Secretory expression and purification of the recombinant duck interleukin-2 in Pichia pastoris

Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular functions and immunity of animals. In this study, the recombinant duck IL-2 (rduIL-2) was secretory expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks and then scaled up in a 5.0-l bioreactor. The result showed that the maximal fresh-cell-weight of 594.1 g/l and the maximal OD600 of 408 were achieved in the bioreactor. The rduIL-2 was purified by two steps of purification procedures, and approximately 311 mg of rduIL-2/L fermentation supernatant was obtained. SDS-PAGE showed that the purified rduIL-2 constituted a homogeneous band of ~16 kDa or ~14 kDa corresponding to the glycosylated or non-glycosylated duIL-2 protein in size, respectively. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay. The result indicated that the rduIL-2 greatly promoted the proliferation of ConA-stimulated lymphocytes in vitro. The P. pastoris expression system described here could provide promising, inexpensive, and large-scale production of the rduIL-2, which lays the foundation for development of novel immunoadjuvants to enhance both the immunity of ducks against various infectious pathogens and vaccine efficacy.     

Cloning,expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>, and purification of soluble recombinant duck interleukin-2

Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular and immunity of animals. In this study, a gene encoding duck IL-2 was cloned and the soluble recombinant duck IL-2 (rDuIL-2) was expressed in Escherichia coli via fusion with glutathione S-transferase (GST). The results indicated that the GST-rDuIL-2 fusion protein expressed in E. coli Origami (DE3) was confirmed to be of about 40 kDa molecular mass by SDS-PAGE and western blotting. In order to produce soluble rDuIL-2 in a low-cost, nontoxic and high-level expression process, lactose was used as a substitute for Isopropyl-β-D-thiogalactopyranoside (IPTG) to induce the above recombinant strain Origami (pGEX-DuIL-2). By optimizing the expression conditions, the production of soluble GST-rDuIL-2 fusion protein was about 29% of total cellular soluble proteins, which was similar with IPTG used as inducer. The soluble GST-rDuIL-2 fusion protein was purified by one-step affinity chromatography, and GST was removed by thrombin. Then rDuIL-2 was purified by a second affinity chromatography. As a result, the 95% pure rduIL-2 was obtained, and the yield of rDuIL-2 was about 10.6 mg/l bacterial culture. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay in vitro. Our study provided a feasible and convenient approach to produce soluble and biologically active rDuIL-2, which would be used as an immunoadjuvants for enhancing vaccine efficacy.     

Injection of duck recombinant follistatin fusion protein into duck muscle tissues stimulates satellite cell proliferation and muscle fiber hypertrophy

Follistatin (FST) can inhibit the expression of myostatin, which is a predominant inhibitor of muscle development. The potential application of myostatin-based technology has been prompted in different ways in agriculture. We previously constructed an expression vector of duck FST and isolated the FST fusion protein. After the protein was purified and refolded, it was added to the medium of duck myoblasts cultured in vitro. The results show that the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide value of the myoblasts in the duck FST treatment group is higher than that in the control group, which indicates that the duck FST fusion protein exhibits the biological activities that can accelerate myoblast proliferation. To further investigate the roles of duck FST on muscle development, we injected the protein into the duck muscle tissues in vivo. The results show that both the duck muscle fiber cross-sectional area and the satellite cell activation frequency are influenced more in the FST treatment group than they are in the control group. In addition to these phenomena, expression of MyoD and Myf5 were increased, and the expression of myostatin was decreased. Together, these results suggest the potential for using duck FST fusion protein to inhibit myostatin activity and subsequently to enhance muscle growth in vivo. The mechanism by which FST regulates muscle development in the duck is similar to that in mammals and fishes.     

