Abstract:Doppel protein (abbreviation Dpl) is a newly recognized Glycosyl phosphatidyl inositol (GPI) anchored and highly glycosylated protein, which is similar to prion protein (PrP) in the chemical structure. The encoding gene of Dpl named PRND locates at the downstream of the prion protein gene (PRNP). These two proteins are different in physiological functions. The expression of Dpl focuses on testis tissue at the adult, and takes an important role in maintaining sperm integrality, normal fertility, and motion ability. We reviewed the biological characters, physiological functions of Dpl and its effects on male reproduction in order to provide theory guidance for the study on physiological function and male reproduction controlling.

Abstract:Nicotinamide adenine nucleotide (NADH), the key cofactor in the metabolic network, plays an essential role in biochemical reaction and physiological function of industrial strains. Manipulation of NADH availability and form is an efficient and easy way to redirect the carbon flux to the target metabolites in industrial strains. We reviewed the physiological function of NADH. Detailed strategies to manipulate NADH availability are addressed. NADH manipulation to enhance metabolic function of industrial strains was discussed and potential solutions were suggested.

Abstract:The a-hemolysin protein of Staphylococcus aureus, which was expressed in Escherichia coli BL21 (DE3) with recombinant pET32a+-a-HL plasmid, was purified with gel filtration chromatography (GFC) and Ni-NTA spin columns. The quality and biological characteristic were compared. First, the purified products were analyzed with SDS-PAGE, and the expected protein band was with a molecular mass of 53 kD. Second, protein concentration was determined by the method of Bradford, and the median hemolytic dose potency (HD50) was finally analyzed with rabbit erythrocyte. The protein purified with GFC was 0.337 mg/mL, its hemolysis activity was 1519 HU/mg, and hemolysin yield was 14.04%. Meanwhile, the protein purified with the Ni-NTA Spin Columns was 0.35 mg/mL, its hemolysis activity was 1463 HU/mg, and hemolysin yield was 17.5%, respectively. The results showed that there is no significant difference in the quality, hemolysis activity and yield of the recombinant proteins purified with Ni-NTA spin columns and GFC.

Abstract:In order to obtain an attenuated vaccine candidate for enteropathogenic Escherichia coli (EPEC) O45, a ler deletion mutant of pig enteropathogenic E. coli (PEPEC) O45 was constructed by using the suicide vector pCVD442, termed as PEPEC O45(Dler). The culture supernatant of PEPEC O45(Dler) deletion mutant was inoculated in vero cell culture. PEPEC O45(Dler) deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the PEPEC O45(Dler) deletion mutant and the virulent strain O45 respectively. Mice were observed daily for clinical signs and weight changes. Test group of mice inoculated with PEPEC O45(Dler) gained weight normally and experienced no clinical signs. In contrast, control group of mice inoculated with virulent strain O45 exhibited weight loss and all died in four days. In another experiment, pregnant mice and pig were orally vaccinated by PEPEC O45(Dler) twice at interval of 14 days respectively. Subsequently, the suckling mice and pig were orally challenged with O45 at 7 days of age respectively. The results showed that 80% of the sucking mice born by vaccinated mice and 75% of the sucking pig born by vaccinated pig were survival; 15% of the sucking mice born by non-vaccinated mice and 10% of the sucking pig born by non-vaccinated pig were survival. This study demonstrated that PEPEC O45(Dler) deletion mutant lost the toxigenicity to vero cell and to be safety to mice and pig. Oral immunization can induce specific immune responses in mice and pig, and this mutant strain could be used as an attenuated vaccine candidate against PEPEC O45.

