Optimization by orthogonal array design and humoral immunity of the bivalent vaccine against Aeromonas hydrophila and Vibrio fluvialis infection in crucian carp (Carassius auratus L.)

Aeromonas hydrophila and Vibrio fluvialis are the causative agents of a serious haemorrhagic septicaemia that affects a wide range of freshwater fish in China. In order to develop a bivalent anti-A. hydrophila and anti-V. fluvialis formalin-killed vaccine to prevent this disease, an orthogonal array design (OAD) method was used to optimize the production conditions, using three factors, each having three levels. The effects of these factors and levels on the relative per cent survival for crucian carp were quantitatively evaluated by analysis of variance. The final optimized formulation was established. The data showed that inactivation temperature had a significant effect on the potency of vaccine, but formalin concentration did not. The bivalent vaccine could elicit a strong humoral response in crucian carp (Carassius auratus L.) against both A. hydrophila and V. fluvialis simultaneously, which peaked at 3 or 5 weeks respectively. Antibody titres remained high until week 12, the end of the experiment, after a single intraperitoneal injection. The verification experiment confirmed that an optimized preparation could provide protection for fish at least against A. hydrophila infection, and did perform better than the non-optimized vaccine judged by the antibody levels and protection rate, suggesting that OAD is of value in the development of improved vaccine formulations.

STUDY ON MICROWAVE CYCLOTRON RESONANCE OF HIGH-MOBILITY GaAs/Al-0.35 Ga-0.65 As TWO-DIMENSIONAL ELECTRON GAS

The properties of electron states in the presence of microwave irradiation play a key role in understanding the oscillations of longitudinal resistance and the zero-resistance states in a high-mobility two-dimensional electron gas(2DEG) in low magnetic field. The properties of electron states in a high-mobility and low-density GaAs/Al0.35Ga0.65As 2DEG in the presence of Ka-band microwave irradiation were studied by reflectance-based optically detected cyclotron resonance(RODCR). The influences of the direction of microwave alternating electronic field, wavelength of the laser, and temperature on RODCR results were discussed. The results show that RODCR measurements provide a convenient and powerful method for studying electron states in 2DEG.

双δ掺杂In_(0.65)Ga_(0.35)As/In_(0.52)Al_(0.48)As赝型高迁移率晶体管材料子带电子特性研究

研究了基于InP基的In_(0.65)Ga_(0.35)As/In_(0.52)Al_(0.48)As赝型高迁移率晶体管材料中纵向磁电阻的Shubniko-de Haas (SdH)振荡效应和霍耳效应,通过对纵向磁电阻SdH振荡的快速傅里叶变换分析,获得了各子带电子的浓度,并因此求得了各子带能级相对于费米能级的位置.联立求解Schrodinger方程和Poisson方程,自洽计算了样品的导带形状、载流子浓度分布以及各子带能级和费米能级位置.理论计算和实验结果很好符合.实验和理论计算均表明,势垒层的掺杂电子几乎全部转移到了量子阱中,转移率在95%以上.

MOVPE生长Al（0.35）Ga（0.65）As/GaAs量子阱结构的光致发光

于2010-11-23批量导入

Synchronism of Holocene East Asian monsoon variations and North Atlantic drift-ice tracers (vol 65；pg 443；2006)

IEECAS SKLLQG

埋藏对65种半干旱草地植物种子萌发特性的影响

比较了半干旱草原科尔沁沙地65种植物新采种子和埋藏1年种子的萌发特性.结果表明:埋藏1年后,有38种植物的种子仍具有活力.新采种子萌发率>80%的植物种子埋藏后萌发率下降;而萌发率<80%的植物种子埋藏后萌发率均有较大幅度的提高.埋藏对种子萌发特性的影响因植物生态类群(杂草、草原植物、草甸植物、沙生植物)而异.种子埋藏1年后,多年生植物萌发率没有明显提高,而1年生植物萌发率显著提高(P<0.05).对于新采种子和埋藏1年种子萌发率间相差10%以下的物种,可根据萌发格局将植物种分为3类:埋藏促进型(如差巴嘎蒿)、埋藏抑制型(如大籽蒿)和埋藏不变型(如鹤虱).

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

Aims: To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins. Methods and Results: A gene (vhhP2) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24 V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non-V. harveyi species, including V. parahaemolyticus and V. alginolyticus. A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2. This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii, which is most closely related to V. harveyi. One of the V. campbellii strains was falsely identified as V. harveyi. Conclusions: vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non-V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi. However, this method can not distinguish some V. campbellii strains from V. harveyi. Significance and Impact of the Study: the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.

