Abstract:Naturally existing Newcastle disease virus (NDV) can specifically execute oncolytic ability in clinical studies. Reports from clinical trials using NDV as oncolytic agents showed promise and warrant results in cancer therapy. In recent years, reverse genetics technology has been used widely in the studies of NDV virology. More recently, the technology was applied to optimize the oncolytic efficacy of NDV, for instance, modification of the F gene, and expression of GM-CSF, IFN-γ, IL-2 or TNF-α. NDV is widely investigated in cancer therapy and will definitely offer a prosperous future for clinical cancer therapeutics. We reviewed the developments of cancer therapy by recombinant NDV using reverse genetics technology, as well as our own experience in this domain.

Abstract:Avian influenza virus Nucleoprotein (NP) is important in viral transcription, replication and determining host specificity of influenza virus. Yeast two-hybrid technique was applied to screen for proteins interacting with virus nucleoprotein, so as to further elucidate the interaction between virus nucleoprotein and cellular proteins, as well as the interaction between virus and host. To explore new proteins interacted with NP protein, a human brain cDNA library was screened using yeast two-hybrid system with NP as the bait. DNA inserts of the positive AD/library plasmids were sequenced. By the BLAST analysis against the GenBank databases seven positive clones resulted in seven genes. Our results could help for the further study on the molecular mechanism of virus replication, transcription and protein-protein interaction. Further investigations were needed to characterize these interactions.

Abstract:We demonstrated the effect of resveratrol on porcine primary preadipocytes apoptosis, to study the intracellular molecular mechanism. Porcine primary preadipocyte was treated with different concentration of resveratrol (0 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L). We used optical microscope and fluorescence microscope to observe morphological changes during apoptosis after Hoechst 33258 Fluorescent dyes staining; and RT-PCR and Western blotting to measure the expression of apoptosis-associated gene sirt1, caspase-3, bcl-2, bax, p53, NF-κB. Primary preadipocyte apoptosis was apparent, accompanied by reduced cell volume, chromatin condensation, and nuclear shrinkage. Compared to the control and low concentration group, high dose group (200 μmol/L) significantly increased the ratio of primary preadipocyte apoptosis. The expression of sirt1, caspase-3, and bax was up-regulated markedly in response to resveratrol; in contrast, apoptotic inhibitor bcl-2, p53, NF-κB down-regulated. We further proved fact that resveratrol can specifically promote the activity of sirt1; moreover, activated sirt1 modulates the activity of caspase-3 and bcl-2 family, involving in transcriptional regulation of p53 and NF-κB through antagonizing factor-induced acetylation. Taken together, our data established resveratrol as new regulator in porcine primary preadipocyte apoptosis via activating the expression of sirt1, modulating activity of apoptotic-associated factor.

Abstract:In order to study ovine follistatin function, we amplified the total of 1 038 base pair of ovine complete follistatin cDNA and cloned into pGEM-T vector by RT-PCR from ovine ovary RNA. After removal of the signal peptide it was subcloned into the pET41a to construct the prokaryotic expression vector, named pFSsig?. SDS-PAGE and Western blotting identified the 66 kDa product of the expression of follistatin cDNA. Based on the complete CDS sequence, we cloned follistatin N-terminal domain and domain 1 with PCR and inserted into pLEX-MCS lentiviral vector, named pFS-N+D1. After package and passage of lentivirus in 293T cells, and then infected sheep primary muscle cells (SPMC). The expression of FS N+D1 in SPMC was assayed by Western blotting. The cell growth curve of the infected SPMC and noninfected control cells displayed that FS N+D1 stablly transfected SPMC proliferated significantly faster than the control cells (P<0.01). Our data inferred that ovine FS N+D1 domain had the function to stimulate sheep muscle cell growth.

Abstract:In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all other groups, while the oil group developed the lowest antibody level. This study provides not only a new rout for production of safe and effective virus like particle subunit vaccine, but also the foundations for peptide presentation and multivalent subunit vaccine design.

Abstract:On the basis of successful cloning the full length hemagglulinin (HA) and neuramidinase (NA) gene and sequence analysis of influenza virus H1N1, part of the gene was ligated into pMETA. Expression vectors pMETA/HA (52–1 557 bp) and pMETA/NA (121–1 263 bp) were constructed and expressed in pMAD16 induced by methanol. Recombinant protein was purified through Ni2+ affinity chromatography. Western blotting and ELISA were used to determine the antigenic activity of the recombinant protein. SDS-PAGE showed that the recombinant capsid gene could be overexpressed in Pichia methanolica. ELISA and Western blotting showed that the recombinant protein had antigenicity.

