Abstract:Cyclic amp receptor protein (CRP) is a global transcriptional factor in many prokaryotes, capable of governing nearly half of the total genes in Escherichia coli. Through the method of error-prone PCR or DNA shuffling, we can first obtain CRP mutant library and then get the expected cell phenotype with enhanced resistance. In this article, we reviewed the following desired phenotype: enhanced tolerance towards oxidative stress, improved osmotolerance, enhanced organic solvent (toluene) tolerance, improved acetate tolerance of E. coli fermentation and improved ethanol tolerance during bio-ethanol production. We then concluded that CRP can also be applied in other host cells to get desired phenotypes. Last, we predicted potential applications of mutant CRP transcriptional factor.

Abstract:Infectious bursal disease virus (IBDV) VP4 plays an important role in immunosuppression of host. In order to develop monoclonal antibodies (McAbs) against VP4, we vaccinated BALB/c mice with His-VP4, screened and subcloned positive clones. We established 4 hybridoma cell lines that stably secreted McAbs against VP4 and named these cell lines 3B3, 3H11, 4C8 and 4G6, respectively. We tested the dissociation constant (Kd) of these McAbs, and found that their Kds were 4.61×10–11, 1.71×10–10, 4.26×10–11, 5.02×10–11, respectively. The isotypes of these McAbs were determined to be IgG1, IgG1, IgG2b and IgG1. These McAbs specifically bound to VP4 in IBDV infected DF-1 cells as demonstrated by Western blotting analysis and fluorescence antibody assay. These McAbs would help to detect IBDV infection and to analyze the biological activities of IBDV VP4.

Abstract:Radiation sensitive protein 23 (RAD23) is a nucleotide excision repair (NER) protein that plays an important role in Ubiquitin-proteasome pathway (UPP). Schistosoma japonicum radiation sensitive protein23 (SjRAD23) cDNA sequences were amplified by PCR and cloned into pET28a (+) vector to construct recombinant expression plasmid pET28a (+)-SjRAD23. The recombinant protein was expressed as both inclusion bodies and the supernatant in Escherichia coli BL21 (DE3) cell. Immunofluorescence observation shows that SjRAD23 was mainly distributed on the tegument surface of the worms. ELISA assay reveals that specific IgG, IgG1 and IgG2a antibodies could be detected in the sera of rSjRAD23 immunized mice. Western blotting analysis shows that the recombinant SjRAD23 could be recognized by serum specific to soluble adult worm antigen of S. japonicum. BALB/c mice vaccinated with rSjRAD23 combined with 206 adjuvant revealed 35.94% worm reduction and 40.59% liver egg reduction when compared with that of the adjuvant control group .This study suggests that rSjRAD23 has the potential as a vaccine against schistosome infection.

Abstract:In order to determine immunogenicity and protective effect in chickens, we used the IBDV (Infectious bursal disease virus)-Vp2/Lactobacillus casei as antigen transfer system. First, the immunized and control chickens were challenged by IBDV/DQ at lethal dose to determine the protective ratio. Second, chickens were orallyand intranasally vaccinated twice with 109 CFU/mL pLA-VP2/L. casei, pLA/L. casei and PBS as negativecontrol and commercial vaccine as positive control. The bursa injury and the lesion score wererecorded post challenge. The level of specific IgG and sIgA in pLA-VP2/L. casei and positive control groups was significantly higher than that in negativecontrol groups. The protection efficacy in pLA-VP2/L. casei oral group was higher than that inintranasal group. The SI. of pLA-VP2/L. casei oral group was significant higher than other groups. The lesion score indicated the pLA-VP2/L. casei was safer than commercial vaccine for bursa. Collectively, the pLA-VP2/L. casei could be a vaccine candidate for IBDV.

Abstract:We amplified genes encoding UDP-glucose dehydrogenase, ecohasB from Escherichia coli and spyhasB from Streptococcus pyogenes. Both ecohasB and spyhasB were inserted into T7 expression vector pRX2 to construct recombinant plasmids pRXEB and pRXSB, and to express in E. coli BL21(DE3). After nickel column purification of UDP-glucose dehydrogenases, the enzymes were characterized. The optimum reaction condition of spyHasB was at 30 ℃ and pH 10. The specific activity reached 12.2 U/mg under optimum condition. The optimum reaction condition of ecoHasB was at 30 ℃ and pH 9. Its specific activity reached 5.55 U/mg under optimum condition. The pmuhasA gene encoding hyaluronic acid synthase was amplified from Pasteurella multocida and ligated with ecohasB and spyhasB to construct the coexpression vectors pBPAEB and pBPASB, respectively. The co-expression vectors were transformed into E. coli BW25113. Hyaluronic acid (HA) was produced by biotransformation and the conditions were optimized. When recombinant strains were used to produce hyaluronic acid, the higher the activity of UDP-glucose dehydrogenase was, the better its stability was, and the higher the HA production could reach. Under the optimal conditions, the yields of HA produced by pBPAEB/BW25113 and pBPASB/BW25113 in shake flasks were 1.52 and 1.70 g/L, respectively, and the production increased more than 2?3 folds as previously reported.

