Abstract:The genetic diversity of 279 indivdiuals from 10 populations in Shandong Province was investigated using inter-simple sequence repeat (ISSR) markers. As a result，116 bands were amplified by 10 informative and reliable primers，of which 101 were polymorphic loci. A relatively high level of genetic diversity was revealed: PPL＝87.07，H_e＝0.2697，H₀＝0.3999 (at the species level); PPL＝64.58，H_e＝0.2004，H₀＝0.3010 (at the population level). A higher level of genetic differentiation was detected among populations with Nei's G_ST analysis and the analysis of molecular variance (AMOVA; G_ST=0.2414，F_ST=0.2224). Habitat fragmentation and gene flow may result in genetic differentiation. UPGMA cluster analysis indicated that the four populations from Linshu，Junan，Tancheng and Feixian grouped together，whereas Laiyang populations clustered in an isolated clade. The results showed that a mixed mating system was possibly the main factor influencing the genetic structure of this species. These results，combined with other information about Castanea mollissima，may provide a valuable basis for proposing conservation strategies.

Abstract:The full length cry2Abgene was cloned by PCR-RFLP method from Bt strain B-Pr-88，which was isolated in China with high toxicity to the Lepidopteran insect pests. Nucleic acid sequence analysis showed that this gene was 1902 base pairs encoding 633 amino acids. This cry gene was named cry2Ab4 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The full open reading frame sequence of the cry2Ab4 gene was amplified with a pair of PCR primers L2ab5/L2ab3 designed according to its DNA sequence，and inserted into the BamHⅠ/EcoRⅠ sites of E. coli expression vector pET21b to obtain the recombinant plasmid pET-2Ab4. The result of SDS-PAGE proved that Cry2Ab4 could be expressed as a 60kD protein in E. coli BL21（DE3）strain induced by IPTG. Bioassay of the expressed product of the cry2Ab4 gene showed that Cry2Ab4 was highly toxic to the larvae of Helicoverpa armigera and Leguminivora glycinivorella，moderately active to the larvae of Plutella xylostella and Chilo suppressalis，but not insecticidal to the larvae of Spodotera exigua and Ostrinia furnacalis. Our result indicated that cry2Ab4 gene could be used as a novel gene for generation of transgenic plants and engineered microorganism.

Abstract:Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse，Stra8 is restrictively expressed in male germ line cells，and its function is related to the development of sperm. In order to investigate the feature of Stra8gene expression，the 1.4kb (-1407～+7) promoter region of Stra8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4kb promoter region，and the reporter gene of EGFP that was regulated by 1.4kb Stra8 promoter. To investigate the specificity of Stra8promoter，the vector pStra8-EGFP was transfected into undifferentiated mouse stem cells such as ES-129，bone marrow mesenchymal stem cell (mMSC) and spermatogonial stem cell (mSSC). The results showed that the expression of GFP was only observed in the mSSC cells，which indicated that Stra8 gene was specially regulated in testis tissue. As the gene marker，vector pStra8-EGFP was then transfected to undifferentiated mMSC cells. After being selected by G418 for 2 weeks，the mMSC cells were induced by retinoic acid. After 12 hours induction，some induced cells started to express GFP protein，which was observed under the fluorescence microscope. At the same time，several stem cell specificity biomarkers such as Oct4，and spermatogonial stem cell biomarkers such as CyclinA2and Stra8 were detected in the induced cells by RT-PCR method. These results showed that the mMSCs would differentiated to spermatognial stem cells after induced by Retinoic Acid.

