Abstract:Recently，cancer therapy with mutiple genes has been attached with great attention. However，at present there is no efficient tool to construct multiple-cistrons. The large sizes and the imbalance in expression of most traditional tools，such as ribosome entry sites (IRESes)，greatly block their wide employment in the construction of multiple cistronic gene therapy vectors. The self-cleaving peptide 2A from foot-and-mouth disease virus (FMDV) has a very small size，and more importantly，high cleavage activity in artifical bicistron，which bring new hope for mutiple genes therapy stategy. In this article，the characteristics and cleavage activities of FMDV 2A will be elucidated，and we further outline its applications in cancer gene therapy.

Abstract:The physiological roles of the glutathione(GSH)/glutathione peroxidase(GPx) system in protecting microbial cells against oxidative stress were reviewed. In eukaryotic model microbeSaccharomyces cerevisiae，this system is obligatory in maintaining the redox balance and defending the oxidative stress. However，The GSH/GPx system only conditionally exists in prokaryotes. Namely，for those prokaryote bacteria containing glutathione reductase and GPx，e.g. Haemophilus influenzae and Lactococcus lactis，by taking up GSH，they might develop a conditional GSH-dependent GPx reduction system，which conferred cells a stronger resistance against oxidative challenge.

Abstract:Cationic liposome is a greatly promising gene carrier. In this paper，the structure trait of cationic liposome was briefly introduced，the mechanism of cationic liposomes mediated gene delivery and the main influencing factor of transfection efficiency in the transfection process were discussed chiefly.

Abstract:The 14-3-3 proteins comprise a family of highly conserved acidic protein with subunit molecular mass 28～33kD and are widely found in different eukaryotic cells. 14-3-3 proteins were the first polypeptides shown to have phosphoserine/threonine (pSer/Thr) binding properties which firmly established its importance in cell signaling. 14-3-3 proteins tend to form dimeric proteins to modulate protein-protein interactions. 14-3-3 proteins have been shown to contribute to the regulation of such crucial cellular processes as metabolism，signal transduction，cell cycle control，cell growth and differentiation，apotosis，protein trafficking，transcription，stress responses and malignant transformation. Many reports link 14-3-3 to disorders，particularly the neurological disorders and cancer. The 14-3-3 test has been used for the diagnosis of prion diseases. 14-3-3 could be exploited for therapeutic purposes. In this review，we discuss the structure，function of 14-3-3 protein and the related research progress in therapeutic applications.

Abstract:Atrazine could be used as the sole carbon，nitrogen and energy sources for growth by strain Arthrobacter sp. AG1，and the atrazine-degrading genes of AG1 were found to be the combination of trzN，atzB and atzC. The atrazine chloride hydrolysase gene trzN was cloned by PCR amplification，whose sequence shared 99% identity with that of Norcardioides sp. C190. Two large plasmids were found in AG1，and trzN and atzB were confirmed to be localized on the larger plasmid pAG1 by the method of southern hybridization. Subculture of AG1 in liquid LB for three generations，34% of the subsequent cells were found to lose degrading activity，however，neither plasmid was lost. PCR amplification results showed that the mutants had only lost the trzN gene instead of atzB and atzC. It was deduced that mutation might be due to the trzN gene deletion from the plasmid. This study provided new evidence that atrazine metabolic genotypes were resulted from horizontal gene transfer between different bacteria under environmental selective pressure.

Abstract:RNA interference（RNAi） is a powerful tool in gene function research. In order to investigate the role of GP2，GP3 and GP4 of porcine reproductive and respiratory syndrome virus(PRRSV) in the viral replication，small interference RNAs (siRNAs) directed to ORF2，ORF3 and ORF4 were designed and 12 short hairpin RNA (shRNA) expression vectors were constructed (designed as 21，22，23，24，31，32，33，34，41，42，43 and 44). Cells treated with shRNA expression vectors were infected by PRRSV. The effective shRNA expression vectors were selected by fluorescent quantatitive PCR (FQ-PCR). The virus titer of supernatant of the cells treated with effective shRNA expression vectors (23，24，31，34 and 41) were reduced by 184 to 4.65 folds compared with that of controls.

Abstract:RNA interference（RNAi） is a powerful tool in gene function research. In order to investigate the role of GP2，GP3 and GP4 of porcine reproductive and respiratory syndrome virus(PRRSV) in the viral replication，small interference RNAs (siRNAs) directed to ORF2，ORF3 and ORF4 were designed and 12 short hairpin RNA (shRNA) expression vectors were constructed (designed as 21，22，23，24，31，32，33，34，41，42，43 and 44). Cells treated with shRNA expression vectors were infected by PRRSV. The effective shRNA expression vectors were selected by fluorescent quantatitive PCR (FQ-PCR). The virus titer of supernatant of the cells treated with effective shRNA expression vectors (23，24，31，34 and 41) were reduced by 184 to 4.65 folds compared with that of controls.