Abstract:Enramycin is a polypeptide antibiotic and new, safe animal feed additive. A new purification process was developed, based on pre-purification by macroporous resin and refining by reversed phase chromatography. AB-8 macroporous resin was used for the pre-purification process of enramycin, with an elution buffer of 0.012 mol/L aqueous HCl solution-methanol (50: 50, V/V). Then, enramycin a and enramycin b were separated effectively by C18 reversed phase chromatography, with a elution buffer of 0.05 mol/L aqueous KH2PO4 solution-acetonitrile (70: 30, V/V, pH 4.5). The purities of enramycin a and enramycin b were up to 98.5% and 98.0%, respectively. The yield reached 29.2%. This study would provide a useful reference for the preparation of enramycin a and enramycin b with ahigh purity.

Abstract:High temperature and humidity stress during seed growth and development of spring soybean can result in seed deterioration in South China. We isolated two genes (GmSBP and GmSBPL) encoding putative SBP proteins from soybean (Glycine max (L.) Merr.) to study their biological functions and response to abiotic stress,. The two SBP proteins are hydrophilic and incomplete membrane ones. Real-time quantitative (RT-PCR) analysis reveals that the expression of the two genes in the developing seeds of the seed deterioration resistant cultivar Xiangdou No. 3 and sensitive cultivar Ningzhen No. 1 was significantly affected by high temperature and humidity treatment. Meanwhile, the levels of sucrose and soluble sugar in the developing seeds of both cultivars were also affected under high temperature and humidity stress. During seed growth and development, the expression of the two genes as well as the levels of sucrose and soluble sugar reached the highest at 30 days after flower. GmSBP2 and GmSBPL were found to be differentially expressed in different soybean tissues. Sub-cellular localization indicated that two genes were located in cytoplasm and cell membrane. Our results indicate that GmSBP2 and GmSBPL might be involved in the response to abiotic stress, which will enrich our understanding of pre-harvest seed deterioration and resistance in soybean from one side.

Abstract:AAV-ITR single strand DNA mini vector (AAV-ITR ssDNA mini vector) is a novel gene expression vector based on AAV-ITR. We have shown efficient gene expression of AAV-ITR ssDNA mini vector in HEK 293T. Here, we studied the efficacy of gene expression of AAV-ITR ssDNA mini vector in vivo. We injected the skeletal muscle of ICR mice separately with equal molars of AAV-ITR ssDNA mini vector, ITR mutated AAV-ITR single strand DNA mini vector (AAV-ITRmm ssDNA mutant vector), AAV-ITR dsDNA and pUC57-minivector-GFP, combined with TurboFect. Florescence microscope analysis of skeletal muscle section shows that AAV-ITR ssDNA mini vector had higher expression efficiency and longer expression period. We extracted DNA from the muscle three months after injection and quantified three vectors by Real-time PCR. RT-PCR analysis shows that there were highest copy numbers of AAV-ITR ssDNA mini vector existing in muscle. Stable existing of AAV-ITR ssDNA mini vector in muscle could be the molecular basis of long term gene expression of the vector. The results suggest that AAV-ITR ssDNA mini vector might be a promising vector for gene therapy.

Abstract:The aim of this study is to clone the mouse T-bet promoter and enhancer, construct and identify the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS for T-bet transcription regulation study and its function in signaling of multiple sclerosis. The promoter and CNS of T-bet were predicted by bioinformatics assay. The predicted fragment of mouse T-bet promoter plus CNS was amplified by PCR and cloned into pGL4.10. The recombinant plasmid pGL4.10-TBX21pr-CNS was transferred into Escherichia coli DH5α. The positive clone was identified by double digestion with KpnⅠand SfiⅠand DNA sequencing. Finally, pGL4.10-TBX21pr-CNS was cotransfected with pRL-TK into 293T cells and Jurkat cells, pRL-TK and pGL4.10 as a control. The luciferase activity in 293T cells (P=0.012 2) and Jurkat cells (P=0.002 2) was higher than that of the control group. A fragment of 1 028 bp mouse T-bet promoter plus 1 308 bp CNS was successfully cloned and the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS was constructed. In 293T cells and Jurkat cells, pGL4.10-TBX21pr-CNS has the promoter functions. This work offers a basic material for the research of T-bet transcription.

