转Hu-IFN-α基因草鱼雌核发育F1的遗传配型分析

The Hu-IFN-α gene, which was transducted into downstream promoter of β-actin gene of common carp (Cyprinus carpio), was recombined by DNA recombination technology. These recombined genes were injected into 1-2 cell fertilized eggs of grass carp (Ctenophatyngodon idellus) by microinjection technology, we gained transgenetic fish by molecular detection methods. In order to analyse the genetic expression of tranHu-IFN-α gene gynogensis F₁, which male individualization were gained by raising methyltestosterone, molecular genetic marker technology was used. In our research, 30 random primers were picked out from 48 and were used into RAPD-PCR, the result indicated that 1 169 clear, steady and repeated DNA finger printing bands were achieved. On the basis of gentic distance matrix among tranHu-IFN-α gene gynogenesis F₁ group, the genetic relationship of gynogenesis F₁ were analysed by UPGMA, the results showed the genetic patterns are close between the 3# male gynogenesis F₁ and the the 23# female of gynogenesis F₁, 5# and 27#, 2# and 28#, 2# and 30#. The data indicated that these group could be served as parent of tranHu-IFN-α grass carp (Ctenophatyngodon idellus) pure line.     

线纹尖塘鳢的形态生物学与核型

This paper reports the morphological character and karyotype of Oxyeleotris lineolatus. Oxyeleotris lineolatus is native in Australia and call ed sleepy cod. It belongs to Oxyeleotris, Eleotridae, Gobioidei, Perciformes in taxonomy. Recently, it was introduced to China and local people were not familiar with it. So we carried out this study. 30 individuals have been observed and some data were recorded. It has a large mouth that is in front and up. The mandible stands out and is longer than the up jaw. There are many rows of thin teeth in up and down jaws. The pelvie fins are located in chest and pectoral fins are large and fanlike. There are two dorsal fins. The tail fin is circular. Gill rakers are sparse and the number of gill rakers is 8-12+3-4. The gas bladder belongs to physoclistaus and its stomach is strong and I-like. The intestine is thick and short and no pyloric caecas. The length of the digestive path is 48.1％-80.3% of the length of body. Its liver has two lobes and the liver weight is 4.1%-7.2% of the body weight. The digestive organs characters are same as the trait of flesheater fish. Its scale belongs to ctenoid scale and its body surface shows several long lines. There is not lateral line in the body. The number of vertebra is 26-27 and it has 10-11 pairs rib. The number of diploid chromosome is 2n=46 and the karyotype formula is 2sm+8st+36t，NF=48. The relative length of chromosome is from 1.37% to 3.48% and it is continuity. No strange size chromosomes relation to sex was observed. The karyotype of Oxyeleotris lineolatus is similar to that of Oxyeletris marmoratus Bleeker from South East Asia and both of them belong to Oxyeleotri. It testifies the correctness of traditional classification on cytology.     

鱼类诺达病毒及其所导致的疾病

In recent years, piscine nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish resulting in high mortality in aquaculture worldwide. Affected fish exhibit a range of neurological signs, such as erratic swimming behaviour with the associated microscopic lesions of necrosis and vacuolation of the central nervous tissues and retina. Numerous roundshaped, unenveloped and 25-30 nm in diameter virus particles were found in the cytoplasm of affected retinal and nerve cells. Nodaviruses have a bipartite genome of positivesense RNA,with RNA1 encoding the RNAdependent RNA polymerase and RNA2 encoding the capsid protein. Both RNA are capped, but not polyadenylated. The family Nodaviridae comprises two genera: Alphanodavirus and Betanodavirus, members of which primarily infect insects and fish, respectively. Therefore, betanodavirus is also named piscine nodavirus. At present, piscine nodaviruses are divided into four genotypes based on partial sequences of the coat protein gene. ELISA and RT-PCR amplification have been developed as specific diagnostic methods for the d etection of the virus. Antibodies to striped jack (Pseudocaranx dentex) nervous necrosis (SJNNV) were found in 65% of plasma samples collected from wild and domestic brood stocks of striped jack, suggesting that the virus is very prevalent. Viral antigens were detected in eggs, larvae, and ovaries of hatcheryreared and wild spawner fish, suggesting both horizontal and vertical modes of transmission of the virus. Selection of nodavirusfree spawners using ELISA for detection of antigens and RT-PCR techniques have successfully reduced incidences of the virus infections in juvenile sea bass (Dicentrarchus labrax),striped jack and barfin flounder (Verasper moseri). The SSN1 and GF cell lines have been successfully used in isolating piscinenodaviruses.Although there are many papers describing the molecular characteristics of betanodavirus, our knowledge of the genomic attributes of these viruses is still limited. Vaccination studies are being undertaken by a number of researchers and need to be fostered. In particular, the use of passive immunization of broodfish with homologous and heterologous, high titre antisera are worthy of investigation.     

