Candidate Multi-Peptide-Vaccine Against Classical Swine Fever Virus Induces Strong Antibody Response with Predefined Specificity

IntroductionClassical swine fever virus(CSFV) is a pestiviruswhich causes significantmortality and morbidity inpigs.An epidemic of CSFV in a high pig densityarea can result in devastating financial losses,because few infected pigs can be effectively cured.In many countries in Europe and Asia,classicalswine fever (CSF) is controlled by vaccinationwith commercially available vaccine strains,suchas the Chinese vaccine strain(C-strain,i.e.,hogcholera lapinized virus) orsome modified-live vir…     

HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

0 IntroductionInbroicguhltat einxpge cDtaNtiAonvafcocri nper eivse ant ninegw aanpdp cruoraicnhg wdiits-heases . Due to the comparative weakness of cellulari mmunity that is purely induced by E7 DNA vac-cine,research on DNA vaccine is now focused onhowto enhance the i mmunogenicity of DNA vac-cine.It has been found in some research that thestrength of cellular i mmunity activity has things todo with the speed of antigen molecules’degrada-tion,i .e.the faster the degradation,the higher th…     

Aboveground biomass and corresponding carbon sequestration ability of four major forest types in south China

We estimated aboveground biomass carbon (TABC) and net carbon accumulation rates (TNEP) for trees in four major forest types based on national forest inventory data collected in 1994-1998 and 1999-2003. The four types were Pinus massoniana forest, Cunninghamia lanceolata forest, hard broad-leaved evergreen forest and soft broad-leaved evergreen forest. We analyzed variations in TABC and TNEP for five stand ages (initiation, young, medium, mature and old). In both time periods, estimated TABC in all four forest types increased consistently with forest stand age and the oldest stage had the largest TABC compared with other stages. Broad-leaved forests (hard and soft) had higher TABC than needle-leaved forests (Pinus massoniana and Cunninghamia lanceolata) for each of the five age stages. The difference of TABC between broad-leaved and needle-leaved forests increased with forest stand age. Comparison of estimated TNEP by age category indicated TNEP increased from the initiation stage to the young stage, and then decreased from the mature stage to old stage in all four forest types. TNEP for any particular stage depended on the forest type; for instance, broad-leaved forests at both the mature and old stages had greater TNEP than in needle-leaved forests. A logistic curve was applied to fit the relationship between TABC and forest stand age. In each period, correlations in all four forest types were all statistically significant (P < 0.01) with R2 > 0.95. TABC was therefore predicted by these regression functions from 2000 to 2050 and the mean TNEP during the predicted period was estimated to be about 41.14, 31.53, 75.50 and 75.68 gCm-2a-1 in Pinus massoniana forest, Cunninghamia lanceolata forest, hard broad-leaved forest and soft broad-leaved forest, respectively. Results from both forest inventory and regression prediction suggest broad-leaved forests are greater carbon sinks, and hence have greater carbon sequestration ability especially in the mature and old stages when compared to needle-leaved forests. Broad-leaved forests should have high levels of carbon sequestration when compared with needle-leaved forests in south China.     

Expression and characterization of HGV E2 cDNA inPichia pastoris

A 559 base pair fragment of cDNA locating at the putative E2 region of GBV-C/HGV was inserted intoPichia pastoris expression vector pPIC9K in the reading frame of α-factor secreting signal peptide. The recombinant expression plasmid pPIC9K-E2 was introduced intoP. pastoris GS 115 with electroporation and recombined with the host genome by homological recombination. The His⁺Mut⁺ recombinant yeasts were selected and cultivated in the BMMY medium. After 3 days induction with 0.5% methanol, the target protein (E2) accumulated up to 30% of total proteins in the supernatant. The expressed E2 protein was proved possessing antigenicity and high specificity with Western blot and ELISA probed with sera from the immunized rabbits and the patients infected by GBV-C/HGV. Biography: Wang Zhuo-hua (1977-), female, Master, research direction: genetic engineering pharmaceuticals.     