Abstract:The Snail transcription factor has been described as a strong repressor of E-cadherin and its stable expression induces epithelial-mesenchymal transitions responsible for the acquisition of motile and invasive properties during tumor progression. A fascinating analogy that has been raised is the seemingly similar and shared characteristics of stem cells and tumorigenic cells，which prompted us to investigate whether the mechanisms of the acquisition of invasiveness during tumor progression are also involved in bone marrow stem cells(MSCs). In this study，we examined whether Snail gene expression acts in the mobility，cytoskeleton and anti-apoptosis of MSCs. Cell Transmigration Assay and Western Blotting were performed to evaluate the cell migratory capability and the related Signaling pathways in MSCs transfected with the Snail expression vector of pCAGGSneo-Snail-HA (MSCs-Sna)，compared with MSCs(MSCs-neo) transducted with the control vector(pCAGGSneo). Actin cytoskeleton by Immunofluorescence and Sub-G1 detection by a FACScan flow cytometer were performed to analyze the cytoskeleton and anti-apoptotic capability of MSCs-Sna. Compared with MSCs-neo，MSCs-Sna show significantly more migration in the transwell migration system (P<0.05). And suppression of PI-3K activation by the specific PI-3K inhibitor，Wortmannin，brought on a reduction in Snail-mediated MSCs migration. In addition，we provide evidences that high expression of Snail inhibited the serum-deprivation triggered apoptosis and cytoskeleton changement of MSCs. These data suggest the possibility of facilitating MSCs migration to injured tissue and subsequent survival and maintenance in the local microenvironment after their transplantation，by investigating and increasing the advantage factors such as Snail high expression in MSCs.

Abstract:PRAS40, a proline-rich Akt substrate of 40kD, is 14-3-3 binding protein. To study the interaction between PRAS40 and 14-3-3 isoforms，We constructed the expression vector pEG-PRAS40 (DNA-binding plasmid) and pJG-PRAS40 (transcriptional activity plasmid) in yeast using gateway cloning technology, then the plasmid of pEG- PRAS40/pJG-PRAS40 was co-transformed into yeast EGY48 strain with each pJG-14-3-3 /pEG-14-3-3 isoform plasmid. The co-transformation were tested by nutrition limitation growth analysis，β-galactosidase color assay was used to study the interaction degree between PRAS40 and 14-3-3 isoforms. We confirmed successfully the construction of pJG-PRAS40 and pEG-PRAS40 with enzyme digestion. four 14-3-3 isoforms were found interacting with PRAS40 using yeast two-hybrid assay, the interaction degree of Epsilon was stronger than beta and zeta, tau was the weakest. Our result will be used to further study the biological function of PRAS40 and 14-3-3 as new drug target.

Abstract:A structure kinetic model was established for describing the relationship among cell growth, genistein formation and substrate utilization in suspension culture of Maackia amurensis. In this model, sucrose uptake, structure components production, intermediate matter changes, cell respiration loss and secondary metabolite (genistein) production were all predicted. The parameter sensitivity test was indicated that intermediate matter self-catalysis rate constant (k_b1), structure component synthesis rate constant (k_b2) and secondary metabolite synthesis rate constant (kp)were the most sensitive parameters for this model. If k_b1, k_b2 or k_p are changed by 10%, the range of the aim function value would be changed by 12.8%, 4.61% and 2.54%, respectively. Adjustment of the other parameters only aroused a change of less than 0.5% in the aim function value. The predicted values were in good agreement with those obtained experimentally.

Abstract:This study is conducted to explore an effective culture method for supporting the embryo development. The cattle fetal ear fibroblasts and the goat fetal ear fibroblasts are transplanted into the enucleated cattle oocytes separately by oocyte intraplasmic nuclear injection method to construct bovine cloned embryos and goat-bovine cloned embryos. The embryos are first cultivated in modified charles rosenkrans 2 amino acid medium (mCR2aa) and modified synthetic oviduct fluid medium (mSOF) separately. Then BSA(8mg/mL) or FBS(10%) can be added to mSOF according to the different culture period. The supplements and orders, added during the first three days and after three days are as follow: BSA and BSA, BSA and FBS, FBS and BSA, FBS and FBS. On the basis of the cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, the best culture way can be screened out. Result: First, cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, cultivated in mSOF solution are all higher than those cultivated in mCR2aa(P<0.05).Second, the cleavage rate and 8/16-cell rate, adding BSA and FBS into mSOF, are in turn 79.8%±7.1%, 49.7%±3.5%, 21.5%±1.8%, and 115.2±4.3 in bovine cloned embryo, and 40.1%±6.3%、29.2%±2.0%、13.4%±2.1% and 100.1±3.0 in goat-bovine cloned embryo, which are significant higher than other culture groups(P<0.05).Conclusion: The goat-bovine cloned embryo can be cultivated by the optimized culture measure of bovine cloned embryo. The best culture ways of bovine cloned embryo and goat-bovine cloned embryo are all to use mSOF supplemented BSA in the first three days and then use mSOF supplemented FBS in the next five days.