Abstract:The cDNA of an i type lysozyme was cloned from Stichopus japonicus (named as SjLys). The DNA fragment of the mature SjLys was subcloned into expression vector of pET-32a (+) to construct the recombinant plasmid of pET32a (+)-SjLys. The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) pLysS and induced by isopropylthio-β-D-galactoside (IPTG). The recombinant protein expressed as inclusion bodies was denatured, partially purified and refolded to be an active form. The bacteriolytic activity of recombinant protein purified by the metal-chelating was 19.2 U/mg. The antibacterial activity of the purified recombinant SjLys (rSjLys) was analyzed. The rSjLys protein displayed inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. In particular, rSjLys had a strong inhibitive activity on Vibrio parahaemolyticus and Pseudomonas aeruginosa, both the most common pathogenic bacteria in the marine animals. The heat-treated rSjLys exhibited more potent activities against all tested bacteria. These results indicated that the S. japonicus lysozyme was the enzyme with combined enzymatic (glycosidase) and non-enzymatic antibacterial action, and it had a wide antibacterial spectrum. Therefore, it is suggested that the S. japonicus lysozyme should be one of the important molecules against pathogens in the innate immunity of sea cucumbers.

Abstract:Δ5-fatty acid desaturase is the key enzyme in synthesis of arachidonic acid. Two specific fragment was cloned from genomic DNA and total cDNA of Phaeodactylum tricornutum through PCR with primer designed according to the reported sequences, respectively 1520 bp and 1410 bp. Comparison of the genomic and cDNA sequences revealed that the Δ5-fatty Acid Desaturase gene from genomic DNA had an 110 bp intron. The 1.4 kb was subcloned into the yeast-E. coli shuttle vector pYES2.0, then an expression recombinant plasmid pYPTD5 contatining target gene was constructed. The plasmid pYPTD5 was transformed into defective mutant INCSc1 of Saccharomyces cerevisiae for expression by electrotransformation method. Dihomo-g-linolenic acid was provided as an exogenous substrate to the yeast cultures, with galactose as inducer. By GC detecting, the recombinant S. cerecisiae had arachidonic acid. The results indicated that high level expression of Δ5-fatty acid desaturase, and the substrate conversion reached 45.9%.

Abstract:Very high gravity (VHG) ethanolic fermentation is a new perspective technology for the fuel ethanol production. Compared with traditional hot cook process in most ethanol plants, uncooked process using milled grain slurry in combination with granular starch hydrolyzing enzymes makes high gravity fermentation much easier to control. In this study, dry solids staging technique was first time reported in uncooked process for fermentation ethanol. We further studied the difference between the new process and the batch fermentation, including different initial fermentation concentrations and different starting times. The results showed that at the same dry solid concentration of 30% and the same enzyme dose at 0.22% (W/W), the final ethanol output of new process was increased to 18.50% ( V/V) from 17.06% (V/V) of the conventional process. This study demonstrated the new application of uncooked fermentation technology.

Abstract:To construct a three-dimensional (3D) model of histidine kinase (HK) YycG protein in Streptococcus pneumoniae and to investigate the interaction between YycG and its substrate ADP for the purpose of providing a theoretical basis for YycG selective inhibitor discovery, we constructed a 3D model of YycG protein by homology modeling, and assessed the reliability of the model using ProCheck and Profile_3D software. Besides, the active-site cavity of YycG and the residues key for substrate interaction were analyzed by Autodock4.0. Sequence alignment indicated that the YycG of S. pneumoniae was homologous to that of Thermotoga maritima. The constructed 3D model of YycG adopted a similar folding pattern to the template and the two matched well. The conservative amino acids in the substrate-binding pocket, such as Asn145, Asn149 and Lys152, as well as the hydrophobic residues at the bottom of the pocket played important role in binding and hydrolyzing substrate ADP. We have successfully constructed a reliable model of YycG protein. The model can be used as a starting point for designing antibacterial drugs.

Abstract:In order to realize over-expression of Burkholderia cepacia (B. cepacia) lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene (T7 RNAP) from the BL21 (DE3) and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene (lipA) promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMΔlipAlipB, pUCPCMΔlipA, pUCPCMΔlipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/mg, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMΔlipB were enhanced to some extent. The specific activities of wild type B. cepacia and B. cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain.