An HTLV-III peptide produced by recombinant DNA is immunoreactive with sera from patients with AIDS

Human T-cell lymphotropic retrovirus type III (HTLV-III), also called lymphadenopathy-associated virus (LAV), has been identified as the aetiological agent of acquired immune deficiency syndrome (AIDS). The sera of most patients with AIDS or AIDS-related complexes, and of asymptomatic individuals infected with HTLV-III, contain antibodies against antigens of HTLV-III. The characterization of these antibodies and their corresponding viral antigens is important not only for understanding immunity against HTLV-III and the pathology of AIDS, but also for the development of diagnostic methods and preventive vaccine for AIDS. Following the successful establishment of a long-term T-cell line permissive for HTLV-III replication, large quantities of virus have been produced, facilitating the purification of viral proteins and the development of mouse monoclonal antibodies against several viral antigens. More recently, the structure of HTLV-III proviral DNA has been elucidated. We now report the production, by genetic engineering methods, of a peptide encoded by a gene segment of HTLV-III. A 1.1-kilobase (kb) EcoRI DNA segment from an isolate of HTLV-III was inserted into a lpp and lac promoter-coupled expression vector, pIN-III-ompA. Escherichia coli transformants of this plasmid produced a peptide of relative molecular mass (Mr) 15,000 (15K) which was strongly immunoreactive with anti-HTLV-III antibodies present in sera from AIDS patients. Lysates of the clones expressing this 15K peptide inhibited the reactivity of the p31 virion protein with AIDS sera, suggesting that it is a fragment of the viral p31 protein. The peptide reacted with sera from all 20 AIDS patients but none of the 8 normal controls tested. These results suggest that the peptide may be useful for detecting anti-HTLV-III antibodies in blood samples.     

Synthetic and biological studies on a cyclopolypeptide of plant origin

Objective： A natural cyclic peptide previously isolated from Citrus medica was synthesized by coupling of tetrapeptide units Boc-Leu-Pro-Trp-Leu-OMe and Boc-Ile-Ala-Ala-Gly-OMe after proper deprotection at carboxyl and amino terminals followed by cyclization of linear octapeptide segment. Methods： Solution phase technique was adopted for the synthesis of cyclooctapeptide-sarcodactylamide. Required tetrapeptide units were prepared by coupling of Boc-protected dipeptides viz. Boc-Leu-Pro-OH and Boc-Ile-Ala-OH with respective dipeptide methyl esters Trp-Leu-OMe and Ala-Gly-OMe. Cyclization of linear octapeptide unit was done by p-nitrophenyl ester method. The structure of synthesized cyclopolypeptide was elucidated by FTIR, ^1H NMR, ^13C NMR, FABMS spectral data and elemental analysis. The newly synthesized peptide was evaluated for dif- ferent pharmacological activities including antimicrobial, anthelmintic and cytotoxic activities. Results： Synthesis of sarcodactylamide was accomplished with 〉78% yield utilizing dicyclohexylcarbodiimide （DCC） as coupling agent. Newly synthesized peptide possessed potent cytotoxic activity against Dalton＇s lymphoma ascites （DLA） and Ehrlich＇s ascites carcinoma （EAC） cell lines, in addition to moderate anthelmintic activity against earthworms Megascoplex konkanensis, Pontoscotex corethruses and Eudrilus sp. Moreover, cyclopolypeptide displayed good antimicrobial activity against pathogenic fungi Candida albicans and Gram-negative bacteria Pseudomonas aeruginosa, in comparison to standard drugs griseofulvin and ciprofloxacin. Conclusion： Solution phase technique employing DCC and triethylamine （TEA） as base proved to be effective for the synthesis of natural cyclooctapeptide. N-methyl morpholine （NMM） was found to be a better base for the cyclization of linear octapeptide unit in comparison to TEA and pyridine.     

Three Candidate Epitope—Vaccines in Combination Inducing High Levels of Multiantibodies Against HIV—1

HIV-1 mutation results in immune evasion, which presents a serious challenge for conventional strategies for developing effective vaccines. So far, much experimental evidence indicates that HIV-1 particles in the blood of patients can be cleaned principally by neutralizing antibodies. Based on these facts, we prepared triple combination of epitope-vaccines with the objective of inducing antibodies with predefined multi-epitope-specificity against HW-1. According to the sequences of three neutralizing epitopes (RILAVERYLKD, ELDKWA and GPGRAFY, designated El, E2, and E3, respectively) on HIV-1 envelope proteins, three epitope-peptides ((E1)2: C-(RILAVERYLKDG)2; (E2)4: C-(ELDKWAG)4; and (E3)2:C-(GPGRAFY)2) were synthesized and then conjugated with carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and used for immunizing rabbits. After the vaccine course, the triple combination of epitope-vaccines induced high levels of predefined multi-epitope-specific antibodies. An immunoblotting-analysis demonstrated that the antibodies could recognize the native epitopes on both gp41 protein and V3 loop peptide. Furthermore, we compared the immune responses of three doses of epitope-peptides in the candidate epitope-vaccine. Strong antibody responses to three epitopes were observed in a dose dependent manner, with increasing dose raising the immune response. This result indicated that immunotolerance did not occur using an epitope vaccine dose of 80 ~tg. Thus, our results demonstrate that epitope-vaccines in combination can synchronously induce high levels of antibodies with predefined multi-epitope-specificity against HIV-1, and may be used to develop effective vaccines against HIV as a new strategy.     