Abstract:Lipase (EC3.1.1.3) from Candida sp.99-125 was immobilized on chitosan by chemical covalence. Lipase was first immobilized to chitosan beads by activating its hydroxyl groups with carbodiimide followed by cross-linking more lipase to the amino groups with glutaraldehyde. In this article, different factors that influenced the immobilization were investigated, and the optimum conditions were ascertained. Comparative studies of organic solvent and thermal stability between free lipase and immobilized lipase were conducted. Immobilization enhanced the lipase stability against changes of temperature and organic solvent. Immobilization lipase can be reused in the synthesis system of palmitate hexadecyl. Operational stability tests indicated that the immobilized lipase occurs after 16 consecutive batches, the conversion rate remained 85%. Such results revealed good potential for recycling under esterification system.

Abstract:In the present study, the genome shuffling was used to improve lipase production of Penicillium expansum. A lipase producing mutant strain-Penicillium expansum FS8486 and a wild type of Aspergillus Tamarii FS-132 isolated from soil of a volcano in Xinjiang were used as the parental strains. After two rounds of genome shuffling, several elite daughter strains were screened. The lipase activity in one of the daughter strains was increased 317% over the starting strain FS8486.Comparisons of the morphology, RAPD (Random Amplification of Polymorphic DNA) polymorphism and the fatty acid compositions between the daughter and the parental strains suggested that the filial generation were generated by genome shuffling. In this study, the genome shuffling used successfully first time in eukaryotic microorganism and increases the production of the desired metabolite in short time, the study will be useful to spread the genome shuffling in eukaryotic microbial breeding.

Abstract:In order to improve the thermostability of the Penicillium expansum Lipase (PEL), the lipase encoding genes was mutated by site-directed mutagenesis. A recombinant vector pAO815-ep8-K55R which contain double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K55R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8- K55R can secret double-mutant lipase PEL-ep8-K55R-GS into the medium when it was induced by Methanol. The yield of the double-mutant lipase is 508u/mL, which is 81% that of the wild type lipase PEL-GS(627u/mL) and 55% that of random-mutant PEL-ep8-GS(924u/mL). The specific activity of double-mutant lipase is 2309.1u/mg, which is similar to random-mutant lipase PEL-ep8-GS and the wild type lipase PEL-GS. The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL-ep8-GS. While the T_{m of the double-mutant lipase is 41.0℃, 2.3℃ higher than the wild type lipase PEL-GS and 0.8% higher than the random-mutant lipase PEL-ep8-GS, indicating that the double-mutant lipase PEL-ep8-K55R-GS has higher thermostability.}

Abstract:The antifungal, anti-bacterical, anti-brine shrimp activities of SD22 isolated from Paenibacillus daejeonensis Bacteria SS02 were studied. The separation steps included ultracentrifugation, ultrafiltration and (NH₄)₂SO₄ fractional precipitation, further purification was performed by SephadexG-75 and DEAE-32 chromatography. Its molecular weight determined by SDS-PAGE was 56.0kD and its isoelectfic point was 6.4．SD22 was thermostable to some extent and stable to ultraviolet, but sensitive to some of the enzyme. SD22 could kill most pathogens from propagation, such asRhizoctonia cerealis,Sclerotinia sclerotiorum Physalospora piricala,Trichodema viride,Gliocladium viride,Curvularia leaf spot, Fusarium sp, Fusarium head blight, Beauveria Bassiana, Escherichia coli, Staphylo-coccus aureus, Bacillus subtilis,Candidal vaginitis, Fusarium oxysporum Schl.emend.Snyder&Hansem et al. The results will be helpful to find out a novel antifungal protein.

