Objective: To construct a lentiviral vector expressing Semaphorin 4D to investigate the role of Semaphorin 4D in angiogenesis. Methods: Semaphorin 4D sequence was amplified from the Jurkat cell cDNA, inserted into pWPI GW vector and identified by DNA sequencing. The packing cell line (human embryonic kidney 293T cell) was cotransfected with pWPI GW/Sema4D, pVSVG and pSPAX. The recombinant lentivirus was packaged and amplified, followed by infection of 293T and HUVEC cells. The expression level of Semaphorin 4D gene was analyzed by Western blot and immunofluresence staining. A Boyden chamber migration assay was performed to establish the biological significance of Semaphorin 4D. Results: The recombinant lentiviral vector expressing Semaphorin 4D was successfully constructed. Western blot and immunofluresence staining results demonstrated that Semaphorin 4D was successfully expressed in 293T and HUVEC cells infected by recombinant lentivirus expressing Semaphorin 4D. Boyden chamber migration assay results showed that enhanced endothelial cell migration is induced by Sema4D-expressing Hela cells.
Overexpression of Growth hormone-releasing hormone (GHRH) can control the synthesis and secretion of the growth hormone (GH) from the hypophysis as well as regulate the GH concentrations in animals and humans. The lentiviral vector system were modified to be a CMV-SP two promoter vector. GHRH cDNAs were cloned into the vector and transfected 293T cells with other three plasmids to package LV-SP-GHRH pseudovirion. Muscle tissue of mice were injected with the virus particles and the animal weight growth were observed. At the same time viral safety was assessed by detection of whether the virus structural genes integrated into the genome of infected cells. The results showed that the body weight gain per day increased 153.85% compared to the control group at 28th day postinjection, and the genome of infected cells without virus structural gene integration. All the results indicated that lentiviral vector mediated GHRH gene can promote animal growth significantly and safely.
It was aimed to construct mpt64-recombinant BCG vaccine to find an improved vaccine to replace BCG and to prevent TB effectively. The gene of MPT64 was amplified by PCR and recombined with shuttle plasmids pYUB295. The recombinant shuttle plasmid was identified by PCR, enzyme digestion and DNA sequencing and then transformed into BCG by electroporation. The antigen-specific antibody levels of mouse immunized with recombinant BCG were evaluated by ELISA and multiplication of mouse lymph-cell was detected by MTS. The protection against M. tuberculosis of recombinant BCG vaccines was tested by their prevention and treatment experiments. PCR, enzyme digestion, DNA sequencing and SDS-PAGE results showed: Thempt64-recombinant BCG was constructed successfully and can express extrinsic MPT64 gene. The immunity experiment showed:extrinsic gene can be expressed in BCG and can stimulate B cell to produce antibody,the antibody level reached the peak after 45 days; The lymph-cells of each group proliferated when stimulated by different antigen, all stimulation index reached 2. The stimulation index of each group had no obviously difference. The prevention experiment of recombinant BCG against M. tuberculosis was indicated: mpt64-recombinant BCG and BCG can extend mean time to death and reduce death rate in 2 month of those mouse, the protection effect of mpt64-recombinant BCG had no difference with BCG. It could be concluded that the mpt64-ecombinant BCG was constructed successfully. The protection effect of mpt64-recombinant BCG had no difference with BCG.