Characterization of the Antigenicity and Immunogenicity of the Escherichia Coli Produced RNase Domain of the Classical Swine Fever Virus Glycoprotein E

E^rns is a highly glycosylated envelope protein of classical swine fever virus (CSFV) with RNase activity. E^rns can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, the RNase domain of the E^rns was produced in Escherichia coli. Its reactivity with CSFV-positive sera and its ability to induce antibodies and to provide protective immunity were then investigated. The serological tests showed that the prokaryotically expressed RNase domain of the E^rns retained its antigenicity and induced high titers of humoral responses. However, only partial protection and a limited amount of neutralizing antibodies were demonstrated by an in vitro neutralization test and an immunization/challenge test. The results suggest that other essential factors rather than simply enhancing the immunogenicity of E^rns should be taken into consideration when E^rns is enrolled as one of the components of a candidate vaccine.     

Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein

Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens: the peptide sequence of amino acids 141-160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freund's adjuvant. The immune response to these peptides is much lower than that to complete virus particles and the same sequence fused to the N terminus of beta-galactosidase did not produce a more potent immunogen than synthetic peptide alone. We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density. The immunogenicity of these structures approaches that of FMDV particles.     

Variable and conserved neutralization antigens of human immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1, HTLV-III/LAV), the retrovirus responsible for acquired immune deficiency syndrome (AIDS), shows a high degree of genetic polymorphism, particularly in the env gene. We have examined sera from rabbits and guinea pigs immunized with gp130, a recombinant env glycoprotein, and sera from HIV-1-infected subjects, to test their capacity to neutralize a panel of genetically divergent HIV-1 isolates. The sera raised against recombinant antigen specifically neutralized the virus strain from which the env gene was cloned (HTLV-IIIB), but not an independent isolate (HTLV-IIIRF). One rabbit serum tested on seven isolates cross-neutralized two at lower titres. In contrast, human sera from Britain and Uganda, chosen for ability to neutralize HTLV-IIIRF, cross-neutralized six other HIV-1 isolates. When serum and isolate were derived from the same subject, the serum was in some cases effective at slightly lower concentrations (higher titres). Human complement did not affect neutralization titres. These findings indicate that genetically diverse HIV-1 isolates carry both variable and widely conserved antigenic epitopes for neutralizing antibodies. The identification of shared epitopes may help the development of protective vaccines.     

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.     

Isolation by phage display and characterization of a single-chain antibody specific for O6-methyldeoxyguanosine

New approaches of making single chain Fv antibodies against O⁶-methyl-2′-deoxyguanosine (O⁶MdG) have been demonstrated by using the phage antibody display system. Using O⁶MdG as an antigen, 21 positive clones were identified by ELISA from this library, one of which, designated H3, specifically binds to O⁶MdG with high affinity. The H3 scFv antibody has an affinity constant (K_aff) of 5.94×10O¹¹(mol/L)^-1. H3 scFv has been successfully used to detect O⁶MdG in DNA hydrolyses from yeast or E. coli cells treated with a DNA methylating agent. To our knowledge, this is the first report of the selection of a specific scFv against DNA adducts. The results demonstrate the potential applications of the phage display technology for the detection of DNA lesions caused by mutagens and carcinogens.     