Abstract:The effect of initial pH values on acetate production was studied in the acidification- homoacetogenesis two-phase coupling system using glucose as the substrate and the heat-treated and activated anaerobic sludge as the inoculum. Substrate degradation, product yield and pH variation during fermentation were examined at various initial pH values （5, 6, 7, 8, 9, 10 and 11）. The results show that initial pH values affect volatile fatty acids and ethanol production not only in the acidification phase itself but also in the homoacetogenesis phase. Ethanol-type fermentation mainly takes place at initial pH 5 while butyrate-type fermentation at initial pH 6 and 7.But acetate is the dominant product at initial pH 8～11. The optimal initial pH value is 10 for acetate production in the two-phase coupling system. At initial pH 8～11, ethanol concentration is highest at the beginning of acidification, but there is a subsequent decline as ethanol is converted to acetate as a result of further metabolism of the microbes.

Abstract:Novel hydrophobic absorbents were synthesized by immobilizing butyl derivative onto the highly cross-linked agarose beads manufatured in China, which are used as matrix. The effect of the spacer arm length (3C, 8C and 10C) and ligand density (from 13 to 45μmol/mL) on the hydrophobicity were investigated using purified Hepatitis B surface antigen (HBsAg) expressed by CHO cell lines. Also considering the effects of salt concentration and pH on HBsAg recovery and purification factor, orthogonal experiment design method was used to evaluated the absorbents. The results showed the butyl-S absorbent with the spacer arm length of C8, the ligand density of 22μmol/mL gel showed the best performance for the separation of HBsAg. Approximately 100% HBsAg recovery and 60 as purification fold were achieved by this media under the operating condition of pH 7.0 and 9% of salt concentrateion.

Abstract:The preparation process and immunogenicity of a novel hepatitis B vaccine containing preS1, preS2 and S epitopes were investigated in this study. APichia pastoris stain GS115-SS1S2 harbouring two chimeric HBsAg gene constructs, SS1 and SS2 was cultivated by high-density fermentation. 300～600mg/L of the expression level was achieved through 48～72h methanol induction. SS1S2 antigen was extracted and purified by silica adsorption, HIC and SEC to 99% purity from the harvested cells. 82mg purified antigen could be achieved from one liter of fermentation culture. The immunogenicity of the purified antigen was evaluated in NIH mice. Three groups of female NIH mice, 14～16g in weight, were injected once intraperitoneally with 2.5, 0.625, 0.156μg of each of the two vaccines: SS1S2 or a commercially available S vaccine. Part of the mice were bled in 30 days after injection to compare the ED₅₀ of the two vaccines. For the SS1S2 vaccine, the ED₅₀ is 0.46, 0.29 and 0.84μg respectively for the preS1, preS2 and S antigens. For the S vaccine, the ED₅₀ is 0.99μg for the S antigen. Another part of the mice were bleed in 7 or 14 days to detect preS1, preS2 and S antibodies. Higher ratios of mice were seroconverted for preS1 and preS2 antibodies as compared to the S antibody in these two time points. These results suggest that the SS1S2 vaccine may be more immunogenic than the conventional S vaccines.