Objective: To enhance immune responses to protein vaccine, IL-17 was investigated as a molecular adjuvant to enhance humoral immune responses, especially cellular immune responses to protein vaccine. Methods: C57BL/6 mice were immunized with OVA alone, or with IL-17A by intramuscular injection. The immunization was performed on week 0, 2. The concentration of the anti-OVA IgG, the stimulated index of T lymphocyte proliferation, and the expressions of IFN-γ, IL-4 and IL-17 in CD+4 T cells and IL-17, IFN-γ in CD+8 T cells, and specific in vivo cytotoxic T lymphocyte (CTL) activity were detected on week 3,4. Results: The results showed that IL-17 as a molecular adjuvant for the protein vaccine, OVA, could enhance immune responses, especially the cellular immune responses. IL-17 could not only enhance the humoral responses and T cell proliferation, but also the expressions of IL-17 in CD+4 and CD+8 T cells, and IFN-γ in CD+8 T cells. Accordingly, the level of CTL in vivo was significantly increased. Conclusion: The results demonstrated that IL-17 as a molecular adjuvant enhanced humoral and cellular immune responses to protein vaccine, especially the CD+8 T cell-immunity. The novel functionality of IL-17 on adaptive immunity may lead to develop new protein vaccine targeted to the cellular immune responses.
Two specific oligonucleotide primers were designed and synthesized according to the sequence of Sus scrofa skeletal α-actin gene, which were used to amplify the α-actin 5'regulating sequence. pcDNA3.1-AF, the muscle-specific expression vector, was constructed by connecting with α-actin 5'regulating sequence, Caenorhabditis elegans ω-3 fatty acid desaturase cDNA and CMV promoter-less pcDNA3.1,and its expression specificity were proved by directly injecting the vector into the quadriceps muscle of mice(4 weeks old). The PCR amplification result showed that a size of 1 815 base pairs fragment was amplifies and the sequencing resulted showed that the fragment had a high identity with original sequence and contained muscle specific elements. The transient expression results indicated that the muscle-specific expression vector could efficiently direct the transcription of ω-3 fatty acid desaturase in the muscle. The study provides the basis for establishing transgenic animal which could specific express ω-3 fatty acid desaturase in the muscle.
Biomaterial surface properties modulate protein adsorption and cell adhesion, proliferation, spreading to elicit diverse cellular responses in biomedical and biotechnological applications. Alkanethiol self-assembled monolayers (SAMs) presenting well-defined chemistries (NH2, COOH, OH and CH3) were employed to analyze the effects of surface chemistry on controlling fibronectin (FN) conformation as well as osteoblasts behaviors. X-ray photoelectron spectroscopy (XPS) is used to confirm the presence of well-formed monolayers of expected physicochemical characteristics, and the hydrophilicity of SAMs was characterized by water contact angle. Immunostaining were applied to indicate conformation of FN. In addition, the effects of SAMs with pre-coated FN on the cytoskeleton organization and proliferation were also examined. Water contact angle data reveals the-CH3groups lead to a hydrophobic surface, while COOH, OH groups lead to a hydrophilic surface and NH2groups a neutral surface. SAMs presenting terminal NH2, COOH, OH and CH3 functionalities modulated adsorbed FN conformation as determined through differences in the binding affinities of monoclonal antibodies raised against the central cell-binding domain,-OH surfaces exhibited a statistically greater HFN 7.1 positive area than the other surfaces. Then, the changes in osteoblasts proliferation and spreading correlated with the FN conformation.
Apoptin is coded by the VP3 gene from chicken anemia virus (CAV), and the protein can induce apoptosis of a large variety of tumor cells. The preparation of polyclonal antibodies specific to VP3 protein was explored. The VP3 gene was obtained from the eukaryotic expression vector pcDNA3.0-VP3. And then, VP3 gene was cloned into the prokaryotic expression vector pET8a. The prokaryotic expression vector pET8a-VP3 was employed to express VP3 protein in E.coli BL21 which was induced by IPTG, and the protein was extracted from the cell and purified. The purified VP3 protein was applied to immunize the New Zealand rabbits combined with the Freunds complete adjuvant or incomplete adjuvant by multi-points subcutaneous injection. ELISA test was performed to measure the titer of the antibody after immunization. Two days after the measurement the whole blood was harvested by cardiac puncture and the serum was separated from the whole blood. The immunglobin IgG was purified by Protein A method from the antiserum. The final purified antibody titers were up to 1 ∶ 243 000. The immunological evaluation of the polyclonal antibody specific binding was assayed by using recombinant adeno-associated virus rAAV-VP3 infected different cell lines. First, it was used to detect the VP3 gene expression by immunofluorescence in human bladder cancer cells T24 and EJ, also in Vero cells. Apoptin was observed in T24, EJ cells, mainly located in the nucleus, whereas in Vero cells were localized in the cytoplasm. Secondly, the specific binding of the purified antibodies to VP3 protein in different human bladder cancer cells were detected by Western blotting. The results have shown that the polyclonal antibody have displayed good effectiveness and binding specificity to apoptin, which will provide a foundation for further clarify the molecular mechanisms of anti-tumor effect and biological characteristics of apoptin.
