Abstract:Yeast flocculation is described as a reversible, asexual and calcium dependent process, in which cells adhere to form flocs by interaction of specific cell surface proteins named flocculins on yeast cells with mannose residues present on the cell wall of adjacent yeast cells. Yeast flocculation provides a very economical and convenient pathway for separation of yeast cells from the fermentation broth or removal of heavy metal ions from effluent. A large number of tandem repeats have been found in genes encoding flocculins, which not only have great regulatory effect on the structure and function of flocculins, generating the diversity of flocculation characteristics, but lead to genetic instability in flocculation as well for driving slippage and recombination reactions within and between FLO genes. Here, the research progress in effect of variation of tandem repeats in FLO genes on flocculation characteristics and genetic stability were reviewed to direct and promote the controllable application of flocculation in industrial fermentation process and environmental remediation

Abstract:Classical swine fever (CSF), an acute and highly contagious disease of swine, is caused by classical swine fever virus. CSF is one of the most devastating diseases to the pig industry worldwide and results in serious economic losses. Currently prophylactic vaccination is still an important strategy for the control of CSF. Live attenuated vaccines (such as C-strain) are safe and effective. However, there are significant changes in the clinical features of CSF, displaying concurrent typical and atypical CSF, and simultaneous inapparent and persistent infections. Immunization failure has been reported frequently and it is difficult to distinguish between wild-type infected and vaccinated animals (DIVA). So there is an urgent need to develop more effective and safer DIVA or marker vaccines for the control of CSF. In this review, some of the most recent advances in new-type vaccines against CSF, including DNA vaccines, live virus-vectored vaccines, protein or peptide-based vaccines, gene-deleted vaccines and chimeric pestivirus-based vaccines, are reviewed and discussed.

Abstract:Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum. Based on our primary study on tegument surface proteins of S. japonicun, a cDNA encoding a 66 kDa calcium-binding protein of S. japonicum (Chinese strain) was cloned, sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S. japonicum. The expression of SjIrV1 were detected by Real-time PCR, using cDNA templates isolated from 7, 14, 21, 28, 35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages, and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms. The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a (+) vector and transformed into competent Escherichia coli BL21 for expression. The recombinant protein was purified using a Ni-NTA purification system, and confirmed by high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS). Western blotting analysis showed that recombinant SjIrV1 (rSjIrV1) could be recognized by the S. japonicum infected mouse serum and the mouse serum specific to rSjIrV1, respectively. Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms. ELISA test revealed that IgG, IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice. The study suggested that rSjIrV1 might play an important role in the development of S. japonicum.

Abstract:We have assessed the relationships between immune trait (antibody titers of Sheep red blood cell, SRBC; Avian influenza, AI; Newcastle disease, ND) and varieties of MHC B-LBII Gene in local chicken breeds (Wenshang Barred chicken, LH; Laiwu Black chicken, LWH; and Jining Bairi chicken, BR). We selected 300 chickens randomly from the three indigenous chicken populations. The variations of MHC B-L BII gene were detected by directly DNA sequencing and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The results indicated that there were about 19?22 nucleotide mutations in the three local breeds, which could affect 16?18 amino acid variations. Another results indicated that there was significantly relationship between seven to eight SNPs of the MHC B-LBII region and some immune traits (P<0.05 or P<0.01). Both locus G97A and locus T138A were found in the three species, which were significantly related to the antibodies of SRBC, ND and AI antibody titers (P<0.05). Among them, the locus G97A was significantly associated with ND antibody titers (P<0.05) in BR chicken, with SRBC antibody titers (P<0.05) in LWH chicken, and with H9 antibody titers (P<0.05) in LH chicken. Furthermore, locus T138A was significantly associated with H9 antibody titers in BR and LH chickens (P<0.05). All those results suggest relationships among the different varieties of MHC B-LBII and immune traits in the three local breeds

Abstract:Spinosad represents a new class of insecticides produced by Saccharopolyspora spinosa. To understand the transcription of the spinosad biosynthetic gene cluster, two promoter detection plasmids based on different reporter genes were constructed and used to detect 9 promoters in the spinosad biosynthetic gene cluster. In addition, the temporal transcriptional profiles of the corresponding genes controlled by the 9 promoters, together with 4 genes outside of the spinosad cluster but are required for the synthesis of sugars in spinosad, were examined by real-time PCR. The results indicate that the 9 spinosad biosynthetic genes were highly expressed at the stationary phase, which coincides with the accumulation of spinosad in the fermentation broth. Of particular note is that the transcription of the 4 sugar synthetic genes showed higher level at the exponential phase, suggesting the expression of sugar synthetic genes is not correlated with the spinosad synthetic genes. The data suggest that spinosad biosynthesis could be improved by engineering the expression pattern of the sugar synthetic genes that lie outside the spinosad gene cluster