Characterization of degQ(Vh), a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50 C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E.coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

Characterization of degQ(Vh), a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50 C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E.coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

The expression of peptidoglycan recognition protein-S1 gene in the scallop Chlamys farreri was enhanced after a second challenge by Listonella anguillarum

A more rapid and powerful response against repeated exposure of same pathogen in vertebrates is usually considered as the reflection of immunological memory, but it is not well understood in invertebrates. In the present Study, the temporal expression profiles of Chlamys farreri peptidoglycan recognition protein-S1 (CfPGRP-S1) gene after two challenges of Listonella anguillarum were examined to evaluate priming response in scallops. The up-regulation of CFPGRP-S1 mRNA occurred 3 h earlier, and the expression level was significant higher (P < 0.05), after the second challenge than that after the first challenge. The preliminary results Provided new insights into invertebrate immunological memory, and they also would be helpful to develop strategies for disease control. (C) 2008 Elsevier Inc. All rights reserved.

The polymorphism of lysozyme gene in Zhikong scallop (Chlamys farreri) and its association with susceptibility/resistance to Listonella anguillarum

Lysozyme functions as a crucial biodefence effector against the infection of bacterial pathogens in innate immunity. The nucleotide sequence polymorphisms in promoter region of a nuclear goose type lysozyme gene from Zhikong scallop Chlamys farreri (designated as CFLysG) were investigated to explore their association with susceptibility/resistance to Listonella anguillarum infection. Eight sites of single nucleotide polymorphisms (SNPs) and two sites of insert-deletion (ins-del) polymorphisms were identified in the promoter region of CFLysG. Two of them, -753 TATCTCGATCAGG ins-del polymorphism and -391 A-G SNP were selected to analyze their distribution in the susceptible and resistant stocks, which were identified according to the survival time after L. anguillarum challenge. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), two genotypes were found at each site, which were ins/del and ins/ins at locus -753, and A/A and A/G at locus -391, respectively. The -753 ins/del genotype was more prevalent in the resistant stock than that in the susceptible stock, 30% vs 16.67% in frequency, but there was no significant difference in the frequency distribution between these two stocks (P=0.15). In contrast, the frequency of -391A/G genotype in the resistant stock was significantly higher (30%) than that in the susceptible stock (7.14%) (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 A/A genotype (96.8%) was significantly higher than those with -391 A/G genotype (64.5%) (P=0.001), which further validate the association between -391 A/G genotype and the resistance of Zhikong scallop to L anguillarum. These results suggested that the -391 A/G could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to L anguillarum. (C) 2008 Elsevier Ltd. All rights reserved.

Immunoprotective analysis of VhhP2, a Vibrio harveyi vaccine candidate

VhhP2 is an Outer membrane protein identified in a pathogenic Vibrio harveyi strain, T4, isolated from diseased fish. When used as a Subunit Vaccine, purified recombinant VhhP2 affords high level of protection upon Japanese flounder against V harveyi challenge. Vaccination with VhhP2 induced the expression of a number of immune-related genes, especially those encoding immunoglobulin M (IgM) and major histocompatibility complex (MHC) II alpha. A VhhP2 surface display system, in the form of the fish commensal strain FIR harboring the vhhP2-expressing plasmid pJVP, was constructed. PF3/pJVP is able to produce and present recombinant VhhP2 on cell surface. Vaccination of fish with live PF3/pJVP via intraperitoneal injection elicited Strong immunoprotection. Vaccination of fish orally with live PF3/pJVP embedded in alginate microspheres also induced effective immunoprotection. In addition, a VhhP2-based surface display system was created, in which VhhP2 serves as a carrier for the Surface delivery of a heterologous Edwardsiella tarda immunogen, Et18, that is fused in-frame to VhhP2. DH5 alpha/pJVP18, which expresses and surface-displays the VhhP2-Et18 chimera, proved to be an effective vaccine that call protect fish against infections by V. harveyi and E. tarda to the extents comparable to those produced by vaccination with purified recombinant VhhP2 and Et18, respectively. These data suggest that VhhP2 may be applied as a vaccine and a vaccine carrier against infections by V. harveyi and other pathogens such as F. tarda. (C) 2009 Elsevier Ltd. All rights reserved.