To facilitate the research on Arabidopsis suecica (A.suecica), a method was presented for de novo assembly of A.suecica transcriptome using short reads produced by Illumina sequencing platform. 23 million sequencing reads were assembled into 125 953 unique sequences with the N50 length of 550 bp and mean size of 331 bp. At the protein level, a total of 96 057 (76.3%) A.suecica transcripts showed significant similarity with transcripts proteins from the other plants in the Nr database. Functional categorization revealed the conservation of genes involved in various biological processes in A.suecica. In addition, simple sequence repeats(SSRs) motifs in the A.suecica transcriptome was identified. The data provides a comprehensive sequence resource available for A.suecica study and demonstrates that the short pair-end reads sequencing allows de novo transcriptome assembly in a allotetraploid species lacking genome information. It is anticipated that the next generation sequencing(NGS) technologies significantly accelerate the research of the transcriptome in both model and non-model organisms. In addition, the strategy for de novo assembly of transcriptome data presented here will be helpful in other similar transcriptome studies.
Objective: To express and purify the peanut major allergen Ara h 2 that obtained by rational sequence reassembly and preliminarily characterize the hypoallergenic of purified recombinant S-Ara h 2 protein. Methods: The Ara h 2 sequence was reassembled and inserted into the expression vector pET-32a(+). The vector was transformed into Origami and the protein expression was induced by IPTG. Ni2+chelating affinity chromatography was used to purify the recombinant S-Ara h 2 protein. The hypoallergenic of S-Ara h 2 was examined by Western blotting and ELISA. Results: The ORF which contained 471 bp and encoded 157 amino acids was authenticated to be S-Ara h 2. The recombinant S-Ara h 2 protein,induced by IPTG,which is consistent with the actual value. The affinity between recombinant S-Ara h 2 protein and IgE antibodies from pooled peanut-allergic patients serum was significant decrease compared with R-Ara h 2 was identified by Western blotting and ELISA. Conclusion: Recombinant S-Ara h 2 protein was obtained with hypoallergenic. It will provide a novel safe vaccine to cure peanut allergy patients by Allergen-specific immunotherapy.
Endophytic fungi, lived in stems and leaves of host plants, were supposed to be effective or novel sources for therapeutic compounds. Camptotheca acuminata Decne is a traditional medical plant in China. About 50 endophytic fungi were isolated from barks of C. acuminata. A new fungus producing camptothecin (CPT) was identified as a Penicillium based on its morphological features. Ten endophytic fungi of C. acuminata were chosen to be amplified using molecular maker sequence related amplified polymorphism (SRAP). The combinations of SRAP primers turned out to be polymorphic and a total of 1 295 polymorphic bands were obtained. These strains were grouped into 3 main clusters and revealed that SRAP could isolate the strains of C. acuminata efficiently. It also provided evidence for discussing the genetic diversities of species in C. acuminata with SRAP molecular method.