绿色微囊藻等不同食物对大型生长和脂类成分的影响

The growth and reproduction of Daphnia magna fed with Microcystis viridis and Chlorella spp respectively and 20% fish oil + 80% yeast and yeast were studied. The intrinsic rate of natural increase of Daphnia magna (rm) was 0.243 fed with Microcystis viridis, 0.301 with Chlorella spp., 0.244 with 20% fish oil + 80% yeast and 0.193 with yeast. The result showed that：Daphnia magna fed with Microcystis viridis had lower growth rates than that fed with Chlorella spp. an d 20% fish oil + 80% yeast which both had full fatty acids. And the Daphnia magna fed with yeast was the lowest. So the fatty acids composition of diet may affect the growth of Daphnia magna. This paper further examined total lipids and main HUFA (EPA and DHA) compositions of the continuous three generations of Daphnia magna (in order to get rid of t he effects of former diet) fed with different of above diets (except the yeast). The result showed that:the three generations of the D. magna fed with Microcystis viridis had the lowest total lipids and the percentages of the HUFA (EPA, and no DHA) have a significant decrease and get the lowest EPA in the final experiment because of the very low HUFA especially EPA and DHA in Microcystis viridis. So the results indicate that Microcystis viridis with low HUFA made the lower growth rate, spawn ing rate and hatching rate of Daphnia magna.     

Accumulation of waterborne selenium by rainbow trout (Salmo gairdneri), eggs,fry and juveniles

The effect of waterborne selenite levels on selenium accumulated by different developmental stages of rainbow trout (Salmo gairdneri) was studied using⁷⁵SeO ₃ ⁼ as a tracer. All stages readily accumulated selenium at both high and low concentrations, but the rate of accumulation increased as the trout developed from the egg to the juvenile feeding stage. The low rate of selenium accumulation by embryos seemed to be related more to a lack of gills than to the presence of a chorion. The bioconcentration factor (the ratio of tissue-to-water concentrations) declined in all groups with increased waterborne selenium levels; accumulation rates appeared limited by cell permeability. At low levels of waterborne selenium (0.4μg/l), the total accumulated was small relative to existing body burdens and unlikely to contribute significantly to the nutritional requirement for selenium. High levels (45.6μg/l), however, caused considerable selenium accumulation and may be sufficient to overcome the effects of low dietary selenium.     

海湾扇贝个体间单向人工授精的分子生物学验证

Hybrid family of Argopecten irradians irradians was created by fertilization between two northern bay scallop individuals. Two families were analyzed in this study. The first family, P_a-P_b, is a pair mating between two scallops named P_a and P_b, while the second one crossed by individuals of Y₁ and P₀. Marker inheritance and segregation were studied in the 10 progenies of each family by randomly amplified polymorphic DNA (RAPD) analysis. 102 RAPD primers were first screened by parental animals of both families. Only the primers with polymorphisms between the two parental animals in each family were select ed for further analysis. In both families, parents and 10 progeny were nalyzed with selected primers. In family P_a-P_b, a total of 122 bands generated from 12 selected primers. 37 of them were polymorphic between two parents. The maternal P_a of this family had 17 molecular markers while paternal Pb had 20 markers. In Y₁-P₀，95 bands were produced by 10 selected primers. 32 bands were polymorphic between maternal Y₁ and paternal P₀, who had 17 and 15 molecular markers respectively. In both families, each progeny analyzed in this study had at least 8 maternal markers and 5 paternal markers. Based on segregation patterns at all markers analyzed, we concluded that none of the progeny analyzed were from self-fertilization, and oneway hybridization between two individuals was successful in both of the two bay scallop families.     