Abstract:Using 50% biogas slurry as basic medium, we investigated the effect of pH on the growth and lipid accumulation of Chlorella vulgaris. Setting two-group experiments, one was only control the initial medium pH, the initial pH was set at 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively. One was control the medium pH constant, set constant pH at 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively. Using HCl and NaOH regulated the pH. Results showed that algae Chlorella vulgaris grows better at pH 6.5 and 7.0, accumulate the lipid at pH 7.0?8.5, so the optimal pH for the growth and the lipid accumulation of Chlorella vulgaris was 7.0. The average removal rate of nitrate from biogas slurry was 95%, phosphate was 97%.

Abstract:The paper dealt with the characterization of polysaccharide of Paecilomyces lilacinus NH-PL-03 strain. First, we extracted and purified exude polysaccharide from the fungal fermentation broth by ethanol depositing method. Second, the proteins were removed by the Sevage method from the crude polysaccharide. Third, the purified polysaccharide (EP-1) was obtained after Superdex G-75 column separation. The results of UV-spectrometer and Sephacryl S-200 HR chromatography experiments showed that the EP-1 was a homogeneous pure polysaccharide with molecular weight of 35.2 kDa. Tested by paper chromatography analysis using the complete hydrolysis by sulfuric acid, we found that the EP-1 comprise single component as glucose. The chemical structure of EP-1 was confirmed as a kind of linear glucan linked by β-(1,3) linkage. The Congo red reaction performed that EP-1 probable presented a triple-helica conformation in the dilute alkali.

Abstract:To optimize a self-replicate Japanese enciphalitis virus (JEV) replicon, and to make it as an efficient vector to express the heterologous protein, we constructed three JEV replicons by PCR-based shortening the length of capsid genes. The vectors remained full or part of C gene, based on the JEV replicon pCTCJEV. Lac Z was selected as the reporter gene to verify the self-replicate ability of these DNA-based replicons. While transfected into the cell lines CME-4, which continuously expressing the JEV structure proteins C-prM-E, the JEV replicons pCMW-2M-1LACZ, pCMW-2M-3LACZ, which remained the first 23aa and 68aa of C protein, can express the reporter protein as the same level as pCMW-2M-LACZ with the full-length C protein. These results illustrated that the JEV replicon vector with 69-nt of the C gene can retain the self-replicate ability, and provide valuable tools to construct a possible vector for a long-lasting JEV RNA virus expression system.

Abstract:DNA polymerase delta-interacting protein 38 (PDIP38) was identified in 2003 as a human DNA polymerase delta interacting protein which plays important roles in DNA repair, mitosis and vascular smooth muscle cells (VSMCs) migration. Our previous study showed that PDIP38 was expressed in mouse embryonic stem (ES) cells and upregulated in protein levels after differentiation from ES cells, while the expression in mRNA levels was not changed. We supposed that microRNA played key roles in the regulation of PDIP38 and the differentiation of ES cells. By bioinformatics assay, we predicted that PDIP38 was a potential target of microRNA-291a-5p (miR-291a-5p). Futhermore, we validated the possibility of miR-291a-5p to regulate the protein expression of PDIP38. Using luciferase reporter assay, realtime PCR and western blot methods, we firstly demonstrated that miR-291a-5p directly inhibited the expression of PDIP38. The present results shed a new light on the study of PDIP38 and miR-291a-5p in the differentiation of ES cells.

Abstract:We converted the TGC codon (307–309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG. The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins. rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.

Abstract:To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector (HDAd) in vitro, we constructed a HDAd encoding enhanced green fluorescent protein (EGFP), denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP (FGAd/EGFP) in vitro. Therefore, we infected A549 cells with 2 000 virus particles (vp) per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.

Abstract:With suspension adapted recombinant Chinese hamster ovary (CHO) cell lines 11G-S expressing human pro-urokinase (pro-UK) as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology. The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture. Among the 10 medium supplements, insulin, transferrin, and putrescine were identified as the most significant factors (P<0.05). The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors. And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated. The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12×106 cells/mL with a maximum pro-UK activity at 5 614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.