Abstract:The structure gene pelA from Thermotoga maritima MSB8 encoding pectate lyase was amplified and ligated into pHsh, resulting pHsh-pelA. Through structural optimization on pHsh-pelA, the ultimate plasmid, pHsh-pelC, which possessed the most appropriate structure and free energy of mRNA, was obtained. Pectate lyase C (PelC) was obtained after expressing pHsh-pelC in Escherichia coli JM109. The optimum activity of PelC was determined at pH 8.5 at 90oC, with a half-life for almost 2 h at 95oC. PelC was stable at the pH range of 8.2-9.8, and was dependent on Ca2+ for activity and stability. The enzyme kept stable for a long time and possessed a high level of activity at 60oC. The kinetic assay using polygalacturonic acid (PGA) as substrate gave Km and Vmax of 0.11 mmol/L and 327 U per mg of protein. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant PelC was about 43 kD, which was exactly the size predicted. The expression vector system of the heat shock plasmid pHsh owned such advantages as high expression level and cheap induction. Moreover, the superior stability of the recombinant enzyme laid the base for large-scale fermentation application.

Abstract:Using Affymetrix’s Chicken Genome Array, we used total RNA isolated from the gonads of male and female chicks at embryonic day 9 to identify the genes differentially expressed between male and female. Statistical results show 19 493 genes expressed in male chick’s embryonic gonads and 19 368 genes expressed in female. There were 145 genes specificity expressed in male and 189 genes in female. The gene ontology classification (GOC) indicated these differentially expressed genes were mainly involved in cellular component, cell process and molecular banding, a part of genes were involved in organelle component, metabolic process, biologic process, catalytic activity and signal transducer activity. Some genes had reported for sex determination and differentiation in birds, such as avian sex-specific avian sex-specific W-linked, chomodomain-helicase-DNA-binding protein 1 and sex determining region Y-box 9. In addition, we also found several genes or hypothetical proteins were unknown function for the gonad differentiation and development, focus to their biological function and expression pattern in further works would provide a beneficial reference for understand the mechanism of sex differentiation and determination in birds.

Abstract:The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG, Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.

Abstract:To investigate the transfer and expression of Snail gene in human bone mesenchymal stem cells (MSCs) and to study effects of Snail gene modification on the CXCR4 expression of human MSCs and their capacity of migration to SDF-1 in vitro. The plasmid PCAGGSneo-Snail-HA or the control vector of PCAGGSneo was transferred into the cells. Fluorescence activated cell sorting analysis, immunofluorescence staining and RT-PCR were used to study the expression of CXCR4 by MSCs. Chemotaxis assays were performed to evaluate the migratory capacity of MSCs-Sna and MSCs-neo to SDF-1 in vitro. For the blocking assay, CXCR4 blocking antibody was added into cell culture. CXCR4 expression was higher in MSCs-Sna than that in MSCs-neo (P<0.05). Chemotaxis assays showed that SDF-1α stimulated migratory activity of MSCs-Sna more than MSCs-neo in vitro (P<0.05). Moreover, the SDF-1a-induced migratory activity of MSCs-Sna was inhibited in a concentration-dependent manner by a CXCR4-blocking antibody. It was concluded that Snail enhanced expression of CXCR4 in MSCs, providing a plausible mechanism for Snail-mediated MSCs transmigration to damaged tissues in vivo where SDF-1 has been shown to be up-regulated as part of injury responses.

Abstract:According to the GenBank sequences (GenBank Accession No. AF539467), one pair of primers was designed to amplify hly gene of Aeromonas hydrophila by PCR. After sequencing, homology analysis indicated that a DNA fragment of 1485 bp was amplified from isolated DNA from Aeromonas hydrophila, and it shared more than 99% homology in nucleotide sequence compared with other reference strains in Genbank. The gene was cloned in pET-28a vector to construct a recombinant plasmid pET-28a-hly, which was transformed into Escherichia coli BL21 (DE3), and the recombinant strain BL21(DE3)(pET-28a-hly) was obtained. The hemolysin was highly expressed when the recombinant strain BL21 (DE3) (pET-28a-hly) was induced by IPTG. The expressed protein was 56 kD as estimated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the expressed Hly protein was confirmed by Western blotting. Mice were immunized with inactivated whole bacteria vaccine and the genetic engineering vaccines showing promise that all these vaccines have a high protective ability. The results showed that the recombinant strain BL21 (DE3)(pET-28a-hly) could be candidate of hemolysin toxoid vaccine to provide protective immunity against diseases caused by Aeromonas hydrophila.