细角螺的繁殖生态条件及繁殖习性

Hemifusus ternatanus(Gmelin), a gastropoda living in the deep water areas of undertide,as well as a delicious seafood, was distributed mainly in Chinese southeast seas and Japanese seas near the coast. In this experiment Hemifusus ternatanus' breeding ecological conditions and its propagation habit were studied in order to provide an experimental foundation for the artificial breeding. The brood stocks of test animals collected from the waters in Taiwan Strait, and then were examinated their growing temperature， salinity, feeding and propagation habit in the laboratory. The re sults showed that the growing temperature for Hemifusus ternatanus ranges from 16 ℃ to 32 ℃, with an optimum temperature between 20 ℃ to 28 ℃ and between 23 ℃ and 28 ℃ for breeding. The optimum growth salinity was 18.3-32.3 though it much adapts to salinity from 13 to 35. They prefer to feed on bivalves particularly those with thin shells and byssusless. Hemifusus ternatanus is dioecious and fertilization finishes inside the body. It was called as egg vesicle procreation that the whole stages of embryo growth were developed within egg vesicle. Larva forms as soon as it leaves egg vesicle, which is called direct occurrence type. The larva became the juvenile after metamorphosis during 20-25 days development. It was first to obtained 233 juveniles with shell height 32-45 mm by an artificial breeding method.     

我国赤潮频发现象分析与海藻栽培生物修复作用

In this paper, the history, main events and present status of red tide (HAB, harmful algal blooms) along China coast in recent years were reviewed and presented. It showed that the HAB's frequency and scale, number of HAB spec ies, percentage of toxic HAB events and the degree of damages to marine environment and economy have sharply increased in China since 1960's. Eutrophication was key factor for high occurrence of red tide. In this paper, main causes of frequent HAB occurrence along China coast was discussed. Many factors might influence the occurrence of red tide, which included weather, climate, coastal current, tidal current, water temperature, salinity, hydrodynamic and nutrient conditions, trace metals and the variation of biological environment. Numerous evidences from all over the world revealed the linkage between the increases in nutrient loading and the occurrences of high biomass blooms. Eutrophication was one of the important causes that involved in high occurrence of HAB. The main sources of nutrients potentially stimulating HABs included terrestrial runoff, aquaculture selfpollution, atmospheric deposition, sea projects and other pollution events in the ocean. Studies showed that the input from land contaminations and the selfpollution of marine aquaculture accelerated eutrophication in coastal waters and were also important impact factors on red tide. Researches suggested that nutrient composition could affect the species composition of phytoplankton as well as the development of some HABs. The changes in nutrient supply ratios, primarily N∶P, often resulted in shifts in red tide species composition. The correlation between cysts and formation of HAB was discussed from the viewpoi nt of transformation of cyst and vegetative cell, the effects of trace elements and other organic substances on the occurrence of HAB were presented also. It indicated that the nutrient control could be an effective way to reduce the risk of red tide occurrence. Seaweed would play an important role for decreasing marine eutrophication. Among the different methods of red tide controlling studied, seaweed biomass has received much attention due to the cost saving, low sensitivity to environmental and impurity factors, the possible contaminant recovery from the biomaterial and its elevated adsorption capacity. Cultivated seaweeds have very high rates of productivity higher than that of seaweed in its natural habits and grow well in water bodies with higher nitrogen and other nutrients. Seaweeds are able to absorb large quantities of nitrogen, phosphorus and carbon dioxide, produce large quanti ties of oxygen, and have excellent effect on decreasing eutrophication. Large amounts of C, N and P are accumulated into seaweed tissues as they accumulate considerable biomass over a period of months or years depending on the cultivation season. When seaweeds are harvested, nutrients are removed from the sea area. An investigation was carried out for inorganic nitrogen and inorganic phosphorus concentration at Lusi Coast, Qidong County, Jiangsu Province in China, where there were about 270 hm2 for Porphyra yezoensis cultivation with eutrophic sea water in recent years. While during Porphyra yezoensis cultivation, from Sep 2003 to May 2004, the concentration of ammonium nitrogen declined form 0.511-0.778 mg·L^-1 to 0.006-0.057 mg·L^-1, nitrite nitrogen concentration declined from 0.010-0.040 mg·L^-1 to 0.001-0.009 mg·L^-1, and nitrate nitrogen concentration declined from 0.466-0.549 mg·L^-1 to 0.286-0.0568 mg·L^-1, the average concentration of inorganic phosphorus declined from 0.024 mg·L^-1 to 0.019 mg·L^-1. Furthermore, during five hours, the concentration of ammonium nitrogen in the seawater declined form 220.88 μmol·L^-1 to 8.59 μmol·L^-1 by cultivated Gracilaria lemanaiformis, and the concentration of ammonium nitrogen declined form 213.84 μmol·L^-1 to zero by cultivated Enteromorpha clathrata. Other bioremediation mechanisms of seaweed inhibiting the red tide microalgae such as nutrients competition and allelopathic effects were also discussed.     