Abstract:Based on the in vitro culturing system developed for epithelial cells in mammary gland of Xinong Saanen dairy goats using tissue explants culture, high density cultivation, and continuous passaging, the cultured epithelial cells were evaluated by growth curve fitting, karyotype analysis, immunofluorescence staining (keratin, epithelial membrane antigen (EMA), vimentin, β-casein), oil red staining and RT-PCR of β-casein gene. The results showed that the growth of epithelial cells with the model number of chromosome of 60 demonstrated a typical ‘S’ shape curve, the positive gene expression of keratin, EMA, vimentin and β-casein was detected, the cytoplasmic lipid droplets were observed following the oil red staining, the cultured cells expressed the mRNA of β-casein. In conclusion, the current in vitro culturing system can obtain the normal mammary gland epithelial cells with the function of secretion.

Abstract:With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.

Abstract:We report here a novel membrane transfer-based DNA detection method, in which alkaline phosphatase labeled gold nanoparticle (AuNP) probes were used as a means to amplify the detection signal. In this method, the capture probe P1, complimentary to the 3￠ end of target DNA, was immobilized on the chip. The multi-component AuNP probes were prepared by co-coating AuNPs with the detecting probe P2, complimentary to the 5￠ end of target DNA, and two biotin-labeled signal probes (T10 and T40) with different lengths. In the presence of target DNA, DNA hybridization led to the attachment of AuNPs on the chip surface where specific DNA sequences were located in a “sandwich” format. Alkaline phosphatase was then introduced to the surface via biotine-streptavidin interaction. By using BCIP/NBT alkaline phosphatase color development kit, a colorimetric DNA detection was achieved through membrane transfer. The signal on the membrane was then detected by the naked eye or an ordinary optical scanner. The method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9:1:1 (T10:T40:P2). The method described here has many desirable advantages including high sensitivity, simple operation, and no need of sophisticated equipment. The method can be potentially used for reliable biosensings.

Abstract:After freeze-drying, the ultrastructure of boar sperms was observed by optical and electron microscopy. The in vitro development ability of the sperm was also examined with intracytoplasmic sperm injection (ICSI). The rate of male pronuclear formation was (68.52%), for cleavage (59.17%) and for blastocyst formation (19.16%) of the trehalose group (0.2 mol/L), significantly higher than those of the 50 mmol/L EDTA group (64.59%, 56.26% and 15.62%) and the control group (35.36%, 52.33% and 8.60%) (P<0.05). After storage for 60, 120 and 180 d at 4°C, no significant difference in the in vitro development was observed (P>0.05). The male pronuclear, cleavage and blastocyst formation after ICSI with freeze-dried spermatozoa incubated for 1 h was superior than those incubated for 2 h (P<0.05). No significant differences in the structures after stored at 4°C or ?20°C (P>0.05) were observed between the trehalose group and EDTA group. The percent of B grade freeze-dried boar spermatozoa in the trehalose group was higher than that of the EDTA group (P<0.05). Based on the ultrastructure observation, main cryogenic damage in freeze-dried boar spermatozoa was swelling, damage or rupture in the sperm acrosome, neck and tail.

Abstract:We developed a high-sensitivity C-reactive protein quantifiable chemiluminescent immunoassay (hs-CRP CLIA). The high-purity native CRP was purified from hepatic cirrhosis patient ascetic fluid by affinity and ion exchange chromatography and used as an immunogen to develop the monoclonal antibodies (mAbs) against CRP. Twenty-two mAbs were identified reactive with CRP in ELISA and 13 of them were reactive in the phosphorycholine ligand capture ELISA. The mAbs 10C5 and 10C11 were selected to develop the hs-CRP CLIA. The linearity and performance of the hs-CRP CLIA was characterized. It was showed not reactive when testing against other serum materials (IgG, hemoglobin and triglyceride). The reliable correlation (R2 > 0.993) was obtained between testing value (RLU/S) and the concentration of human serum CRP calibrator. The linearity fell in the range of 0.04?20.38 mg/L. The assay has good accuracy and reproducibility, the mean recovery was 99% and the precision of the intra- and inter assay was CVs (4.2%?5.8%) and (9.0%?11.5%), respectively. In testing of 90 human sera, this assay performed well and correlated comparably with a commercial hs-CRP ELISA kit. Thus, hs-CRP CLIA is an accurate, reliable, quantifiable assay for detection of high-sensitive C-reactive protein in serum, it may be useful to improve the risk assessment of cardiovascular disease and the prognosis of inflammatory bowel disease.

Abstract:To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.