Abstract:In the present study, we constructed a prokaryotic expression vector containing SOX4 protein encoding sequences. The GST-SOX4 soluble protein was expressed in Escherichia coli DH5a and purified by glutathione sepharose-4B. The purified recombinant protein was used to immunize Balb/C mice and the monoclonal antibody against SOX4 was prepared by using hybridoma technique. The titer of the antibody was determined as 1×10-5 by indirect ELISA. The specificity of the antibody was verified by Western blotting analysis. The monoclonal antibody specifically recognized the overexpressed exogenous SOX4 protein as well as endogenous SOX4 protein. The expression level of SOX4 protein in different cell lines and mouse tissues was detected by using the antibody. Differential expression of the protein was demonstrated by Western blotting. The data indicated that the antibody was specific. The antibody can be used as an important tool for further exploration of the role of SOX4 in tumorigenesis.

Abstract:In this study, we amplified human lysosomal acid b-glucosidase (GlcCerase) gene by RT-PCR from human placenta, and analyzed the sequence of the PCR product cloned in pMD-19T. The gene identity was 99% comparable to that of the reported human GlcCerase cDNA sequence in GenBank. The GlcCerase gene digested with Xho I was subcloned into eukaryotic express vector pEGFP-C1 to generate recombinant expression vector pEGFP-GlcCerase. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pEGFP-GlcCerase into COS7 cells by liposome. GlcCerase mRNA was expressed and the activity of GlcCerase was also detected in COS7 cells. This study would lay a foundation for the function of GlcCerase and its production by transgenic bioreactor.

Abstract:Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4°C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operate in the cell.

Abstract:In order to express and purify the catalytic domain of SHP-1/SHP-2 (named as D1C and D2C respectively) and determine their kinetics, the constructed D1C and D2C plasmids were transformed into Escherichia coli BL21 and the expression was induced with IPTG. The harvested cells were suspended in extraction buffer. After sonication, the solution was applied to HPLC and the results were confirmed by SDS-PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic phosphotyrosine (pY) as substrate by malachite green method and analyzed by Lineweaver-Burk plot calculation. From this study, we found D1C and D2C were expressed successfully in soluble state in Escherichia coli BL21 and purified efficiently with HPLC system. The molecular weight of D1C was 34.6 kD, and its Michaelis constant (Km) was 2.04 mmol catalytic constant (Kcat) was 44.98 s, specific constant (Kcat/Km) was 22.05 L/(mmol·s); the molecular weigh of D2C was 35.3 kD, and its Michaelis constant (Km) was 2.47 mmol, catalytic constant (Kcat) was 27.45 s, specific constant (Kcat/Km) was L/(mmol·s). The enzyme activity of D1C is stronger than that of D2C.

Abstract:To investigate the inhibitory effect and anti-cancer mechanism of adenovirus mediated IL-24 gene expression on the human U251 glioma cell. U251 glioma cells were infected with Ad-IL-24 at various multiplicity of infection (MOIs). Cell proliferation was determined by MTT assay. Cell apoptosis was detected by flow cytometry and Hochest staining. The transcription of apoptosis-related genes was analyzed by reverse transcription-PCR (RT-PCR), and the expression of Cleaved Caspase-3 was analyzed by Western blotting. The result showed that the growth of U251 glioma cells was significantly inhibited by Ad-IL-24 at the MOI of 100. The apoptotic rate of U251 glioma cells was 42% 72 h after infection with Ad-IL-24. Four days after infection, the growth of the U251 glioma cells was inhibited to 50%. RT-PCR showed that Ad-IL-24 not only up-regulated expression of bax/ bcl-2, ICE, C-myc, p53 and down-regulated the expression of HIF-1α, but also enhanced Caspase-3 activation, eventually resulting apoptosis. Taken together, these results suggest that infection of U251 glioma cells with Ad-IL-24 can inhibit growth and induce apoptosis significantly by the regulation of apoptosis- related genes.