湛江沿海五种石斑鱼线粒体DNA的RFLP分析

In this paper, genetic diversity of intraspecies, and genetic relationship of interspecies in Epinephelus spp. (E. merra, E. fario, E. awoara, E. akaara and E. septemfasciatus) were assessed by using mitochondrial DNA restriction fragment length polymorphisms (mtDNA RFLPs).The samples were collected from the coastal area of Zhanjiang,Guangdong province. MtDNA was extracted from the fresh liver tissue by applying a difference centrifugation procedures. Using 17 restriction enzymes with 5-or 6-bp recognition sites, the purified mtDNA was cleaved by single enzymes. These enzymes included BamH Ⅰ，Bgl Ⅰ，Bgl Ⅱ，Dra Ⅰ，EcoR Ⅰ，EcoR Ⅴ，Hind Ⅲ，Kpn Ⅰ，Mlu Ⅰ，Pst Ⅰ，Pvu Ⅱ，Sal Ⅰ，Sca Ⅰ，Sma Ⅰ，Sty Ⅰ，Xba Ⅰ and Xho Ⅰ. The phylogenetic analysis was done using the Neighbor joining(NJ) method and Unweighted pairgroup method with arithmetic mean(UPGMA) method. Genetic diversity indices such as haplotype diversity (h), average genetic distance between haplotypes (P) and nucleotide diversity (π) were calculated using Nei and Li's segment method to quantify the genetic diversity within species. There were 8, 5, 8, 5 and 2 haplotypes detected within E. merra, E. fario, E.awoara, E.akaara and E. septemfasciatus, respectively. The haplotype diversity (h) was 0.8943, 0.6186, 0.9242, 0.6927 and 0.1820,respectively. The average genetic distance between haplotypes (P) was 0.62%±0.31%, 0.64%±0.37%, 1.12%±0.55%,0.72%±0.42% and 0.45%, respectively. And the nucleotide diversity (π) was 0.22%, 0.13%, 0.46%, 0.17% and 0.04%, respectively. The wild groupers in the Zhanjiang Coastal Area exhibited a relative higher level of genetic diversity. The net genetic distance between species (P_net) was 0.0694（E. merra - E. fario）,0.1337（E. merra - E. awoara）,0.1090 E. merra -E. akaara）, 0.1286（E. merra - E. septemfasciatus）,0.1590（E. fario -E. awoara）,0.0825（E. fario -E. akaara）,0.1153（E. fario - E. septemfasciatus）,0.1131 E. awoara - E. akaara）,0.0724（E. awoara -E. septemfasciatus） and 0.1336（E. akaara - E. septemfasciatus）. Both NJ and UPGMA methods yielded an identical phylogenetic tree for the five species. The E. merra and E. fario first clustered together, then joined with E. akaara, and finally clustered with E. awoara and E. septemfasciatus.     