Abstract:The coordination compound of L-hydroxyproline(Hyp)-Zn(Ⅱ) was synthesized with Hyp and zinc sulfate as raw materials in water medium, coordination Synthesizing Mechanism and Antioxidant Activity of Hyp-Zn(Ⅱ)coordination compound has been researched. Compared with Hyp, the infrared spectrogram of Hyp-Zn(Ⅱ) coordination compound emerge a new absorption peak at 1100cm^－1. Conclusion could be obtained that there exists a coordination effect between Hyp and ZnSO₄; TG and DSC curve of Hyp and Hyp-Zn(Ⅱ) coordination compound were analysed. Compared with Hyp, the peak of Hyp-Zn(Ⅱ) disappear at 290℃ and 375℃. This phenomenon confirmed the front conclusion; At the NMR graph of Hyp-Zn(Ⅱ)coordination compound, the disappearance of the α-carboxyl-hydrogen and α-hydroxyl- hydrogen's peak at 3.5～3.9ppm could indicate that combination's position of Hyp isα-carboxyl and α-hydroxyl; Structure of Hyp-Zn(Ⅱ) coordination compound were exosyndrome by the Atomic Force microscopy. It is showed that centr-atom Zn(Ⅱ) was surrounded by several Hyp at Hyp-Zn(Ⅱ) coordination compound's phase diagram; The proportion of Hyp-Zn(Ⅱ) coordination compound was determined by dialysis experiment, the proportion is 4∶1; Above-mentioned determination and exosyndrome indicated that the molecular formula of Hyp-Zn(Ⅱ) coordination compound is Zn(Hyp)₄·H₂O. The results indicated that the Hyp-Zn(Ⅱ) coordination compound can inhibit hydroxyl free radicals of Zn (Ⅱ), and the Percentage of Inhibition is 75.5% ;the total antioxidant activities of Hyp-Zn(Ⅱ) coordination compound is 80.167u/mL, the anti-superoxide activities of Hyp-Zn(Ⅱ) coordination compound is 53.19u/mL.

Abstract:To set up a new rapid and efficient way for the preparation of specific monoclonal antibodies (MAbs) with plasmid DNA immunization.Methods 　The fusion gene of IL-2 signal peptide and profilin 1 by ‘overlapping PCR' was obtained and inserted into the vector pVAX1 to construct the recombinant plasmid pVAX-IL2-prof1. Another fusion gene of IgG kappa chain signal peptide and profilin 1 by ‘no template PCR' was obtained and inserted into the vector pVAX1 to construct the recombinant plasmid pVAX-Igκ-prof1. BALB/c mice were single intrasplenic immunized with plasmid DNA. Results After cell fusion and hybridomas screening by indirect ELISA, we charactered two mAbs (1D8A2B4 and 4B12A5E3) against profilin 1. The MAbs isotype were determined as IgM and IgG3. Conclusion A single intrasplenic plasmid DNA immunization is rapid and efficient and can be used as a helpful tool for the production of specific mAb.

Abstract:Bacillus pumilus xylanase was cloned and sequenced. Based on the tertiary structure that originated from homology modeling, the potential active pocket was searched and ligand-protein docking was performed using relative softwares. The information extracted from the molecular docking is analyzed; several amino acid residues might play a vital role in the xylanase catalytic reaction are obtained to instruct the further modification of xylanase directed-evolution.

Abstract:Infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, carries a small nonstructural protein (NS). In this study, vvIBDV Gx_VP5 genes were cloned into plasmid pET30a(+) and expressed in E.coli with IPTG inducing. BALB／c mice were immunized with the purified recombinant fusion protein． SP2/0 myeloma cells and spleen cells of BALB/c mice were fused by PEG（MW1500）, three hybridoma cell lines were examined by indirect ELISA and clone for three times by limited dilution, and were named as 4B4, 6D12, 3E8. The subtype of the monoclonal antibodies were IgG1 with a subtype identified ELISA kit, and light chains were kappa. The ascites titers of monoclonal antibodies were 5×10⁴ 3.5×10⁴, 3×10⁴ by indirect ELISA, respectively. Indirect ELISA and Western blot results showed that the monoclonal antibodies only acted with VP5 protein, IF analysis indicated that three monoclonal antibodies acted with IBDV Gt. There were specific fluorescence in detected Vero E6 cells which transient expressed VP5 protein by IFA. Therefore, monoclonal antibodies specific to IBDV VP5 proteins are specific method for detected VP5 proteins, and base on establish stabilize expressed VP5 protein Vero cell lines to research IBDV VP5 protein function.

