soluble hsp60 protein is expressed by the recombinant plasmid-transfected E. coli. Key words: Chlamydia trachomatis; Heat shock protein 60; Recombinant fusion proteins; Geneclone

Cloning, expression and functional analyses of human platelet-derived growth factor-B chain peptide for wound repair of cat corneal endothelial cells

To investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application. Total RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively. PDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells. The construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.

CDNA cloning, expression and bioactivity of porcine BAFF

B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF) is critical for B cell survival, maturation and T cell activation by acting through its three receptors, BAFF-R, BCMA and TACI. In the present study, a porcine BAFF cDNA, designated pBAFF, was cloned by RT–PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pBAFF consists of 805 bp with a 702 bp open reading frame, encoding 233 amino acids. The deduced amino acid sequence contains a predicted transmembrane domain and a putative furin protease cleavage site corresponding to other identified BAFF homologues. The amino acid similarity between the functional soluble parts of pBAFF and human BAFF (hBAFF) or chicken BAFF (cBAFF) is 93% and 85%, respectively, with identity at the amino acid level was 88% and 76%, respectively. The characteristic of the three-cysteine residues of BAFF is conserved in pBAFF. RT-PCR showed that BAFF is expressed in many tissues in the pig, including spleen, liver, lung, heart, intestine, kidney, thymus and PBLs. Recombinant soluble pBAFF (psBAFF) fused with His 6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. In vitro, purified psBAFF co-stimulates the proliferation of not only porcine B cells but also human B cells. In addition, hsBAFF binds to porcine B cells and has a positive effect on their proliferation. These findings indicate pBAFF plays an important role in proliferation of porcine B cells and functional cross-reactivity occurs between porcine and human BAFF. In vitro expression of bioactive psBAFF provides the basis for further investigation of its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and an immunotherapeutic in pig. It also provides the basis for investigations on the role of BAFF in this important domestic species and an animal model for human diseases.

Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate

To construct a new target-oriented conjugate of humanized carcinoembryonic antigen (CEA) specific single chain variable fragment (scFv) and mitomycin (MMC) against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells. The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamHI and EcoRI in its upstream and downstream. PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamHI and EcoRI, and connected to an expression vector which also has the restriction enzyme cleavage sites BamHI and EcoR. Expression of the reaction was induced by isopropy-beta-D-thiogalactoside (IPTG). Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay (ELISA). The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry (FCM). Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) confirmed that this vector correctly expressed the fusion protein. ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent manner. The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.