Histopathological analysis of metastatic melanoma deposits in patients receiving adoptive immunotherapy with tumor-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TIL) from a wide range of human and murine tumors can be expanded in vitro using interleukin-2 (IL-2). These TIL are cytolytic T lymphocytes with in vivo and in vitro antitumor activity in mice and in humans. TIL from human melanoma can recognize autologous tumor in an MHC-restricted fashion, localize in vivo after¹¹¹In labeling, and mediate regression of large metastatic deposits. Although studied extensively in vitro, less is known in vivo about TIL activity associated with tumor regression. This study was undertaken, in association with a study of TIL localization, to investigate mechanisms of TIL action by evaluating histopathological changes that occur at the tumor site during TIL administration. A total of 106 pre- and post-treatment pathological specimens from 25 patients enrolled in phase II TIL treatment and¹¹¹In-TIL imaging protocols were examined blindly by a single pathologist. Histological subtype, lymphocytic infiltration, melanin content, vascularity, and necrosis were documented for each tumor specimen. Average baseline and post-treatment parameters were compared. Any significant changes were evaluated for correlation with clinical response and¹¹¹In-TIL localization to tumor. Melanin content and vascularity of the tumor did not change as a result of therapy or correlate with either response or TIL localization. However, both increased lymphocytic infiltration and tumor necrosis were present after TIL administration (P=0.044 and 0.032 respectively). Furthermore, increases in lymphocytic infiltration correlated with tumor imaging using¹¹¹In-TIL, and with the percentage of¹¹¹In-labeled injectate present per gram of tumor specimen (P=0.036 and 0.0041 respectively). This suggests that TIL either account for the increased lymphocytes directly, or localize to tumor and recruit endogenous lymphocytes. We were unable to demonstrate any pretreatment histopathological predictors of response or variables that significantly correlated with subsequent clinical response, although peak and average values of necrosis were higher in responding patients compared to non-responding patients.     

A comparison of in vivo catalysis by creatine kinase in avian skeletal muscles with different fibre composition

The maximum activity of creatine kinase in vitro is similar in the pectoralis major muscle of the chicken and the duck. However, the flux (phosphocreatine to ATP) as measured by 31P saturation transfer NMR in vivo is almost 2-fold higher in the duck. This apparent discrepancy can be accounted for by the differences in the cytosolic free ADP concentrations in resting muscle.     

Localization of HSP90 binding sites in the human hepatitis B virus polymerase

The fact that HSP90 proteins and their chaperonin partners play an important role in epsilon RNA binding of duck HBV Pol protein during duck HBV replication has been reported. To elucidate the molecular basis of HBV Pol/HSP90 interaction, we have characterized the HSP90 interaction to HBV Pol. We found that human HBV Pol protein upon synthesis in rabbit reticulocyte lysate formed a complex with HSP90 in vitro as duck HBV Pol did. In addition, HSP90 protein was copurified with MBP/POL protein expressed in HepG2 cells, suggesting that human HBV Pol protein is associated with HSP90 in vivo. To localize the HSP90 interaction site region, several deletion mutants of HBV Pol translated in vitro were immunoprecipitated with anti-HSP90 antibody. The result indicates that C-terminal regions of the TP and RT domains interact with HSP90 independently.     

Peptide variants of viral CTL epitopes mediate positive selection and emigration of Ag-specific thymocytes in vivo

During development, thymocytes carrying TCRs mediating low-affinity interactions with MHC-bound self-peptides are positively selected for export into the mature peripheral T lymphocyte pool. Thus, exogenous administration of certain altered peptide ligands (APL) with reduced TCR affinity relative to cognate Ags may provide a tool to elicit maturation of desired TCR specificities. To test this "thymic vaccination" concept, we designed APL of the viral CTL epitopes gp33-41 and vesicular stomatitis virus nucleoprotein octapeptide N52-59 relevant for the lymphocytic choriomeningitis virus-specific P14- and vesicular stomatitis virus-specific N15-TCRs, respectively, and examined their effects on thymocytes in vivo using irradiation chimeras. Injection of APL into irradiated congenic (Ly-5.1) mice, reconstituted with T cell progenitors from the bone marrow of P14 RAG2(-/-) (Ly-5.2) or N15 RAG2(-/-) (Ly-5.2) transgenic mice, resulted in positive selection of T cells expressing the relevant specificity. Moreover, the variants led to export of virus-specific T cells to lymph nodes, but without inducing T cell proliferation. These findings show that the mature T cell repertoire can be altered by in vivo peptide administration through manipulation of thymic selection.     