Abstract:BmNPV bacmid constructed recently and Red recombinant system were used to rapidly disrupted Bombyx mori nucleopolyhedrovirus (BmNPV) orf60 in Escherichia coli (E. coli) BW25113. BmNPV bacmid isolated from E. coli BmDH10Bac was electroporated into E. coli BW25113，which harbors plasmid pKD46 encoding λ Red recombinase，to produce E. coli BW25113-Bac，which could be used for gene deletion of BmNPV. A linear fragment was amplified by PCR from plasmid pKD3 (containing a chloramphenicol acetyltransferase gene cat) using a pair of primers with length of 63bp，which had 45 bp homologous to the orf60 gene and 18bp homologous to cat sequences. The linear fragment was electroporated into E. coli BW25113-Bac and homologous recombination occurred between the linear fragment and orf60with the help of λ Red recombinase. Three specific primer pairs were used to confirm the replacement of orf60 by cat gene. Western blot analysis showed that orf60was not expressed in BmN cells infected with knockout bacmid.

Abstract:Taking the genome DNA of Infectious Bovine Rhinotracheitis Virus (IBRV) as the template，the gG gene was amplified with PCR and cloned into the T cloning vector pMD18-T. After being identified by restriction digestion and DNA sequencing，the insert was subcloned into the expression vector pGEX-KG. Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot assay showed that this gene was expressed as both soluble form and inclusion body by the transformed E. coli BL21 strain (DE3). The fusion protein was purified and used as the coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay (ELISA). Comparison between this gG-ELISA and commercial IBRV gB-ELISA Kit (IDEXX) was made in the detection of 380 cow serum samples. The results demonstrated an agreement of 92%. By using this novel gG-ELISA，1248 cow serum samples were tested and the average positive rate of IBRV antibodies for imported cows is 21.7%，while the positive rate ranged greatly from 0.0%～41.5% for Hubei local Chinese Black and White Dairy Cows.

Abstract:A completely synthetic gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype A (AHc，1287bp，429aa，～50kD) was constructed with oligonucleotides. After expressed in Escherichia coli，soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%～53% of soluble total proteins) and the purified yield was more than 30mg/L by one-step purification. Then，the purified AHc was used to vaccinate Balb/c mice，which developed a strong and specific immune response as expected following administration of AHc protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble，stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab，but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.

Abstract:The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR，then the pPIC9K-IL-18 of fusion expression vector was constructed by inserting IL-18 fragment，and was transformed to GS115 by electroporation，multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol，SDS-PAGE was used to analyze expression product，fusion protein was purified by Sephadex G-100 column，bioactivity of IL-18 was measured by MTT assays. Experiment results show fusion protein of pIL-18 secreted by GS115，expression reaches the secretion peak of 160mg/L at 72h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in Pichia pastoris.

Abstract:The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR，then the pPIC9K-IL-18 of fusion expression vector was constructed by inserting IL-18 fragment，and was transformed to GS115 by electroporation，multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol，SDS-PAGE was used to analyze expression product，fusion protein was purified by Sephadex G-100 column，bioactivity of IL-18 was measured by MTT assays. Experiment results show fusion protein of pIL-18 secreted by GS115，expression reaches the secretion peak of 160mg/L at 72h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in Pichia pastoris.

Abstract:In order to produce relatively large amounts of recombinant human intestinal trefoil factor and assess its biological activity. The expression plasmid pPIC9-hITF containing AOX1 promotor and the sequences of secreting signal peptides was transformed into the yeast cells. Then through selection，positive transformants were cultivated in fermentation basal salts medium in a 5L fermenter to obtain large amount product with low cost. The secreted peptides were then purified by a combination of ionic exchange chromatography and molecular sieve. To verify the product，electrospray mass spectrometry analyses was used to determine the structure of rhITF and Western Blotting was performed to test the immunological activity. Furthermore，the biological activity of the peptide was examined by experiments from cell to tissue. The nucleotide sequence of rhITF was the same as expected. With a 5_L fermenter, 253mg of hITF was isolated at the purity of 96% from 3.5L of yeast fermentation broth. The expression level for recombinant human ITF in this yeast system was 73.33mg/L. In our study，we provided a way to gain a production among milligram to gram of recombinant human ITF by the use of a yeast expression system. As human ITF are difficult to purify in any significant amount from tissue extraction，the way described may become a valuable tool in obtaining pure peptide for further studies of trefoil peptide function.