Abstract:DNA recombinase FLP gene exists on the 2μ plasmid of Saccharomyces cerevisiae. Recombinase FLP could recognize an FRT site composed of 34bp and function the sequences for exchange, recombination, deletion and reversion between the two orientated FRT sites. These functions are highly recognized by molecular biologists and biotechnology engineers for theoretic and applicable technology studies. This work constructed a prokaryotic over-expressed vector harboring FLP gene nominated as pQE30-flpe and established its over-expression culture system in which recombinase FLP could be efficiently expressed in E. coli strain M15. Purification procedures for high purity and active FLP are established through combination of ammonium sulfate precipitation with a 0.5～1.0mL micro-column technique of Ni affinity chromatography with gradient elution. To verify the recombinase activity of purified FLP, substrate vectors, sequence donor vector (pUC18-FRT-gfp-FRT) and sequence accepting vector (pET30a-FRT) are constructed with various number, orientation of FRTs harboring the GFP gene for the expression of visible assay of the functions of recombination, exchange and deletion. Results showed that the system not only over expressed recombinase FLP in prokaryotic E.coli, but also efficiently purified the enzyme with a higher activity of the function of recombination, exchange and deletion. The system and the method are easily implemented and feasibly manipulated for theoretic study and biotechnology application.

Abstract:Interferon α gene was cloned from genomic DNA of Chinese Luxi yellow cattle by PCR, and the PCR product was inserted into vector pET32a(+) to make a recombinant plasmid pET32a(+)/BoIFN-α. The expression of BoIFN-α in Escherichia coli was induced by addition of IPTG. Sequence analysis showed that the Chinese Luxi yellow cattle IFN-α gene is composed of 498 nucleotides, encoding a mature polypeptide of 166 amino acids. Compared with other BoIFN-α subtypes, it shares the highest identity of 97.6% to the C-subtype. SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 40kD and the recombinant proteins accounted for 26.7% of the whole proteins.The expressed product was purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and its antiviral activities were tested on MDBK/VSV cell system. Its antiviral activities were 5×10⁵u/mg on MDBK/VSV cell system. The results showed that the expression plasmid was successfully constructed and BoIFN-α C2 protein was expressed in Escherichia coli. Moreover the purification had good effects on antiviral activities.

Abstract:In this study, polyacrylicacid precipitation alkalescence protein from Momordica charantia L. seeds was studied, and the effect of conditions on experiment was also evaluated. Isoelectric precipitation is achieved by adjusting the pH of a protein solution and is based on that a protein's solubility is at minimum at its pI. The sample was titrated to pH 6.0 with citric acid, and 14.62% proteins were precipitated. With hydrochloric acid to pH 4.0, 32.49% proteins were precipitated. With the acetic acid to pH 6.0 and pH 4.0, 26.17% and 38.72% proteins were precipitated, respectively. In the 1 mL Bitter melon seeds extraction(pH 4.0) adjusted by acetic acid, hydrochloric acid and citric acid, the optimum dosage of PAA（1%）precipiting alkalescency protein (pI 8.65～9.30）was 100μL, 120μL and 100μL respectively. The respective extraction (1mL) was titrated to pH 5.0, pH 4.0, and pH 3.0 by acetic acid. After isoelectric precipitation, the PAA precipitation protein was performed. When concentration of PAA （1%） was 160μL/mL, the protein decreased in the supernatant was 33.77% at pH 5.0 , and 43.56% at pH 3.0. When concentration of PAA （1%） was 120μL/mL, the protein decreased in the supernatant was 30.83% at pH 4.0. PAA-Protein complex could redissolve in alkaline conditions (pH＞9.0) and the protein most easilly redissolved when the NaCl was 3.0%. The bitter melon seeds extraction after PAA purification flowed throμgh the Sephadex G-75 columns. The peaks Ⅰand Ⅱ were obtained after 175min and 300min, respectively. SDS-PAGE and IEF analysis showed that the molecule weight from peaks Ⅰwas 30kD with pI 9.5, peaks Ⅱ10kD with pI 9.3.