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.     

Administration of human recombinant IL-7 to normal and irradiated mice increases the numbers of lymphocytes and some immature cells of the myeloid lineage.

In vitro experiments performed by several investigators have demonstrated that IL-7 is a growth factor for immature B lymphocytes, thymocytes, and mature T lymphocytes. To evaluate the potential therapeutic use for human rIL-7 (rhuIL-7) as a hematopoietin, we have studied the in vivo hematopoietic effects of rhuIL-7 in mice. In these experiments, sublethally irradiated and normal mice were treated with or without rhuIL-7 for up to 26 days. Administration of rhuIL-7 significantly increased the white blood cell count in the peripheral blood and spleen in both normal and irradiated mice. Treatment with rhuIL-7 also accelerated lymphocytic recovery in irradiated mice. Precursor and mature B lymphocytes showed the greatest expansion in response to rhuIL-7 administration, with smaller increases in T lymphocytes being observed. In mice recovering from high dose irradiation, rhuIL-7 treatment resulted in preferential expansion of CD8+ T lymphocytes and more rapid normalization of the CD4/CD8 ratios. Differential analysis of peripheral blood smears demonstrated that rhuIL-7 also increased the numbers of immature granulocytes in both normal and irradiated mice. Moreover, administration of rhuIL-7 to normal, irradiated, cyclophosphamide-pretreated, or 5-fluorouracil-pretreated mice increased the number of acetylcholinesterase-positive megakaryocytes in the spleen, but not the bone marrow. Therefore, although the major in vivo effects of rhuIL-7 were on cells of the lymphocytic lineage, rhuIL-7 also increased the numbers of some immature cells of the myeloid lineage.     

不同种雏鸭建立鸭乙肝病毒感染模型及抗病毒效果的实验

目的探讨不同种雏鸭建立鸭乙肝病毒感染模型的影响因素,观察应用该模型抗病毒的效果。方法采集鸭血清,应用PCR方法定性检测鸭血清中病毒DNA;定量PCR方法检测鸭血清中病毒DNA载量变化;用抗病毒药物处理,观察其在鸭DHBV感染模型中的抗病毒效果。结果不同种鸭DHBV自然感染率不同,樱桃谷鸭为8.75%,湖北麻鸭两个批次分别为17.80%和10.68%;静脉注射和腹腔注射两途径均能致雏鸭感染DHBV,静脉注射感染率80%,腹腔注射感染率65%;鸭感染DHBV后,体内病毒载量维持在106～108copies/mL,可持续20 d以上;抗病毒药物处理后,在不同DHBV模型中其抗病毒效果变化趋势一致。结论鸭的种类和人工感染途径可影响DHBV感染率;雏鸭感染DHBV后其体内有持续性的病毒血症;DHBV感染模型是药物抗病毒研究较好模型。     

Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta2 crystallin

Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.     

In vivo inhibition of duck hepatitis B virus replication and gene expression by phosphorothioate modified antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotide strategies have been employed in a variety of eukaryotic systems both to understand normal gene function and to block gene expression. Pharmacologically, 'code blockers' are ideal agents for antitumour and antimicrobial treatments because of their specific mode of action. Here we report the inhibition of duck hepatitis B virus (DHBV) by antisense oligodeoxynucleotides in primary duck hepatocyte cultures in vitro as well as in DHBV-infected Pekin ducks in vivo. The most effective antisense oligodeoxynucleotide was directed against the 5' region of the pre-S gene and resulted in a complete inhibition of viral replication and gene expression in vitro and in vivo. These results demonstrate the application of antisense oligodeoxynucleotides in vivo and exemplify their potential as human antiviral therapeutics.     

The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern.