Abstract:The specific expression of TGH and HSL genes in different tissues of Bamei pig was investigated by RT-PCR and Western blot in this study. The result of RT-PCR showed that the expression of HSL could be detected in all these seven tissues examined，and which was higher expressed in fat，lower in heart，liver，lung，spleen and kidney. Expression of TGH gene could also be detected in seven tissues，and higher in liver and fat，lower in heart and kidney and lowest in spleen and lung. The result of Western blot showed that，HSL gene was highest expressed in epiploica fat and subcutaneous fat，higher in other tissues，but couldn't be detected in kidney. Expression of TGH was detected in epiploica fat，subcutaneous fat，liver，lung and spleen，and highest in fat and liver，but it hadn't be found in heart and kidney. These results suggested that both HSL and TGH could be regulated by post-transcriptional，and their function was involved in different tissues.

Abstract:The expression of plant gene is controlled by its promoter. The isolation and the function analysis of promoter are important for studying the genetic engineering and the regulation expression of plant genes. In this paper, we cloned a promoter, Os252, which was predicted to be highly expressed in the stem of rice from the EST database. After the construction of the Os252::GUS expression vector, it was transformed into rice. The integration of transgenes into transgenic rice genome was confirmed through PCR analysis. X-Gluc staining showed that Os252 can promote GUS gene expression in leaf, stem and matured seed. GUS enzyme activities driven by Os252 promoter in leaf and seed are about 190% and 250% of that driven by the 35S promoter. Thus, the Os252 promoter can be applied for rice genetic engineering.

Abstract:The dhaB gene encoding glycerol dehydratase and dhaG dhaF gene encoding glycerol dehydratase reactivating factor from Citrobacter freundii were amplified by PCR. The temperature control expression vector pHsh harboring yqhD, vdhaB, dhaG and dhaF gene was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-dhaG-dhaF-yqhD). The results from SDS-PAGE analysis show that the recombinant product was consistent with the molecular weight predicted from gene sequence. The fermentation result show that the yield of 1,3-propanediol was increased by 28% compared with E. coli JM109(pHsh-dhaB-yqhD).

Abstract:Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type Ⅲ secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system，causing necrosis or apoptosis. popN gene is the first gene in the popNoperon of TTSS gene cluster. To investigate its function，popN gene deletion mutant was generated in this study，and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However，no secreted effector proteins were detectable when the popN^-- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons，effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN^- mutant.

Abstract:The gene for LPTS is originally cloned as a human liver-related putative tumor suppressor (LPTS) gene that encodes a full length protein of 328 amino acids (LPTS-L). LPTS-L is also identified as a telomerase inhibitor to regulate telomere length in the cells. To facilitate the functional and structural studies of LPTS-L protein，the cDNA for LPTS-L was cloned into the expression vector pET-24 in frame to generate a recombinant plasmid pET-24-LPTS. The LPTS-L protein was expressed in E.coli BL21 solublely，and purified by Ni Sepharose affinity chromatography which，however，is not fit for large scale protein purification. The gene of LPTS-L was then PCR amplified to remove the 6×His tag，and cloned into pET-24a. The non-fusion protein of LPTS-L was expressed in E.coli BL21，and purified by phosphocellulose P11 chromatography. The purity of LPTS-L protein was about 55% after that procedure，and arrived at 80% after second purification by Sephadex G-100 chromatography. Western Blotting analysis showed that the band reflects the specific binding of anti-LPTS antiserum against the purified LPTS-L protein. The TRAP assay was performed to detect the telomerase inhibitory activity of LPTS-L protein in vitro. It was observed that the purified LPTS-L inhibited the activity of telomerase greatly，similarly with that of LPTS-L protein purified by Ni Sepharose 4B. Our results suggest that phosphocellulose P11 plus Sephadex G-100 chromatography could substitute for Ni Sepharose 4B affinity chromatography for preparation of purified LPTS-L protein. Through this study，a technique for preparation of LPTS-L protein in a large scale is established.

Abstract:Spider silk is a natural protein fibroin with excellent character as it is light and tenacious. It has a wild potential applications in the biomedical field due to its good biocompatibility and degradation. Arginine-glycine-aspartic acid (RGD) is a highly conserved amino acid sequence of many adhesion protein. Biological materials binding with RGD peptide in the surface can promote cells adhesion， migration and proliferation.Our lab had constructed the 16 muhimers with the introduced RGD peptide codons which involve cell adhesion for the first time. It was found that the mechanical capability of the 16 mulimer protein was very limited because of the big gap in molecular weight with nature spider proteins when it was used to made biomaterial scaffold.In this paper,based on the 16 multimers of the highly,repetitive sequence of spider dragline silk and with RGD peptide condons which has been constructed by our lab forestall,it was used to construct the 32 and 64 multimers sequence of spider dragline silk by the strategy of “head to tail". The 32 and 64 multimers were ligated into prokaryotic expression vector pET-30a，and then the Bl21(DE3)pLysS. The fragments were in agreement with the desired through digestion,agarose gel electrophoresis respectively.By registration into the GenBank data-base,the serial numbers of DQ469929 and DQ837297 were gained respectively. The expression of recombinant protein was introduced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 102kD and 196.6kD in agreement with the desired respectively. It was the first time for the high polymer spider dragline silk protein expressed in prokaryotic biology. Furthermore，a larger quantity of synthetical proteins with high density fermentation were searched after,and a suit of high efficient purification methods for 32 multimers protein were established.

