中国对虾非包涵体型杆状病毒核衣壳的分离纯化

An effective procedure for isolation and purification of nucleocapsids of Penaeus chinensis non-occluded baculovirus (PcNOBV) which has destroyed the Chinese shrimp industry since 1993 was described. Gill, stomach, gut and cuticle epidermis under exoskeleton were excised from cultured P. chinensis diseased with typical white spot syndrome and homogenized in liquid nitrogen with TNE buffer containing PMSF and β-ME. The homogenized mixture was filtered through a 0.45μm millipore filter membrane to remove cell debris and ultracentrifuged to pellet the remaining material.The pellet was suspended in PMTNE buffer and laid onto a handmade CsCl gradient. An obvious viral band was observed in the middle of the gradient. Large amounts of virus nucleocapsids were visualized under electron microscope consistently corresponding to the milk-colored viral band. The viral envelope was all lost after purification. The nucleocapsid was bacilliform averaging 80±13nm×380±24nm in size. The negatively stained PcNOBV nucleocapsids revealed 13-16 conspicuous stripes located periodically perpendicular to the longitudinal axis of the nucleocapsids. Six to seven capsomers of 9 nm in diameter were visualized on each side of the stripe.     

伪狂犬病病毒湖北株糖蛋白gD基因的克隆及序列测定

According to the sequence of gD gene of PRV Rice strain, the primers of 22bp were designed.Using PRV genomic DNA of Hubei and Shuangcheng virus strains which infected BHK 21 cell separately as template, the gD gene of PRV was amplified sucessfully by PCR and cloned into pGEM T vector. Restriction enzyme analysis showed that the cloned gD gene at SmaⅠ,SalⅠ,KpnⅠ,PvuⅡ sites was the same as that of PRV Rice strain. The gD gene consisted of 1,263 nucleotides including an open reading frame spanning 1,197 nucleotieds which could encode a protein of 398 amino acids. The ORF didn′t include an amino acid sequence directing N linked glycosylation (NXT or NXS). Comparison of our complete Hubei strain gD gene sequence with the Rice strain gD gene sequence showed that the nucleotide and deduced amino acid homology were about 97% and there was an 12 basepair deletion in 835 846 nucleotide sites that coded Arg Pro Arg Pro. A region of the amino acid sequence and the positions of the cysteine residues of PRV HB gD were homologous to HSV I glycoprotein D. This work laid foundation for PRV gene immunization and studying PRV sub unit vaccine.     

蜘蛛基因组DNA Cosmid文库构建和拖丝蛋白基因的克隆

The genomic DNA Cosmid library was constructed from Nephila clavipes spider muscle using SuperCos 1 Cosmid as a vector.The title of library was >5×10 4 cfu/μg ligated DNA.On the basis of published sequence from a partial cDNA sequence of the 3′end of the dragline silk gene, we designed and synthesized 3 oligonucleotides.Oligonucleotides were labeled with non radioactive digoxigenin dUTP and detected with chemilluminescent substrate.56 positive recombinants were screen from the Cosmid library using DIG Oligo 2 as a probe.DNA dot hybridization using DIG Oligo 1 and DIG Oligo 3 as the probes, respectively, 3 positive signals were identified from 56 colonies.They were appeared the same pattern when DNA from the colones digested by restriction enzymes.The spider dragline silk gene was confirmed again by Southern blot hybridization.     

荧光素标记的庚型肝炎病毒基因探针的制备及应用

The sequences of HGV gene that had been reported were checked and analyzed. After comparison of HGV seqences from different virus strains, a gene fragment of 31 nucleotides (7121-7152 nt) served as a probe was labeled by fluorescein-N₆-dd ATP with terminal transferase. The specificity of HGV probe was tested by dot-blot hybridization with HCV RNA—related virus RNA, C.T DNA, HGV-RNA, and Southern-blot hybridization with RT-PCR product of HGV. Positive results obstained only from the HGV-RNA of positive sera that was confirmed by nested RT-PCR, all others were negative. The sensitivity of fluorescein-labeled probe was examined. The result showed that the fluorescein-labeled probe was able to check≥1.56 pg of target genome. The conformity of HGV-RNA detection using RT-PCR and HGV gene probe was 97.9%. The experiment suggested that fluorescein-labeled probe can be used in clinic detection and epidemic study.     