A set of wild-type and mutant human, woodchuck, and duck hepatitis viral core proteins have been prepared and used to study the free thiol groups and the disulfide bonding pattern present within the core particle. Human (HBcAg) and woodchuck (WHcAg) core proteins contain 4 cysteine residues, whereas duck (DHcAg) core protein contains a single cysteine residue. Each of the cysteines of HBcAg has been eliminated, either singly or in combinations, by a two-step mutagenesis procedure. All of the proteins were shown to have very similar physical and immunochemical properties. All assemble into essentially identical core particle structures. Therefore disulfide bonds are not essential for core particle formation. No intra-chain disulfide bonds occur. Cys107 is a free thiol buried within the particle structure, whereas Cys48 is present partly as a free sulfhydryl which is exposed at the surface of the particle. Cys61 is always and Cys48 is partly involved in interchain disulfide bonds with the identical residues of another monomer, whereas Cys183 is always involved in a disulfide bond with the Cys183 of a different monomer. WHcAg has the same pattern of bonding, whereas DHcAg lacks any disulfide bonds, and the single free sulfhydryl, Cys153 which is equivalent to Cys107 of HBcAg, is buried.     

In vivo regulation of monomer-tetramer conversion of pyruvate kinase subtype M2 by glucose is mediated via fructose 1,6-bisphosphate

The activity of pyruvate kinase, subtype M2 (PKM2), is known to be increased by fructose 1,6-bisphosphate (Fru-1,6-P2), one of the metabolites in the glycolytic pathway. Recently, we have shown that in vitro, Fru-1,6-P2 activated the association of monomer to form the tetrameric PKM2. To ascertain whether this mode of regulation also occurs in vivo, we prepared monomer-specific monoclonal antibody and quantified the monomer formation in situ in cultured cells by immunocytochemistry. The intracellular Fru-1,6-P2 was manipulated by the glucose concentration in the media. At the physiological concentration of glucose (4-6 mM), 30-35% of PK existed as a monomer. However, PKM2 was dissociated into monomer within minutes after cells were deprived of glucose. The maximal level of monomer was detected after 1 h at 37 degrees C. Monomer was rapidly (within minutes) converted to tetramer after addition of glucose. Furthermore, when cells cultured in 10 mM of glucose were treated with cytochalasin B, an inhibitor of the glucose transporter, a maximal level of monomer was detected within 20-30 min. Determination of Fru-1,6-P2 indicated that its intracellular concentration decreased concomitantly with the reduction in glucose concentration in the medium. These results indicate that monomer-tetramer inter-conversion is a major in vivo cellular regulatory mechanism in response to changes in the extracellular glucose concentration via Fru-1,6-P2.     

Rapid conversion of effector mechanisms from NK to T cells during virus-induced lysis of allogeneic implants in vivo

Viral infections can strongly stimulate both NK cell and allospecific CD8 T cell responses, and these same effector cells can lyse allogeneic cell lines in vitro. However, the impact of viral infections on the effector systems mediating rejection of allogeneic tissues in vivo has not been fully explored. Using in vivo cytotoxicity assays, we evaluated the effector systems mediating the rejection of CFSE-labeled allogeneic splenocytes after an infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus. Naive B6 mice predominantly used a NK cell-effector mechanism to reject allogeneic splenocytes because they rejected BALB/C (H2(d)) splenocytes but not CBA (H2(k)) splenocytes, and the rejection was prevented by immunodepletion of NK1.1(+) or Ly49D(+) NK cells. This rapid and efficient in vivo cytotoxicity assay recapitulated the specificity of NK cell-mediated rejection seen in longer duration in vivo assays. However, as early as 1 day after infection with lymphocytic choriomeningitis virus, a CD8 T cell-dependent mechanism participated in the rejection process and a broader range of tissue haplotypes (e.g., H2(k)) was susceptible. The CD8 T cell-mediated in vivo rejection process was vigorous at a time postinfection (day 3) when NK cell effector functions are peaking, indicating that the effector systems used in vivo differed from those observed with in vitro assays measuring the killing of allogeneic cells. This rapid generation of allospecific CTL activity during a viral infection preceded the peak of viral epitope-specific T cell responses, as detected by in vivo or in vitro cytotoxicity assays.     