Abstract:Comparing to Chitosan, Chitosan-oligosaccharides have several special functions, such as water-soluble, antitumor activity, immunostimulating effects, and antimicrobial activity. The chitosan-oligosaccharide, the molecular weight of which was about 5000, was used as research model. According to the agarose gel electrophoresis and UV spectrophotometer it was proved that electrostatic interaction was playing a very important role in the formation process of chitosan-oligosaccharide/DNA complex. The potential of adsorbing DNA on chitosan-oligosaccharide was analyzed by gel electrophoresis and UV spectrophotometer, and it was indicated that chitosan-oligosaccharide can improve the storage and structure stability of DNA. To check its protection ability to DNA by DNase I digestive experiment, the result showed that chitosan-oligosaccharide could load with plasmid effectively and protect DNA from being digested by DNase I. It was proved that chitosan-oligosacchide was safe and effective for gene delivery and will have a very good future in the field of gene therapy.

Abstract:Bakers yeast is an important additive among the products which improves bread quality and for present time is being produced in different countries by batch, fed batch or continuous cultures. Saccharomyces cerevisiae is used in fermentation of starch in dough, giving a favourable taste and produces a variety of vitamins and proteins.The main ingredient in yeast production is carbon source such as beet molasses, cane molasses, and so on. Since beet molasses has other major function as in high yield alcohol production and also due to the bioenvironmental issues and related wastewater treatment, the use of other carbohydrate sources may be considered. One of these carbohydrate sources is date which is wasted a great deal annually in this country (Iran). In this study, the capability of date to act as a suitable carbon sources was investigated. The waste date turned into juice and consequently production and growth rate of Sacchromyces cervisiae were studied with this juice. A maximum possible yield of 50% was obtained by the optimum medium (P3), at pH 3.4, 30℃, 1.4vvm aeration rate and agitation of 500r/min.

Abstract:Male germ stem cells(mGSCs)，which is in testis after sex differentiation, derive from primordial germ cells. In this study, bovine mGSCs were isolated from testis of 20 weeks fetuses. Number of CD9 positive cells of the cells through two-steps adhering plates velocity different was 95.8% by flow cytometer. The carina-type cells clones and the plane-type cells clones appeared in co-cultured system. One cells lines had been successively maintained for 4 passages, and the cells clusters showed AKP positive staining. The cells clusters showed nest-shape in third passage showed SSEA1 and Oct-4 positive staining. These cells can also spontaneously differentiate into c-kit positive staining germ cells，and the cells were directional induced to forma-actin positive staining cardiac-like cells cluster and NF positive staining neuron-like cells. The conclusion showed that male germ stem cells from 20 weeks bovine fetuses could be in vitro formed like embryonic stem cells.

Abstract:Stable transformants for mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a newly screening vector plasmid pMCEscan, which carrying aFRT-neo^*-IRES-k₂tPA fusion gene and a DHFR gene, we screened colonies by k₂tPA expression level. We selected 7 clones that expressed high level of k₂tPA and carried one copy of the plasmid in their chromosomes. These clones showed in high level k₂tPA production without amplification. So we targeted reporter gene (k₂2tPA) to test the basal expression ability of these cells clones. The clone, 8-1, showed the same effect to high base expression level. In this clone, the FRT-neo^*-IRES-tPA gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a FRT-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal FRT by FLP recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. Using the site-specific integration CHO/dhfr^－－ cell line 8-1, the expression level of k₂tPA could amount to 17.1μg/10⁶cell·24h.