Abstract:Glucose oxidase (GOD) is an important industrial enzyme with many potential applications. In order to increase the production and productivity of GOD by recombinant Pichia pastoris GS115, we investigated the feeding strategies of mixed carbon sources during induction phase, based on results of the optimization of initial cell and methanol concentration on GOD production. The optimal initial cell and methanol concentration were 100 g/L and 18 g/L. During induction phase, the mixed-carbon-sources strategies showed that glycerol, sorbitol or mannitol co-feeding with methanol could enhance GOD production. With mannitol co-feeding (20:1(W/W)), the maximum GOD production and maximum GOD productivity reached 711.3 U/mL and 4.60 U/(mL·h) after an induction period of 156 h. Compared to the control, the enhancements of GOD production and productivity were 66.3% and 67.9%, respectively. Meanwhile, we found an appropriate mannitol co-feeding strategy that would not inhibit the expression of promote. The activity of alcohol oxidase was 8.8 U/g, which was enhanced by 69.2% compared to the control (5.2 U/g). We can use the same optimization process to improve the production of other proteins from recombinant Pichia pastoris by changing the fermentation parameters

Abstract:To realize the simultaneous fermentation of xylose and glucose, ptsG (one of the glucose-PTS genes) was deleted from the engineered ethanologenic Escherichia coli SZ470 (?pflB, ?frdABCD, ?ackA, ?ldhA), resulting in loss of glucose effect in the mutant SZ470P (?ptsG). When tested in 5% mixture of glucose (2.5%) and xylose (2.5%), SZ470P simultaneously used glucose (13 g/L) and xylose (20 g/L) whereas the parent strain SZ470 sequentially used glucose (25?g/L) then xylose (5 g/L). Upon completion of the fermentation, both strains achieved similar product yield of 89%. SZ470P produced 15.01 g/L of ethanol, which was 14.32% higher than that produced by SZ470 (12.86 g/L). Deleting ptsG gene enabled the mutant strain SZ470P to simultaneously use both glucose and xylose and achieve better ethanol production

Abstract:We studied the function and mechanism of miR-24 in regulating β-like globin gene expression. We first detected the expression of miR-24 during erythroid differentiation and also detected the globin gene expression in miR-24 overexpressing K562 cells through q-PCR. Dual-luciferase reporter assay and Western blotting were used to identify target genes of miR-24. “Rescue experiment” was further used to investigate the regulation of miR-24 on globin gene expression whether depending on targeting Sp1 or not. We found that miR-24 increased during hemin-induced K562 cells and EPO-induced HPCs (hematopoietic progenitor cells) erythroid differentiation. Overexpression of miR-24 in K562 cells promoted the ε- and γ-globin gene expression during hemin-induced erythroid differentiation through targeting the negative globin regulator Sp1. These results suggested that miR-24 can improve the expression of β-like globin gene through targeting Sp1

Abstract:To enhance the penetration of P53 into tumor cells by fusion it with the cell penetrating peptide 9R. The fusion gene of 9R-p53 was cloned into the expression vector. The fusion protein, CPPs-P53, was expressed and purified. We detected the rate of cell growth inhibition and apoptosis by MTT and Annexin-V-FITC/PI double stained method respectively for measuring its effect on tumor cells. CPPs-P53 and P53 were successfully expressed and purified, the purity of both proteins reached up to 90%. MTT assay showed that the cell growth inhibition by CPPs-P53 was more efficient than P53, and the rate of cell growth inhibition is dose-dependent. The apoptosis experiment showed that P53 could induce apoptosis of tumor cells. Compared with the P53, CPPs-P53 had a more significant effect in inducing cell apoptosis (**P<0.01). The CPPs-P53 shows more significant effects than P53 in inhibiting cell growth and inducing apoptosis on tumor cells

Abstract:The 11 kDa protein, a small nonstructural protein of parvovirus B19, may play important roles in viral replication cycle. To investigate the effect of 11 kDa protein on the NF-κB signaling pathway, we first prepared the poly-antiserum using GST-11 kDa fusion protein purified via prokaryotic expression system, and demonstrated that the 11?kDa protein mainly localized in cytoplasm when expressed in Hela cells. Meanwhile, luciferase activity assay and Western blotting assay showed that 11 kDa up-regulated the transcriptional activity of NF-κB and induced the degradation of IκB-α in Hela cells. Moreover, the 11 kDa protein activated the IL6 promoter, which is probably through the NF-κB pathway. Taken together, these results suggested that 11 kDa protein may contribute to activating inflammatory factors through participating in the cell signaling pathway