Structural and functional similarities of delta-crystallin messenger ribonucleic acids from duck and chicken lenses

delta-Crystallin of the embryonic duck lens was compared with that of the embryonic chicken lens with respect to polypeptide composition, synthesis, and messenger ribonucleic acid (mRNA) sequences. Labeling experiments with [35S]methionine revealed that the duck delta-crystallin is composed of minor amounts of polypeptides with molecular weights near 50000 (50K) and 49000 (49K) and much greater amounts of polypeptides with molecular weights near 48000 (48K) and 47000 (47K), as judged by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. All four sizes of polypeptides were synthesized in similar relative proportions as found in vivo in a rabbit reticulocytes lysate supplemented with delta-crystallin mRNA isolated from the embryonic duck lens. Synthesis of the 48K and 47K delta-crystallin polypeptides was differentially reduced in duck lenses cultured in the presence of ouabain. This is similar to the differential reduction of synthesis of the lower molecular weight delta-crystallin peptides in embryonic chicken lenses demonstrated previously. R loops formed between duck or chicken delta-crystallin mRNA and a cloned chicken delta-crystallin cDNA and heteroduplexes formed between duck or chicken delta-crystallin mRNA and cloned chicken genomic DNAs containing delta-crystallin sequences showed that, except for the putative 5' leader sequence, the duck and chicken delta-crystallin mRNAs have extremely similar nucleotide sequences. These data indicate considerable conservation of delta-crystallin throughout the approximately 100 million years of divergence between ducks and chickens. The findings also suggest a possible relationship between the structure of delta-crystallin mRNA and the differential reduction in synthesis of the lower molecular weight delta-crystallin polypeptides in ouabain-treated lenses of ducks and chickens.     

免耕稻-鸭生态种养技术的环境经济学分析

采用田间试验及环境经济学方法研究免耕稻-鸭生态种养技术的生态、经济效益。试验结果表明:农户采用免耕稻-鸭生态种养技术对稻田杂草的控制效果显著;在晚稻分蘖盛期和孕穗期,采用免耕稻-鸭生态种养技术对稻二化螟防效达100%,稻纵卷叶螟发生率分别比免耕抛秧不养鸭的稻田低48.05%、93.55%,免耕抛秧养鸭稻田中水稻纹枯病的病株率比免耕抛秧不养鸭的稻田分别低48.15%、38.21%;在稻田甲烷排放高峰期(晚稻分蘖始期-分蘖盛期),免耕抛秧养鸭对甲烷排放的控制效果明显,分别比翻耕抛秧不养鸭稻田、免耕抛秧不养鸭稻田的甲烷排放量减少4.723g/m2、2.333g/m2,晚稻整个生育期间,免耕抛秧养鸭稻田甲烷排放量比免耕抛秧不养鸭稻田减少3.37g/m2,比翻耕抛秧不养鸭稻田减少5.59g/m2;免耕可节约灌溉用水1300m3/hm2。环境经济学分析结果表明:采用免耕稻-鸭生态种养技术的农户比采用免耕抛秧不养鸭技术或采用翻耕抛秧不养鸭技术的农户分别增加财务净收益2166yuan/hm2、4207yuan/hm2;免耕抛秧养鸭获得的经济净效益为4062yuan/hm2,而免耕抛秧不养鸭、翻耕抛秧不养鸭的经济净效益分别为1592yuan/hm2、-997yuan/hm2。免耕稻-鸭生态种养技术既能充分发挥稻-鸭复合生态系统的生态和经济效益,又能较好地克服免耕给生态环境带来的不利影响,是一种很有发展潜力的可持续农业生产模式,具有良好的推广和发展前景。     

Nonhyperbolic kinetics of Na,K-ATPase--a new viewpoint]

The kinetics of the 130 kDa monomer obtained by treatment of duck salt gland Na,K-ATPase with C12E8 was compared with that of the membrane-bound enzyme. The shapes of the substrate-velocity curves for the membrane-bound and solubilized forms were quite different: a hyperbolic one for the monomeric Na,K-ATPase and a nonhyperbolic one for the native enzyme. A reaction scheme for ATP hydrolysis based on a comparative analysis of kinetic properties of these two forms is proposed. Experimental evidence in favour of this hypothesis is presented.