The physiological mechanisms in response to Verticillium wilt were analyzed by comparing the physiological indicators of Solanum torvum and Suqi eggplant infected with Verticillium. dahliae Kleb. The results showed that: (1) S. torvum exhibits the strong capability of egodefense and self-rehabilitation, compared with the cultivar Suqi. (2) The active oxygen scavenging system (such as: SOD, POD, CAT enzymes activity) in S. torvum was higher than in Suqi during the treatment. MDA content in S. torvum decreased after treatment, but increased in the Suqi. The results suggested that infection might activate active oxygen scavenging system in vivo, then producing certain defensive substances (such as lignin and chlorogenic acid et al.) quickly, while reducing the accumulation of MDA in the body. (3) The response time of the physiological indicators in S. torvum in response to V. dahliae was different: Activity of POD, PAL and content of soluble protein in S. torvum change rapidly within 12h, and then, change in the activity of SOD, PPO, CAT and MDA content. Eventually, S. torvum resist Verticillium. Wilt infection effectively by combinations of each enzyme.
CRISPR is a novel type of microbial defense system, which is unique in that it is invader-specific, adaptive and heritable. It is a recent breakthrough in understanding host-virus interactions. Bioinformatics methods including BLAST, multiple sequence alignment, and RNA structure prediction was used to analyze the CRISPR structures of 24 Bacillus cereus group genomes. CRISPR existed in 42% strains. Two types of RNA secondary structures derived from the repeat sequences were predicted, and demonstrated that stem-loop secondary structure might function in mediating the interaction between foreign genetic elements and CAS-encoded proteins. The sequence homologous among 31% spacer, phage, plasmid and the genomes of Bacillus cereus group further verified that spacer was likely to come from the exogenous mobile genetic factor. As most of the Bacillus cereus group strains contain multiple plasmids and prophages, the CRISPR research in Bacillus cereus group by this study would be help to reveal relationship between host strains with plasmid or host strains with phage.
Monascus can produce several kinds of beneficial secondary metabolites. But Monascus can also produce citrinin that is harmful to mammals because of nephrotoxic and hepatoxic activity. Thus it is essential to control the production of this toxin in order to increase the safety of Monascus-related product. Extensive research on citrinin pathway and genes involving in this toxin biosynthesis is conducted. Six genes responsible for citrinin biosynthesis are clusted within 21kb DNA region. A gene(orf7) which locates in the vicinity of orf5 was cloned. The orf7 gene disruption mutants in M. aurantiacus As3.4384 were constructed. The citrinin yield of the orf7 gene disruption mutants was analyzed by HPLC and the pigments were analyzed by UV spectrophotometer. From 13th to 19th day of cultivation, the yield of citrinin of orf7 disruptant increased 142.4% compared to wild-type As3.4384. So it was confirmed that orf7 was related to metabolism of citrinin.
Recombinant E.coli cells were immobilized with calcium alginate for the isomerization of D-galactose to D-tagatose. The influences of immobilization process and reaction conditions on the catalytic efficiency and D-tagatose production stability of the immobilized cells are investigated. The optimal conditions were as follows: the temperature, pH and mental ion were 65℃,6.5 and 1mmol/L Mn2+, respectively. The optimal substrate (D-galactose) and cell concentrations of 100 g/L and 40 g/L were selected, respectively. Stability of the alginate beads under high temperatures was enhanced after cross-linked by 0.3% glutaraldehyde for 30min. The effect of molar ratio between borate and substrate on isomerization was studied, it was found that borate could change the initial chemical equilibrium of D-galactose isomerization and lead to a high production of D-tagatose. After conversion for 24h, the highest conversion rate for D-tagatose using D-galactose as the substrate reached to 65.8% by immobilized recombinant E.coli cells, and the average conversion rate of eight repeated batch conversions was 60.6%, which laid the foundation for industrial production of D-tagatose.