Extraction of methyl red from industrial wastewater using xylene as an extractant

A laboratory study on the liquid–liquid extraction system has been carried out for the removal and recovery of methyl red from aqueous solutions using xylene as an extractant. The concentration of methyl red has been studied in the range of 1.72 × 10⁻⁴ to 1.72 × 10⁻³ mol L⁻¹. The efficiency of dye extraction increased with increasing time of extraction. Distribution ratio is reasonably high (D = 49) even in the presence of inorganic salts. Extracted dye in the organic phase has been stripped into sodium hydroxide solution. The organic solvent is reused for reextraction of the dye from aqueous solution. The efficiency of reused organic solvent was maintained up to 10 runs. Loading capacity of dye has been found to be 14.32 mg. Under optimized conditions, real textile wastewater has been studied and the results are satisfactory.     

CFD simulation of fluidization quality in the three-dimensional fluidized bed

Multiphase computational fluid dynamics （CFD） has become an alternative method to experimental investigation for predicting the fluid dynamics in gas-solid fluidized beds. The model of Brandani and Zhang, which contains additional terms in both the gas- and solidphase momentum equations, is employed to explore homogeneous fluidization of Geldart type A particles and bubbling fluidization of Geldart type B particles in three-dimensional gas-fluidized beds. In this model, only a correlation for drag force is necessary to close the governing equations. Two kinds of solids, i.e., fine alumina powder （dp= 60 μm and pp = 1500 kg/m^3） and sand （dp = 610 μm and pp = 2500 kg/m^3）, are numerically simulated in a rectangular duct of 0.2 m （long） × 0.2 m （wide）× 0.5 m （high） size. The results show good agreement with the classic theory of Geldart.     

Specific immune responses induced by a multi-epitope antigen of hepatitis C virus in mice and rabbits

Five highly conserved and immunogenic epitopes of hepatitis C virus (HCV) have been chosen to form a multi-epitope antigen gene and fused with β-galactosidase gene to express a hybrid GZ-PCX antigen, which could be specifically recognized by human HCV sera. High level of anti-GZ-PCX IgG has been induced when mice or rabbits were immunized with GZ-PCX antigen emulsificated with complete Freund’s adjuvant or mixed with killed attenuatedSalmonella typhimurium SL3261. The specific anti-GZ-PCX IgG reached a high titer of 10^-6, which remained for several months. Specific cytotoxic T lymphocyte (CTL) effects, delayed type hypersensitivity reaction (DTH) and proliferation of peripheral lymphocytes have been induced by GZ-PCX antigen or synthetic peptides. High level of anti-GZ-PCX slgG has been detected in mice’s intestinal washing fluids, which indicates that the antigen induced mucosal immunity as well as systematic immunity. The studies show that the HCV multi-epitope antigen induces high level of specific immune responses without obvious toxicity, which might be able to provide protectivity to any HCV genotypes and isolates.     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

巨尾桉株行配置造林试验研究

巨尾桉是最速生高产的桉树，在相同的条件下造林，单位面积株数相等（１６６５株／ｈｍ２），采取四种株行配置造林方式，生长差异非常明显，株行距１ｍ×６ｍ的，定植后３年５个月，平均树高１３．７２ｍ，平均胸径１０．１４ｃｍ，蓄积量６８．７６ｍ３／ｈｍ２，长势名列前茅。     

Spike protein homology between the SARS-associated virus and murine hepatitis virus implies existence of a putative receptor-binding region

Coronavirus has been determined to be the cause of the recent outbreak of severe acute respiratory syndrome (SARS). Human coronavirus 229E had been studied well and its receptor-binding domain was restricted to aa417-547 of S protein. However, this region has no homology with the newly separated SARS-associated virus (Hong Kong isolate CUHK-W1). Then we analyzed the phyiogenesis of S1 subunit of the coronavirus spike protein (SARS-associatedvirus, Hong Kong isolate CUHK-W1). Interestingly, thehighest homology between murine hepatitis virus (MHV)and SARS-associated coronavirus was found. And the important sites (aa62-65 and aa214-216) on the spike proteinof MHV with receptor-binding capacity were highly conservative in comparison with the newly separated SARS-associated virus (the corresponding sites are aa51-54 and aa195-197). These results from bioinformatics analysis mighthelp us to study the receptor-binding sites of SARS-associated virus and the mechanism of the virus entry into the target cell, and design antiviral drugs and potent vaccines.     

棒状杆菌诱发质粒简报

用溴化乙锭诱发质粒，溴化乙锭不同浓度对棒状杆菌基因组起不同作用，低浓度无作用；高浓度消除质粒，适中浓度能诱发质粒，本试验诱发出４种大小不同质粒，转种５代质粒消失。     

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.