Abstract:To use Agrobacterium rhizogenes-induced hairy roots to create new germplasm of Dianthus caryophyllus, we transformed D. caryophyllus with A. rhizogenes by leaf disc for plant regeneration from hairy roots. The white hairy roots could be induced from the basal surface of leaf explants of D. caryophyllus 12 days after inoculation with A. rhizogenes ATCC15834. The percentage of the rooting leaf explants was about 90% 21 days after inoculation. The hairy roots could grow rapidly and autonomously in liquid or solid phytohormone-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and silica gel thin-layer chromatography of opines from D. caryophyllus hairy roots. Hairy roots could form light green callus after cultured on MS+6-BA 1.0?3.0 mg/L +NAA 0.1?0.2 mg/L for 15 days. The optimum medium for adventitious shoots formation was MS + 6-BA 2.0 mg/L +NAA 0.02 mg/L, where the rate of adventitious shoot induction was 100% after cultured for 6 weeks. The mean number of adventitious shoot per callus was 30?40. The adventitious shoots can form roots when cultured on phytohormone-free 1/2 MS or 1/2 MS +0.5 mg/L NAA for 10 days. When the rooted plantlets transplanted in the substrate mixed with perlite sand and peat (volume ratio of 1:2), the survival rate was above 95%.

Abstract:Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.

Abstract:RNA-seq can help us quickly obtain the whole transcriptome sequences of species under different conditions. Many Unigenes that are assembled by raw reads always do not contain complete open reading frame (ORF). In addition, it also has some redundancy in transcriptome library. Some Unigenes in the library, although belong to one transcript, cannot be assembled without overlapping. We found five incomplete Unigenes annotated ammonium transporter (AMT) from Salicornia europaea transcriptome, in which two Unigenes (Uni4 and Uni5) had identical expression patterns across four transcriptomes. The two Unigenes may come from one transcript. Analyzing the Unigene position of transcript by NCBI blastx, we found that Uni4 and Uni5 respectively located in 5′ end and 3′ end compared with the reference transcript, and an unknown gap of 120 bp may exist in a hypothetic transcript to which Uni4 and Uni5 both belong. To verify the hypothesis, single forward primer and single reverse primers were respectively designed on Uni4 and Uni5, and a fragment with about 800 bp was generated by PCR. Then it was sequenced and aligned with Uni4 and Uni5. Finally, we assembled a sequence with 1 667 bp, which contains a complete ORF (1 482 bp, coding 494 amino acids). It belongs to amt1 subfamily and was named Seamt1 via the phylogenetic analysis. It was pointed by bioinformatics tools that SeAMT1 protein conformed to the AMT characteristics of other species. This work clustered expression pattern to explore the Unigenes of one transcript, and the feasibility of this method was validated through the other two groups of Unigenes. The handy method will benefit extension and assembling of Unigene in transcriptome, it also helps achieve the complete ORF and gene function.

Abstract:We expressed 17β-hydroxysteroid dehydrogenase10(17β-hsd10) recombinant protein, prepared anti-17β- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system pET15b-17β-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-β-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17β-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25×104 and 2.5×104 respectively. The concentration of 0.1 μg/mL of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.

Abstract:Cephalosporins are widely used antibiotics owing to their broad activity spectra and low toxicity. Many of these medically important compounds are made chemically from 7-aminodeacetoxycephalosporanic acid. At present, this intermediate is made by synthetic ring-expansion of the inexpensive penicillin G to form G-7-ADCA, followed by enzymatic removal of the side chain to obtain 7-ADCA. The chemical synthetic process is expensive, complicated and environmentally unfriendly. Environmentally compatible enzymatic process is favorable compared with chemical synthesis. In our previous research, metabolic engineered E. coli strain (H7/PG15) was constructed and used as whole-cell biocatalyst for the production of G-7-ADC with penicillin G as substrate. The whole-cell biocatalysis was studied by single factor experiment, including the composition of substrates and the conversion conditions (OD600, pH, concentration of penicillin G, MOPS, glucose, time and FeSO4). After optimization, 15 mmol/L of G-7-ADCA was obtained. The process is convenient, efficient and economic. This work would facilitate the industrial manufacturing and further product research.

Abstract:Adenovirus vectors are promising delivery systems for gene therapy. We established a new process for clinic trial of recombinant adenovirus vectors using a novel disposable bioreactor. The suspension HEK293 cells were inoculated into a 5 L disposable bioreactor with parameters control of pH, DO, agitation and temperature. After 6 days of a fed-batch culture, the final cell density reached 2.0×106 cells/mL. The culture was infected with Ad-IFNγ at an MOI of 30. The harvest was performed at approximately 48 h post-infection and crude viral lysate was obtained after 3 freeze/thaw cycles and centrifugation. The maximum titers of crude viral lysate was 1.49×1013 Infectious units (IFU) and the bulk product specific was 3 800 IFU/cell. Purified Ad-IFNγ by anion-exchange chromatography and the final recovery of infectious unit reached 35.9%. The result demonstrates that an efficient and stable process of producing Ad-IFNγ using a disposable fed-batch bioreactor is established.

Abstract:To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6×His?tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 ℃ and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because?of?its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.