Some environmental stresses, i.e., high osmotic stress, fluctuating temperature and ethanol concentration, will greatly influence the viability and capability of the yeast S.cerevisiae strains during ethanol fermentation. In this study, we constructed a series of yeast strains with improved stress tolerances and ethanol fermentation performance through trehalose metabolism engineering combined with hybridization-based whole genome recombination. Firstly, two haploid strains , Z1 and Z2 isolated from the diploid strain Zd4, were engineered to enhance the intracellular trehalose by (1) overexpression of trehalose-6-phosphate synthase gene TPS1 (Z1ptps1 and Z2ptps1), (2) deletion of acidic trehalase gene ATH1 (Z1Δath1 and Z2Δath1), and (3) TPS1 overexpression combined with ATH1 deletion (Z1pTΔA and Z2pTΔA). We then obtained four recombination strains (Z12, Z12ptps1, Z12Δath1 and Z12 pTΔA) through the hybridization of Z1 and Z2, and their engineered strains. The results of high-gravity fermentation (270 g/L glucose) showed that TPS1 overexpression combined with ATH1 deletion had a distinct advantage in the improvement of stress tolerance over the single genetic manipulation. Compared to the original strain Zd4 and Z12, the strain Z12pTΔA(the hybrid of Z1pTΔA and Z2pTΔA) improved the fermentation rate by 11.4% and ethanol yield by 7.0%, while the strain Z12 without metabolic engineering only increased the main fermentation rate by 4.8% and ethanol yield by 2.8%. These improvements of fermentation performance consisted with their tolerances of the constructed strains under the conditions with osmotic pressure, high temperature and high concentration of ethanol. The combination of trehalose metabolic engineering and genome recombinant technology could effectively improve the stress tolerance and the ethanol fermentation performance of the industrial S.cerevisiae strains is demonstrated, and an innovative strategy for industrial yeast breeding is proposed.
Objective: To construct a rabbit source high-capacity and good-diversity ribosome display single chain antibody (scFv) library of FMDV. Methods: The isolated spleen cells from rabbit immuned by the FMDV Virus,and extracted RNA for amplifying rabbit VH gene, VL gene and Ck gene as the spacer by RT-PCR,and then connect VH-VL gene by overlapping extension PCR (referred to as SOE-PCR). After that,the elements for ribosome display such as T7 promoter and ribosome binding site were introduced after SOE products were amplified. Finally, the ribosome display scFv library template was constructed in vitro. Moreover, the ribosome display templates were connected to pMD18-T vector into E.coli DH5α, and the positive clones were picked sequencing to identify the ScFv assembly. Results: We successfully constructed a rabbit source ribosome display scFv library whose capacity is 8.21 × 1013 of FMDV. Conclusion: The rabbit-derived high-capacity ribosome display antibody library can be a good platform for screening specific foot and mouth disease, and lay a good experimental basis for the measuring test of FMDV.
Objective: To establish a colloidal gold immunochromatography assay for rapid quantitative detection of specific antibody to Venezuelan Equine Encephalitis Virus(VEEV) using the expressed E2 recombination protein. Method: The expressed E2 recombination protein was expressed in the form of inclusion body which used Isopropyl-β-D-thiogalactopyranoside (IPTG) as revulsant. The expressed inclusion body was denatured, renatured and dialyzed. With colloidal gold labeling Staphylococcal protein A and immune-chromatography technics, The colloidal gold immunochromatography assay method(GICA) was prepared and applied to detect fast the VEEV serum. The VEEV antigen was coated on the test region. This study evaluates the sensitivity, specificity and stability of this method. Results: SDS-PAGE revealed the VEEV expression product had a 40 kDa protein, as expected. And the purity of the optimized inclusion body was above 95%.The detection can be completed within 20min with this method with sensitivity of the established method is the same as R&D the antibody of VEEV ELISA. The other homologous virus and the viruses which had the similar symptom had nonspecific reaction. The test strip could save two weeks in 37℃ with no changed results. Ninety-two blood serum samples were tested by means of gold-immunochromatography and ELISA, the rate of coincidence is 96. 7%, when compared with that of ELISA. Conclusion: The inclusion body of the VEEV can be significantly and stably improved after denaturation and renaturation. So the inclusion body can be used as a candidate for many testing methods. The GICA can detect the antibody of VEEV fast, sensitive, specific and stably in samples, and suitable for field detection.