嗜水气单胞菌外膜蛋白基因ompTS的克隆与序列分析

根据已发表的外膜蛋白基因ompⅡ的核苷酸序列设计引物，从分离 自患红底板病的中华鳖的嗜水气单胞菌中扩增得到了ompTS基因，对ompTS 基因进行序列分析，发现其与ompⅡ基因的核苷酸序列有83.5%的同源性。ompTS基因最长的开放阅读框（ORF）为1068nt，编码由355个氨基酸组成，分子量为38.9kDa的蛋白质OmpTS，其氨基酸序列的前20个氨基酸残基可能组成信号肽。由ompTS基因的编码氨基酸序列与其它细菌外膜蛋白的氨基酸序列的比较结果，进一步证实细菌外膜蛋白氨基酸序列的N端存在高度保守区。根据序列分析结果推测，ompTS基因很可能是一个新的基因，编码38.9kDa的嗜水气单胞菌外膜蛋白OmpTS，该蛋白质在膜中极有可能形成孔道，具备与孔蛋白相似的性质。     

嗜水气单胞菌J-1株丝氨酸蛋白酶基因克隆与序列分析

根据已发表的气单胞菌胞外蛋白酶基因核苷酸序列，设计和合成了一对引物，以嗜水气单胞菌AhJ—1的基因组DNA为模板，通过PCR技术，扩增到约900bp的丝氨酸蛋白酶基因片段，并克隆到质粒载体pGEM—T中进行测序和分析，结果表明扩增的丝氨酸蛋白酶基因片段与已发表的嗜水气单胞菌丝氨酸蛋白酶Ahe2的同源性有87％，扩增片段编码343个氨基酸，推测的分子量为35700，计算机软件分析表明编码的氨基酸有较高的抗原性，可作为核酸疫苗的侯选基因片段。     

日本沼虾cytMnSOD和mtMnSOD基因全长cDNA克隆及表达

利用RACE技术从日本沼虾肝胰腺中克隆了cytMnSOD和mtMnSOD基因cDNA全长序列。cytMnSOD基因cDNA全长1 233 bp,开放阅读框为858 bp,编码286个氨基酸,N端含有60个氨基酸残基组成的延伸区;mtMnSOD基因cDNA全长1 113 bp,开放阅读框为654 bp,编码218个氨基酸,N端含有20个氨基酸残基组成的信号肽;cytMnSOD和mtMnSOD预测蛋白分子量及等电点分别为31.33、24.05 ku和5.62、7.12。日本沼虾cytMnSOD推导的氨基酸序列与其mtMnSOD的相似性为40%,二者均含有MnSOD的特征肽段(DVWEHAYY)、4个Mn2+结合位点和2个N-糖基化位点。Real-time PCR结果表明,cytMnSOD和mtMnSOD在日本沼虾肝胰腺、肌肉、血细胞、大颚器官、卵巢和鳃等组织均有表达,其中肝胰腺表达量最高;肝胰腺cytMnSOD和mtMnSOD基因的表达量在蜕皮间期最高,蜕皮后期和蜕皮前期较低。嗜水气单胞菌刺激后3 h,肝胰腺cytMnSOD和mtMnSOD的表达量显著增加,推测MnSOD是参与机体免疫防御反应的一种重要分子。     

实时荧光定量PCR扩增特异性vapA基因检测杀鲑气单胞菌

为实现杀鲑气单胞菌早期快速准确定量检测,研究旨在建立杀鲑气单胞菌的SYBR Green Ⅰ实时荧光定量PCR(Real-time PCR)检测方法。根据GenBank中杀鲑气单胞菌毒力阵列蛋白基因(vapA)保守序列设计并合成一对特异性引物,对其特异性、灵敏度、可重复性和应用性进行评价。结果显示,研究设计的引物具有良好的种间特异性,仅对杀鲑气单胞菌及其亚种有阳性扩增,与其他细菌不发生交叉反应。构建的Real-time PCR标准曲线质粒拷贝数与循环阈值呈良好的线性关系,扩增所得标准曲线分别为y=–4.8345x+42.535,相关系数R~2为0.998,最低检测限为34拷贝/μL,较常规PCR的灵敏度高出约1000倍。应用建立的方法检测人工感染的虹鳟病样,15个被检样品呈阳性反应,与细菌常规鉴定方法结果一致。研究表明,所建立的基于实时荧光定量PCR技术的杀鲑气单胞菌检测方法快速、特异、灵敏,可用于临床诊断和疫病监测。     

团头鲂tf和tfr1a基因启动子克隆及转录调控分析

为探索鱼类转铁蛋白基因tf和转铁蛋白受体基因tfr1a的转录调控机制,本实验以团头鲂为研究对象,在其全基因组数据库中获取tf和tfr1a基因序列,对2个基因候选启动子区转录因子结合位点及CpG岛进行预测,通过PCR方法克隆得到tf和tfr1a基因近端启动子区不同长度片段,连接至pGL3-Basic/pEGFP-1载体,瞬时转染入Hela细胞,并采用双荧光素酶报告基因检测系统进行检测。结果发现,团头鲂tf基因启动子区无CpG岛位点,而tfr1a基因启动子区有2个CpG岛位点。成功构建9个tf和10个tfr1a不同长度启动子片段的重组质粒,经双荧光素酶报告基因系统检测发现,tf启动子核心区域为-268^+56 bp,且-1 308^-1 102 bp片段可能存在正调控该基因表达的转录因子结合位点;tfr1a启动子核心区域为-224^+48 bp,且+48^+92 bp可能存在抑制该基因转录的负调控元件,而-1 229^-1 219 bp区域可能存在促进tfr1a基因表达的正调控转录因子结合位点。     

Cloning and characterization of the haemolysin determinants from Aeromonas hydrophila

Abstract. Two haemolysin genes (AHH4 and AHH-2) of Aeromonas hydrophila ATCC7966 were cloned into a plasmid vector in Escherichia coli K-12. An open reading frame (ORF) of the AHH-1 haemolysin gene was 1734 base pairs (bp). and corresponded to a protein of 577 amino acid residues. Analysis of the deduced amino sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The sequence also included the -10 region and the -35 region of a promoter, and a ribosome- binding site upstream from the ORF. The termination site was located downstream from the ORF. The haemolysin was a thermolabile protein with the predicted molecular mass of 60 kDa. The AHH-1 gene is distributed in various A. hydrophila and A. salmonicida strains. The nucleotide sequence of a 981 bp ORF of the AHH-2 gene was encoded with the predicted molecular mass of 377 kDa polypeptides. The homology of the nucleotide sequence was very low between the AHH-1 and AHH-2 genes, and also with the aerolysin gene cloned by Howard & Buckley (19S6). No leader peptide was found in the N-terminal region of the ORF of the AHH2 gene. The AHH-2 gene was detected in the original strain ATCC7966, but was not detected in other tested strains of A. hydrophila and A. salmonicida.     

河南华溪蟹生殖调控分子VASA的原核表达、抗体制备及免疫鉴定

为了研究生殖调控分子VASA在河南华溪蟹性腺发育过程中的表达模式和功能,本研究制备了VASA蛋白特异的多克隆抗体。选取河南华溪蟹vasa基因813 bp的特异区段,克隆到p ET32a载体,构建了原核表达载体p ET32a-Shvasa,转化至大肠杆菌BL21,经IPTG诱导表达和SDS-PAGE检测。结果显示,47 ku的VASA融合蛋白在菌液上清液中大量存在。VASA融合蛋白经Ni-NTA His-Bind亲和层析柱分离纯化后,免疫新西兰大白兔,制备了河南华溪蟹VASA蛋白的多克隆抗体。ELISA检测显示,VASA蛋白多克隆抗体的效价高达1.0×105。进一步通过免疫吸附实验和Western blot方法鉴定抗体特异性,研究表明,获得的多克隆抗体不仅能识别VASA融合蛋白,而且能特异识别河南华溪蟹卵巢中的天然VASA蛋白。研究为鉴定河南华溪蟹及其他蟹类生殖细胞提供了有效手段,为进一步解析十足目动物VASA蛋白的功能提供了分子基础。     

溶藻弧菌外膜蛋白（Va-OMP）的免疫原性及免疫保护性

利用初步制备的溶藻弧菌外膜蛋白（outer membrane protein of Vibrio alginolyticus, Va-OMP）、溶藻弧菌蛋白分子量58 kD外膜蛋白（Va-OMP58）和溶藻弧菌（Vibrio alginolyticus, Va）灭活菌苗、溶藻弧菌脂多糖（lipopolysaccharides of Vibrio alginolyticus, Va-LPS）菌苗等进行主动保护性和被动保护性的实验比较，VaOMP、Va-OMP58免疫组小鼠免疫后用活菌攻击，免疫保护率为62.5%～86.7%,而Va-LPS组和Va灭活菌苗组的免疫保护率为50%～62.5%，对照组的免疫保护率仅为6.7%。用不同抗血清免疫小鼠，活菌攻击后，Va-OMP组存活率达到53.3%和66.7%，VaOMP58组次之，存活率为40%和50%，Va灭活菌组的存活率为33.3%和46.7%，对照组为13.3%。与对照组相比较，Va-OMP组差异较显著，保护效果较好。溶藻弧菌外膜蛋白和蛋白分子量58 kD外膜蛋白的保护性效果高于溶藻弧菌灭活菌苗和溶藻弧菌脂多糖的保护性效果，证明外膜蛋白（outer membrane protein, OMP）具有较强的免疫原性。迟发性超敏反应试验也证明，OMP能诱发变态反应，间接证明OMP具有较强的引起细胞介导免疫反应的能力。因此实验证明，Va-OMP、Va-OMP⁵⁸是能在小鼠产生体液免疫和细胞介导免疫的保护性抗原。     

鳗源嗜水气单胞菌β—溶血素基因的克隆及表达

应用PCR技术,从1株鳗源嗜水气单胞菌ML316中扩增得到β 溶血素基因AHL316HEM,将AHL316HEM克隆到pGEM TEasyVector,经分析验证后重组到pcDNA3.0中,构建了重组质粒PDLH。转化重组质粒PDLH的大肠杆菌(Escherichiacoli)DH5α能在血琼脂培养皿中形成明显的β 溶血斑,重组菌纯化的胞外产物溶血价为1.28×104HU·mg-1,同时重组菌的胞外产物能被原始菌株的高免血清特异性地识别。结果证实,克隆到鳗源嗜水气单胞菌β 溶血素基因,重组质粒PDLH能够表达具有天然生物学活性的β 溶血素,为构建嗜水气单胞菌核酸疫苗奠定了基础。     

食蚊鱼CYP19a基因的克隆与序列分析

硬骨鱼类CYP19基因与生物的性别分化和激素调节相关,因此可开发用来探究环境激素污染与基因表达的关系。本研究首次克隆和分析了食蚊鱼CYP19a cDNA的全系列,为将CYP19基因作为监测环境激素生物标志物的研究提供了全面的实验数据。根据CYP19a基因c DNA保守区域设计引物,扩增保守区域并测序。采用RACE法扩增食蚊鱼CYP19a基因c DNA序列全长,对其蛋白序列进行同源性分析,并将序列应用于CYP19a mRNA转录水平的RT-PCR法检测中。成功克隆食蚊鱼CYP19a基因全长,获得CYP19a基因总长为2020 bp,ORF为238~1791 bp,共编码518个氨基酸,对其编码的蛋白质进行有关信号肽、跨膜螺旋、亲水性/疏水性、一级结构、二级结构和三级结构分析,与其他硬骨鱼类底鰆、青鰆、平鲷、鲫、鲤和斑马鱼的性腺CYP19a基因作同源性比较,其基因相似度分别为93%、84%、84%、71%、71%和66%。用MEGA6.0软件对19个物种的CYP19a基因进行聚类分析,食蚊鱼CYP19a基因与底鰆、青鰆同源性最高,说明芳香化酶在进化上相对保守。确定从食蚊鱼性腺所克隆的CYP19a基因是芳香化酶基因,证明食蚊鱼的芳香化酶是由CYP19a和CYP19b两种基因编码的。食蚊鱼卵巢芳香化酶具有3个高度保守的片段,并具有催化活性。