Abstract:Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3⁺CD4⁺+ T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.

Abstract:High resolution two-dimensional polyacrylamide gel electrophoresis, followed by computer-assisted analysis, was applied to investigate the fat body of silkworm, Bombyx mori. 722 spots were obtained by silver stain from 18cm，pH 3～10 gel. Most of them were distributed in the area from 15 kD to 90 kD with pH 4～8. The matrix-assisted laser desorption ionization time of flight mass spectrometry were applied for identifying the major spots of the 2D map. A total of 41 spots were excised to identify by a combination of MALDI-TOF MS after digested with trypsin. The result showed among the 34 proteins identified, plenty of them were involved in metabolism and immunity. Additionally, Heat shock proteins, 30K proteins and Actin were also detected in the fat body of silkworm. The result will provide a useful tool for understanding the role of fat body in silkworm.

Abstract:Pectin lyases from Aspergillus oryzae and Aspergillus niger are usually used for the production of traditional fermented foods, but these fungi produce less pectinases under natural conditions. The cDNA coding mature Pel1 (without signal peptide) was amplified from Aspergillus oryzae by RT-PCR. Pel1 cDNA was cloned into pET-28a (+) expression vector, then was transformed into E. coli Turner (DE3) placⅠcells to express Pel1 with 6-His tag. For improving the efficiency of Pel1 expression in E. coli, the conditions of expressing the Pel1 in E. coli were optimized. E. coli Turner (DE3) placⅠcells with pET-28a (+)-pel1 was first cultivated at 37℃, 220 r/min until OD₆₀₀ reached about 0.8. Then, cultivation broth was added with 0.05～0.1mmol/L IPTG and continuously incubated at 15℃, at 170 r/min for 60 h for expressing of Pel1. The recombinant expressed Pel1 activity could reach 400u/mL medium, which is 4000_fold of Pel1 produced naturally by A. oryzae and superior than known recombinant amount of pectin lyases expressed in different fungi expression systems.

Abstract:Redox electrode was used to control redox potential at four different levels(-50mV,-100mV,-150mV,-230mV) for the study of ethanol fermentation. The result showed that there was notably influence on the yield of ethanol ,the formation of glycerol, the secretion of organic acid，biomass and the death of cell by controlling redox potential at different levels. For example: the biomass of ORP at -50mV was 1.26,1.86,2.59 times higher than ORP at -100mV、-150mV、-230mV respectively, the final glycerol concentration was 1.2,1.1,1.7 times higher but final ethanol concentration was 0.87,0.49,0.51 times lower compared to the latest three ORP control level. And take biomass, ethanol yield, glycerol concentration, and unconsumed glucose into consider, we get the conclusion that it is very favorable for ethanol fermentation by control ORP at 150mV. So it give us a apocalypse that we can use redox electrode to control the ethanol fermentation exactly on bioreactor scale.

Abstract:The diatom Nitzschia laevis is a good alternative source of eicosapentaenoic acid (EPA). Besides strategies for high cell density culture, EPA productivity may be further improved by herbicides. The effect of the herbicide quizalofop-p-ethyl on the growth and EPA production was studied in this paper. As the solvent of the herbicide, DMSO was proved to inhibit the growth and EPA production of N. laevis. The concentration of DMSO in the medium should not exceed 0.2%. Quizalofop-p-ethyl could cause morphology damage to the N. laevis cells. With the increasing concentration of quizalofop-p-ethyl from 0mmol/L to 0.4mmol/L, the dry cell weight production decreased, while at the same time, the lipid content of the dry cell mass increased. When treated with 0.1mmol/L quizalofop-p-ethyl, the EPA content increased from 3.00% to 3.58% (of dry cell weight, DW), and the proportion of EPA (20:5) in total fatty acids (TFA) increased from 25.15% to 32.88%. These results indicated that the herbicide quizalofop-p-ethyl could stimulate the accumulation of EPA; therefore it might be useful for selecting algae colonies that overproduce EPA.