Objective: To develop a generic method for the determination of protein content in alhydrogel (aluminum) absorbed vaccine formulation using o-phthalaldehyde (OPA). Method: based on the theory that OPA can react with amino acids under alkaline conditions and give strong fluorescence, in the presence of 2-mercaptoethanol, a fluorescent method without antigen extraction was developed and the specificity, linearity range, accuracy and precision were evaluated. The integrity of the vaccine formulation was also investigated by SDS-PAGE. Results: a good linearity was obtained from 0.02mg/ml to 0.16mg/ml of antigen content, the accuracy of 90%~115% and CV% less than 15% were achieved. Little protein disassociation was also observed after CpG was added into the formulation. Conclusion: the OPA fluorescent method was simple and can be further used not only in the laboratory but also in routine quality control of the vaccine formulation with alhydrogel.
Two types of stem cells in muscle: satellite cells and muscle-derived stem cells (MDSCs) have been identified. MDSCs, a predecessor of the satellite cell, are considered to possess a higher regeneration capacity and to exhibit better cell survival and a broader range of multilineage capabilities. MDSCs are not only able to differentiate into mesodermal cell types including the myogenic, adipogenic, osteogenic, chondrogenic, endothelial, and hematopoietic lineages, but also possess the potential to break germ layer commitment and differentiate into ectodermal and endodermal lineages under certain conditions. The progress on isolation, identification, plasticity, and the current clinical applications of MDSCs are reviewed.
Caenorhabditis elegans(C. elegans) has been widely used as a model organism in the research of nervous system, aging mechanisms and programmed cell death, owing to its advantages of simple tissue structure, easily culture, short lifecycle. Different from higher organisms, C. elegans is lack of adaptive immune, only the innate immune plays an important role in anti-bacteria, anti-oxidative stress and so on. Four immune-related signal transduction pathway in C. elegans, including insulin-receptor-like pathway, transforming growth factor β(TGF-β) pathway, mitogen activated protein kinases(MAPK) pathway and programmed cell death(PCD) pathway play major roles in various conditions of environment. Meanwhile, the innate immune system of C. elegans is conservative in many respects, which provides new idea for research of immune mechanism in higher organisms. Accordingly.The progress on innate immune signal transduction pathway in C. elegans is reviewed, expecting to provide some reference for investigating innate immune related to higher organisms including mankinds.
With advantages of easy-to-operate, low-cost, low copies and preferential integration of defined T-DNA into transcriptionally active regions of chromosomes without vector DNAs, Agrobacterium is employed for genetic modification of plants routinely. However, until recently, still there are a great many species recalcitrant to Agrobacterium-mediated transformation. Data suggest that the infection capability is designed by virulence (vir) genes of Ti plasmid outside of A. tumefaciens genome. Among all vir genes, virA and virG express constitutively, while other vir genes need phenolic compounds for induction. Besides, carbohydrates can enhance vir induction by phenolic compounds, while low phosphate and acidic pH environmental conditions may also increase induced expression of vir genes. In order to better utilize Agrobacterium and improve its efficiency for applications in research and biotechnology, molecular mechanisms for vir induction by factors such as phenolic compounds, carbohydrates, low phosphate, acidic pH conditions and incubation temperature are discussed.
Legume lectins, the largest lectin family in plants, were the most investigated ones in terms of their biological properties. Most legume lectins were assembled into dimers and tetramers under normal physiological conditions. The oligomer molecules provide a very high sugar specificity and macromolecular stability to the legume lectins. Besides the function of plant storage, legume lectins are identified by glycoprotein, glycopeptide and carbohydrate of biological membrane, and acted as symbiosis neurogen between plant and surrounding microorganisms.The research progresses of the legume lectins with respect to structure and function, as well as applications in biology, agriculture and medicine are reviewed.