Abstract:To prepare recombinant human retinol binding protein 4 (RBP4) by using the baculovirus expression system and to detect its immunogenicity, the fusion DNA fragment of secretory signal peptide SS64 and human RBP4 gene was subcloned into a baculovirus transfer vector pFastBac-dual(pFBd), and the corresponding recombinant transfer plasmid was transformed into E. coli strain DH10bac, after transposition recombinant shuttle bacmid was screened out. The logarithmic phase Sf9 cells were transfected with the recombinant bacmid and then the recombinant baculovirus containing hRBP4 expression box were generated. After amplification of recombinant baculovirus, the recombinant baculovirus seeds were obtained. To express human RBP4, logarithmic phase Sf9 cells were infected with the virus seeds and SDS-PAGE and Western blotting were used to detect and identify the expression. Finally, to prepare a batch of RBP4 protein, logarithmic phase Sf9 cells in suspension culture were infected with recombinant baculovirus seeds and the supernatant was harvested after 120 hours post-infection for purification. Finally for preparation of polyclonal antibody and evaluation of immunogenicity, the recombinant hRBP4 from insect cells and from E. coli were immunized rabbits. Restriction enzyme digestion and sequencing confirmed that the recombinant baculovirus transfer plasmid was constructed correctly, and subsequently recombinant RBP4-bacmid was generated successfully. SDS-PAGE and Western blotting analysis suggested that human RBP4 protein was highly expressed in Sf9 cells with the molecular weight of approximately 23 kDa. The recombinant RBP4 protein could be secreted into the medium efficiently, and the expression level was calculated amount of 100 mg/L. Finally the rabbit antiserum was harvested after recombinant RBP4 immunization, therein the titer of antiserum against baculovirus recombinant RBP4 is 1:100 000 whereas the titer of antiserum against E. coli recombinant RBP4 is only 1:10?000. Overall, human RBP4 was high efficiently expressed successfully with good antigenicity in baculovirus system, and high affinity antiserum was obtained. A solid foundation was laid for the next step of the preparation of human serum RBP4 detection kit

Abstract:We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism

Abstract:PARP1 is an important part of DNA repair machinery. In recent years, PARP1 as novel anti-cancer therapeutic target has been broadly explored. In this study, we expressed hPARP1 enzyme in the baculovirus system and tested its activity. We inserted hPARP1 gene into the pFastBacTM1, a baculovirus transfer vector and then transformed it into DH10Bac containing a shuttle vector of Bacmid. After co-transfecting the recombinant plasmid into Sf9 insect cells, the expressed hPARP1 was purified by 3-aminobezamide affinity chromatography. The expression of hPARP1 was visualized by SDS-PAGE and Western blotting; the activity of expressed and purified hPARP1 was confirmed by the reaction of consumption of NAD+ by hPARP1 in vitro. After the purification by 3-aminobezamide affinity column, 3.2 mg protein was obtained and its specific activity was 1.988 nmol/(min?μg)

Abstract:As an effective vehicle for bio-research and for gene therapy, Lentiviral Vector (LV) has been drawn large attention in recent years. However, transcriptional read-through limits its application. In order to understand?the extend of LV read-through in chromosome, a reliable method to assess transcriptional read-through rate is needed. Here, we report the method as follows: 293T cells were transfected with the lentiviral transfer vectors which borne with two LTRs at its two ends in order to mimic the state of “proviral vectors” in chromosome. Using the primers specific for 3'U5 and 3'U3, read-through and total transcripts were reverse transcribed, respectively. These two cDNAs were quantified by realtime PCR using the primers and probe specific for 5'end of 3'U3. Read-through rate was then calculated by the division of the two. Meanwhile, read-through product of green fluorescence protein was also analyzed by Fluorescence Activated Cell Sorter. They both reciprocally proved the principal and confirmed that self-inactivated LV appeared higher read-through rate than the wild type one. The method described in this article, therefore, provides a useful technique to study how to reduce read-through rate, and improve the bio-safety of?LV

Abstract:Activities of 58 miRNAs for BHK21, HEK293, and Vero cell lines were screened using the high-throughput miRNA activity profiling method. miR-206 activity was found specifically high in BHK21. Considering miR-206 was recognized as a muscle-specific miRNA, we further detected the expression and activity level of miR-206 in BHK21 cells, with myoblast cells C2C12 as positive control and HEK293 cells as negative control. Then, we induced BHK21 by culturing with medium containing 2% horse serum (HS) and tested expression level of slow skeletal myosin heavy chain (MHC), activity and expression levels of miR-206, and expression level of Connexin43 (Cx43) which was reported negatively regulated by miR-206. Results demonstrated that activity and expression levels of miR-206 were both higher in BHK21 cells than in C2C12 cells. After induction of HS, MHC expression level was increased in BHK21 cells. The activity and expression levels of miR-206 were further enhanced. The Cx43 expression level was decreased. These results suggested that BHK21 had the characters of myoblast cells. In conclusion, we firstly discovered that miR-206 activity was specifically high in BHK21 cells, suggesting that BHK21 cells were derived from interstitial cells other than parenchyma cells of kidney from miRNA point of view. Our study also indicated that BHK21 cells were able to be used as models in vitro for research of miR-206 function

Abstract:An enzyme-displaying yeast as a whole-cell biocatalyst is an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, lipase-displaying Pichia pastoris whole-cell was used as a biocatalyst to synthesize diisooctyl adipate in the non-aqueous system. The maximum productivity of diisooctyl adipate was obtained as 85.0% in a 10 mL reaction system. The yield could be reached as high as 97.8% when the reaction system was scaled up to 200 mL. The purity obtained is 98.2% after vacuum distillation. Thus, the lipase-displaying P. pastoris whole-cell biocatalyst was promising in commercial application for diisooctyl adipate synthesis in non-aqueous phase