Abstract:Male germ-line stem cells (mGSCs) have the capability of self-renewal and latent capability of differentiation. mGSCs is the unique diploid immortal cell which can transfer genetic information to filial generation. The combination of transgenic technology and mGSCs heterotransplanting will supply new opportunities and paths to cloning animal, transgenic animal and gene therapy of some human hereditary disease. We studied the isolation and cultivation of mGSCs that were isolated and purified from 5-6 month old bovine fetal testis, new born bovine testis by adopting mixed enzymes digestion and different attaching velocities methods. The results showed that sertoli cells were indispensable to mGSC’s proliferation and differentiation in vitro. The sertoli cells in logarithmic phase have a significant effect on mGSC’s attaching, proliferation and differentiation. Co-culture with Sertoli cells, mGSCs differentiated to long sperm after 16 days. A preliminary system for mGSC’s inducing differentiation was established.

Abstract:We observed the effects of IKVAV self-assembling peptide nanofiber scaffold (SAPNS) on olfactory ensheathing cells (OECs). The IKVAV molecules were triggered to self- assemble to interconnected nanofibers hydrogel by adjusting pH of solution and adding of DMEM/F12 culture medium. Atomic Force Microscopy (AFM) showed that self-assembly hydrogel was consisted of the interconnected nanofibers, which varied from three nm to five nm in diameter and hundreds nanometer in length. The primary OECs were isolated from rat olfactory bulb and purified by differential adhesion twice. At days 12, the purity of OECs was 85% according to immunostaining of P75 NGFR antibody. OECs were cultured with IKVAV peptide. The adhesion, viability and proliferation of OECs were observed with inverted microscope, Calcein-AM /PI staining and Cell Counting Kit-8. OECs cultured on IKVAV SAPNS grew well and the viable cell count was 95%. IKVAV SAPNS can promote the adhesion of OECs and did no hinder the proliferation of OECs. IKVAV SAPNS nanofiber gel has good biocompatibility and bioactivity for OECs. It can serve as a good nerve tissue engineering scaffold.

Abstract:To construct the lentiviral RNA interference (RNAi) vector of rat CXCR4 gene, three target sequences were selected according to rat CXCR4 mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After phosphorylation and annealing, these double-strand DNA were cloned to Bgl II and Hind III sites of pSUPER. Then the product pRiCXCR4 was confirmed by electrophoresis and sequencing. Next, CXCR4 shRNA was cloned to a transfer vector of lentivirus, pNL-EGFP, and pNL-RiCXCR4-EGFP was produced. It was confirmed by digestion and sequencing that CXCR4 shRNA expression structure was correctly cloned to pSUPER and pNL-EGFP respectively. Three plasmids, pNL-RiCXCR4-EGFP, pHELPER and pVSVG were cotransfected into 293T to package lentivirus particles. The functional titer of obtained virus was determined by flow cytometry after transduction in 293T, the resulting functional titer of unconcentrated virus and concentrated virus were 6.4×104 TU/mL and 6.9×106 TU/mL respectively. After the rat mesenchymal stem cells (rMSCs) were transduced with the constructed lentiviral vectors, real-time RT-PCR, Western blotting and flow cytometry were used to evaluate the level of CXCR4 expression. Compared with control group, the CXCR4 mRNA expression were obviously suppressed in all three experimental groups (rMSCs-CXCR4a, rMSCs-CXCR4b, rMSCs-CXCR4c), especially the expression rate in rMSCs-CXCR4b group was reduced by 95.6%. The RNAi lentivirus vector of rat CXCR4 gene has been constructed successfully. This greatly facilitates the further studies such as evaluation the role of CXCR4 in rMSCs recruitment to damaged tissue.

Abstract:To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5’ phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E.coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes.