Abstract The aim of the study is to observe the regulation role of sulfatase 2 gene fragment in vivo expression on mouse myelodepression induced by 5-fluorouracil injection. The recombinant pcDNA3.1-Sulf2 eukaryotic expression vector was constructed using gene cloning method，mice bone marrow depression and regeneration model was established by injection of 5-fluorouracil (250mg/kg) via mice tail vein. 50μg of pcDNA3.1-Sulf2 recombinant eukaryotic expression vector (experimental group) was injected into the tibial muscle of each mouse, then introduced into muscle cells for expression by electroporation on the first day after 5-fluorouracil injection, so did the control group which injected and electroporated with the same quantity of pcDNA3.1 blank plasmid . Mice peripheral blood cells and platelets were calculated via automatic blood cells analyzer, and bone marrow cells per leg of each mouse was counted by hemacytometer on day 0 before injection of 5-fluorouracil and on day 3, day 7,day 11,day 14 after injection of 5-fluorouracil respectively. Colony forming test were carried out between the experimental group and control group on day 7 and day 14. The numbers of peripheral blood cells and platelets in experimental group on day 7 were significantly lower than those in the control group respectively ［(1216.7±457.9)μl, n=6 versus(1691.7±228.9)μl, n=6; and (8.1±5.4)×104/μl, n=6 versus (14.7±2.1)×104/μl, n=6, respectively, p<0.05］.The total bone marrow cells per single leg on day 7 in experimental group were (94.2±21.1)×104 (n=6)，and significantly lower than the control group (173±59.9)×104(n=6, p<0.05), but there was a significantly higher numbers of bone marrow cells per leg on day 11 in experimental group (585±337.9)×104(n=6) than in the control group (255±65.3)×104(n=6, p<0.05). Colony forming tests showed that the total colonies per 10000 of bone marrow cells on day 7 of experimental group was at 9±8.4 (n=4), significantly lower than that in control group 39±12.2(n=4, p<0.05), while there was no statistical significance between experimental group (23.3±7.6) per 1000 of bone marrow cells(n=4) and control group (28±6.1) per 1000 of bone marrow cells (n=3, p>0.05) on day 14. The results indicated that there is a protective effect of sulfatase 2 gene expression on the regeneration of mouse bone marrow depressed by 5-fluorouracil.
Abstract Phospholipase C gamma-1(PLC-γ1) is a nerve-enriched protein containing SH2-SH2-SH3 domain. It has been demonstrated that the SH2 domain of PLC-γ1 regulates the phosphorylation of PLC -γ1 through interaction with receptor tyrosin kinase , and the SH3 domain of which interacts with sosorvinculinto mediate Ras signaling and regulate actin polymerization. To understand better this potential functional role and produce the monoclonal antibody against SH2-SH2-SH3 of PLC-γ1，using RT-PCR from adult human cortex ,the functional domain（SH2-SH2-SH3 domain）ofPLC-γ1 was cloned to pGEX-2T fusion vector and transformed into E.coli BL21 host cells. SDS-PAGEand Western blot analysis revealed that the induced recombinant protein by IPTG was expressed successfully at 33℃ but weakly at 37℃. Furthermore, the recombinant protein was degraded at 37℃ . The GST fusion protein extract was immobilised on glutathione sepharose 4B bead and eluted with glutathion. Taken together, the results indicated that a lower culture batch(33℃) was utilized to produce recombinant protein easily. The fragment expression of SH2-SH2-SH3 at higher temperature(37℃), however, suggests that PLC-γ1 degradation was involved not only in eukaryotic cell but also in prokarytic cell(BL21 strain). The identification of cleaved site of SH2-SH2-SH3 domain by peptide sequencing analysis will be done in the future
Abstract The BALB/c mice were immuned with purified recombinant fusion protein （ORF2-V1）from swine Hepatitis E virus. The myeloma cell line SP2/0 was fused with the spleen of the immuned BALB/c mice, and the positive clones which produced McAbs against ORF2-V1 were screened by ELISA. The titers of the six McAbs in the supernatant culture supernatant were 1∶1.60×103～1∶3.20×103, respectively , and the titers in the ascites of the mice were 1∶1.28×106～1∶2.56×106, respectively, detected by ELISA. And six McAbs only could react with ORF2-V1, and but not react with other recombinant fusion protein from ORF2. Among them , The A549 cell culture affected with HEV was carried out comparative studies, which is detection of pathogene with indirect immunofluorescence by using monoclonal antibody against HEV ORF2-V1 protein and polyclonal antiserum against HEV. The result shows that McAb against HEV ORF2-V1 protein, its speciality is high. Six monoclonal antibody belonged to IgG1, The titers of the six McAbs in the supernatant culture supernatant were 1∶1.60×103～1∶3.20×103, and the titers in the ascites of the mice were 1∶1.28×106～1∶2.56×106. The six strains of McAbs were specific for three different kinds of epitopes on HEV. Among them, McAb-γH1, McAb-BC4 and McAbCH8 direct to different epitopes, respectively．The epitope recognized by McAb-γH1 overlapped partly that of McAb-BC4．McAb-αC11 and McAb-αC12 were identical to McAb b-γF8．The epitope of which was supposed to be locateed in the overlapping region between epitope-γH1 and epitope-BC4．
Objective Establish mass-scale purification technology of cell-derived influenza virus. Methods A microcarrier-based process was used to produce human influenza virus in serum free-adapted Vero cells. The virus was purified in a sequence of downstream processing steps including inactivation, clarification, anion exchange chromatography, affinity chromatography and size-exclusion chromatography. Results The recovery of HA reached 102%. 95.3% total protein, including 99.77% host cell protein, and 99.99% host cell DNA were removed during downstream processing. Conclusion This research provides a high effective purification technology for cell-derived influenza virus.
Abstract: Objective: To screen gravity related genes of silkworm Embryogenesis. Methods: Suppression subtractive hybridization (SSH) was used to isolate gravity related genes of silkworm embryogenesis in simulated weightlessness and normal gravity environment. Differential expression genes were cloned, sequenced and analyzed on homology. Results: 34 gravity related genes were obtained using SSH. 16 genes were up regulated in simulated weightlessness environment, including 15 unknown genes and one known gene maintaining the stability of mRNA. 18 genes were down regulated in simulated weightlessness environment, including 3 unknown genes, 4 contig genes, 5 submitted and unknown function genes in SilkBase, and 6 protein synthesis genes. Conclusion: Simulated weightlessness environment affects the expression of genes playing a role in maintaining the stability of mRNA and synthesizing protein during the period of silkworm embryogenesis.
In this study, the PGA gene of Bacillus megaterium 1.1741 was amplified by PCR, using primers designed on the base of PGA gene sequences of Bacillus megaterium in GenBank. Then the amplified PGA gene was cloned into pYES2 (amp+ ura+) vector, with T7lac promoter as the control. The constructed recombinant plasmid, named as pYES2-PGA, was transformed into Saccharomyces cerevisiae H158 (his- trp- leu- ura-) according to the LiAc/SSDNA/PEG method. PGA activity was detected in the culture liquid of recombinant Saccharomyces cerevisiae without penylacetic acid as the inducer, and the highest PGA activity was 0.75 U/ml. The sequence of PGA gene (present study) showed a high homology of 97.1%, 99.8% and 99.8% with three other strains of Bacillus megaterium L04471.1, U07682.1 and Z37542 in GenBank, respectively.
Soft rot caused by Erwinia carotovara.subsp.carotovora (Ecc) is a very serious disease in the world. Owing to the absence of natural resistant resources, the work moves slowly. Arabidopsis thaliana became the main target material for studying the mechanism of resistance to Ecc. In this paper, we selected 29 Arabidopsis accessions, then established the disease classification standards, and took the disease index at 48 hours post inoculated as the evaluation criteria of resistance to Ecc. The results showed that the ecotype CS906 was resistant and the ecotype CS20 was highly susceptive. By comparing gene expression changes of AOS, ERF1-1, PR1, PDF1.2 and PAL1 between the resistant and susceptible accessions at different time points after inoculation, we found that SA、ET and MJ signal transduction pathways were involved in the defense response of Arabidopsis to Ecc, and gene expression model was similar in CS906 and CS20, but the expression levels of genes in CS906 were higher than that in CS20. Upper results will facilitate the study on the resistance mechanism of Arabidopsis to Ecc.
Two ORFs in Staphylococcus aureus genomes were predicted to encode nucleases. One is encoded the nuclease A (SNase) without a gene name (termed nuc1); the other is encoded a thermonuclease (TNase) with a name of nuc (termed nuc2). To further investigate the function of nuc2 in S.aureus, the resulting plasmid, pBT2Δnuc1 was successfully constructed. The recombinant plasmid pBT2?nuc1 was introduced into S .aureus RN4220 by electroporation. Using the resistant marker for seven passages of culture, the recombination rate was about 2% (7/345). A nuc1-deleted mutant of S. aureus strain RN4220 (termed RN4220Δnuc1) was successfully constructed and conformed by PCR and RT-PCR.
Targeting rRNA of bacteria is a new strategy for antibiotic agent development. The rRNA such as mRNA are naturally self-folded molecules which expose only limited accessible target-sites for binding. These accessible sites are pivotal for designing the effective antisense oligonucleotides, ribozymes, and DNAzymes. MAST, an RNA accessible site screening method, illustrated 6 accessible sites on 16S rRNA by immobilizing 16S rRNA and hybridizing with oligonucleotide library. 5 of the accessible sites were identified valid, and the antisense oligonucleotides targeted to which showed inhibition effectiveness on the proliferation. Among the 5 target sites, one showed the priority of accessibility. Ribozyme designed to this site showed obvious inhibition to the growth when induced expressing in the transfection E. coli.
In the reductive branch, glycerol is first dehydrated to 3-hydroxypropionaldehyde that is then reduced to 1,3-PD under the consumption of reducing power (NADH). If over-expression of the gene dhaB encoding glycerol dehydrase is achieved,the reducing power will be scarce and 3-hydroxypropionaldehyde will be accumulated,which is disadvantage to produce 1,3-propanediol.The structure gene yqhD from E.coli encoding 1,3-propanediol oxidoreductase isoenzyme（under the consumption of reducing power (NADPH)）and the gene dhaB encoding glycerol dehydrase from Klebsiella pneumoniae was amplified using PCR method.The two gene were transferred into expression vector pEtac to construct a novel recombinant Klebsiella pneumoniae (pEtac-dhaB-tac-yqhD).Over-expression of yqhD and dhaB in Klebsiella pneumoniae was achieved with pEtac-dhaB-tac-yqhD.The fermentation result on aerobic conversion showed the increase of 20 % of 1,3-propanediol yield by Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD) was obtained compared with Klebsiella pneumoniae.The main by-products,acetic acid and butanediol decreased estrogen receptors 30% obviously.
Preliminary screening study on 21 fungi strains were carried out to identify those capable of producing lipids using N-acetyl-D-glucosamine (NAG) as carbon source. It was found that 7 strains can assimilate NAG effectively, and 3 of them can accumulate substantial amount of lipids. Under shaking flask culture conditions, cellular lipid content for C. albidus ATCC 56298 and T. fermentans CICC 1368 reached 67% and 48%, respectively. Gas chromatography analysis indicated that compositional fatty acids of these microbial lipid samples contained mainly palmic acid, stearic acid and oleic acid, closely resembling those of vegetable oil. This work further extends feedstock options for microbial lipid fermentation and provides new ideas for exploration of chitin and related renewable materials.
【Objective】Prepare monoclonal antibody(MAb) using SP2/0 cell isolated from tumor in live mouse, and compare the efficiency of cell fusion with conventional method.【Methods】The SP2/0 cell were hypodermic injected in the back of the SPF level BALB/c mouse which were 8 week old. The SP2/0 cells were re-prepared by isolation from the tumor when it reached 2~3cm diameter. The fusion efficiency of SP2/0 cell from live tumor and from cell culture was compared by fusing with spleen cells. The relative affinity of MAbs made by two methods were also compared. 【Results】We have made six fusions. The fusion rate is 70.4% using SP2/0 cell from live tumor, while that is 44.6% using SP2/0 cell from cell culture. The relative affinity of MAbs made by two method were all above 1:100000. 【Conclusion】 We can significantly improve the fusion efficiency by fusion spleen cell with SP2/0 cell from live tumor.
Endo-type chitosanase (ChiN) was isolated from the chitosanase fermentation broth by membrane separation techniques and the conditions that oligo-chitosan was prepared by ChiN were also optimized. The number average molecular weight of prepared oligo-chitosan is about 1200. Analysis by the methods of TLC and HPLC, no monosaccharide exists in the enzymatic product. So we confirm that the major enzymatic products are oligo-chitosan. Oligo-chitosan can be produced rapidly and inexpensively by enzyme-catalyzed reaction which has laid a basis for the large-scare preparation of oligo-chitosan.
The new intein-mediated PHB purify protein system is a high expression, automatic cutting, for purification, low-cost protein purification system,it is conducive to large-scale protein purification. This research choose human antibacterial peptide LL-37 as the purification objects,which is poison to prokaryotic cell.We construct intein-mediated PHB purified human antimicrobial peptide LL-37 system through genetic engineering technology and use this system to purify LL-37. The results show that this system can highly express LL-37 fusion protein and purifiy the product as same size with expectations
To develop a rapid and convenient site-directed mutagenesis method based on homologous recombination and Designed Restriction Endonuclease Assisted Mutagenesis (DREAM). Methods: Two inverse PCR primers containing the desired mutation were designed with complimentary 5' sequences for homologous recombination and a restriction site introduced by DREAM design for rapid mutant screening. Full-length plasmid DNA was amplified with the above primers with a high-fidelity thermostable DNA polymerase capable of long-rang amplification. The amplified full-length plasmid PCR products were transformed into competent E. coli and they were able to circularize due to the terminal homologous sequences. The mutants were screen by convenient restriction digestion. Results: With this strategy we successfully performed the site-directed mutagenesis on a plasmid over 8 kb in length. Conclusion: This site-directed mutagenesis method contains only one step, and no mutation kits or any special regents are needed. Introduction of a restriction site enables convenient and reliable mutant screening. The use of a high-fidelity thermostable DNA polymerase guarantees that most of the mutants are free of unwanted mutations and its capacity of long-rang amplification makes this method applicable to most plasmids.
To obtain recombinant soluble HP0527-C protein of Helicobacter pylori ( H. pylori ) NCTC 11637 , with biological activity . Methods : The hp0527-c coding region was amplified from the Helicobacter pylori NCTC11637 by PCR then was subcloned into expression vector pET-28a for sequence analysis.Recombination plasmid was used to transformed E. coli BL21 cells and fusion protein HP0527-C expression was induced by IPTG. The products was purified by Ni-NTA chromatography column andid entified by SDS-PAGE and Western blot. The protein, was co-cultured with BGC-823 cells, the expression of IL-8 mRNA in cells could be determined by reverse transcriptase polymerase chain reaction(RT-PCR). Rime time PCR was also applied to determine the mRNA expression level of IL-8.The proliferation inhibition activity of HP0527-C was detected by MTT mothod. Results:The target protein expressed in E.coli BL21(DE3) have the same Mr as that of expected and could be recognized by Western blot. The expressed product reached a purity of 98% after Ni2＋-NTA column chromatography. The protein after dialyzed and annealed ,was co-cultured with BGC-823cells, the expression of IL-8 mRNA in BGC-823 cells could be determined. A real-time PCR analysis on IL-8 at different time showed that, the expression level of IL-8 peaked in 24h.The protien of different concentration co-cultured with BGC-823 cells for different times, found that the protein in low concentration promote the proliferation of cells, to achieve 100ug/ml concentration, it inhibits proliferation of cells along with multiplication of the concentration of the protein; Conclusion: A 1494 base pairs long hp0527-c gene, which encodes a product of 497 amino acid, was obtained using PCR method and was subcloned into expression vector pET-28a for sequence analysis..It is indicated that we have obtained the correct hp0527-c gene and expressed in E.coli . BL21(DE3) We obtained high purified protein by Ni2＋-NTA column chromatography, Recombinantthe HP0527-C protein with biological activity was obtained .This proepective research laid solid foundation for further research on its biological activity and function.
Chemokine receptor 5 (CCR5 ),as a member of the G protein-coupled receptor （GPCR）superfamily，is a membrane protein on cell surface and one of the major of coreceptors for HIV-1infection.CCR5 has became a molecular target for the novel drugs against HIV-1,and Antagonists for CCR5 could be grouped as following, chemokine derivatives,small molecule non-peptide compounds,molecular antibodies and peptides. These compounds with high anti-viral activity and affinity are at different stages, and some have been under clinical studies.This review is about the development research in the different kind of CCR5 antagonists.
The technology of somatic nuclear transfer have broadly and attractively applicable prospect, but applying it in various fields was limited by extremely low efficiency. Many researchers made lot of efforts in various kinds of animals, in order to improve the efficiency of somatic cell nuclear transfer. In this article, we briefly summarized the experience of first successfully cloned animal derived from somatic cell nuclear transfer; moreover, presented some ideas about how to improve the efficiency of somatic cell nuclear transfer.
Fed-batch culture is the predominant mode of the current animal cell culture process for many recombinant protein production. Fed-batch culture operation is mainly based on the nutrient continuous consumption and demand of cells to design continuous or semi-continuous concentration feed medium to maintain or support high-density cell growth and improve volumetric productivity of target protein in the reactor. The main methods to improve production efficiency of fed-batch cells culture include the optimization of medium design, selection and optimization of feed strategy and regulation of cell metabolism.
Gene targeted mutagenesis and replacement can be used to modify gene sequence in genomic background without position effect or insertion inactivation in transgenic plants. Targeted mutagenesis organism has little biosafety concerns free of transgenes or marker genes. Gene targeted mutagenesis and replacement in high plants now appears to be a potential tool for gene functional analysis in situ, crops genetic improvement and molecular design. Zinc finger nuclease （ZFN）is most important and would be widely used in gene targeted mutagenesis and replacement through introducing double-strand breaks in genome. In this paper, strategies for gene targeted mutagenesis and replacement in plants is discussed. ZFN is described in detail from its structure, operation model and application in plants. Developmental prospect of ZFN in plant gene targeted mutagenesis and replacement is also discussed.
Plant insect-resistant genetic engineering has opened a new way to prevent and cure against the insect pest in crop and wood productions. With the progresses in studies, many insect-resistant genes were cloned and characterized. This review describes the classification of most insect-resistant genes from plant, the functional mechanism and the applications of the plant insect-resistant genes, the problems and prospects for using the plant insect-resistant genes in genetic engineering.