PCR检测中国对虾暴发性流行病毒靶基因的克隆和序列分析

An explosive epidemic disease of shrimp had occurred in China and the south Pacific coast. The causative agent is a new baculovirus called hypodermal and hematopoietic necrosis baculovirus (HHNBV) in China, and the pathogen is very much similar to Japanese panaeid rod-shaped DNA virus (PRDV) which caused mass mortality of shrimp. In order to distinguish both HHNBV and PRDV at molecular level, we have sequenced the PCR-targeted DNA fragment of HHNBV. One pair of PCR primers were prepared based on the PRDV DNA. The sequence of the PCR-targeted DNA of HHNBV is 975bp in length, and shares 99.7% homology with the PRDV. The sequence of PCR-targeted DNA of HHNBV-XIA differs from PRDV DNA in deleting three nucleotides (CAT) at 510～512 site. These characteristics of the sequence classified HHNBV and PRDV as strains of different genotypes.     

严重急性呼吸综合征(SARS)冠状病毒刺突蛋白基因片段的表达及其初步应用

SARS virus spike gene fra gment was expressed by Ecoli expr ession systemThe fragment enclose s major neutralization epitope of the virusThe expressed protein wa s purified and an ELISA method was set upBy using the recombinant,tw elve patients' sera were detected The recombinant SARS coronavirus s pike protein offers an efficient wa y for serological diagnosis and is useful for epidemiological survey and vaccin e development     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library of Streptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_(TH4) as probe. The experiments revealed that this DNA fragment consists of saw D gene and a 1.4 kb Pvu Ⅱ fragment which can accelerate mycelium formation of S. ansochromogerms. The nucleofide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was desiguated as samfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded by hppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) in Rhodococcus globerulus. The function of samfR gene was studied using strategy of gene disruption, and the resulting samfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped     

生态制品对提高中国对虾出池仔虾成活率和生长率的初步研究

根据微生态平衡原理,应用三种微生物菌群（系从中国对虾成虾消化道中分离筛选出来的）制成生态制品作为饲料添加剂,对中国对虾出池仔虾进行投喂,实验结果表明：三种生态制品不汉都能显著地提高仔虾的成活率,而且对仔虾的生长也有一定的促进作用。本文对出池仔虾死亡率较高的原因,微生态制剂的促生长机理,以及直接利用出池仔虾进行养成或放流的可能性等问题进行了讨论。     

中国对虾球状病毒垂直传播的初步研究

应用透射电镜技术在越冬中国对虾雌性亲虾体内检测出一种球状病毒,直径为80nm左右。人工感染实验证实该病毒颗粒具有感染性。对带毒亲虾在室内隔离条件下进行了暂养催产,并对其所产的卵子、幼虫和的虾进行了隔离培育。带毒亲虾的卵子孵化率、幼虫成活率和幼虾生长速度均明显低于不带毒亲虾的子代。电镜观察发现．在带毒亲虾卵巢、各期幼虫和幼虾的细胞内存在球状病毒颗粒,在刚产的卵子的卵黄颗粒中可看到一种似球状病毒颗粒。研究表明：带球状病毒的对虾呈隐性感染,在胁迫条件下带毒幼虾可暴发病害,带毒亲虾可正常发育产卵,病毒可能通过卵巢、卵子进行垂直传播。     

中国对虾呼肠孤样病毒感染的电镜观察

呼肠孤病毒在自然界广泛存在于脊椎动物、无脊椎动物和植物中.水产动物呼肠孤病毒感染曾见于鱼、贝、蟹.近几年随着对对虾病毒病害研究的日益重视,在对虾中也发现呼肠孤病毒的感染.Tsing等[1]最早于1987年在法国南部养殖的日本对虾(P.japonicus)幼虾中发现呼肠孤病毒感染.Krol等[2]于1990年在试验感染的南美白对虾(P.vannamei)中发现呼肠孤样病毒与对虾杆状病毒混合感染.中国大陆养殖的中国对虾自1993年全面暴发流行病以来,许多学者进行了对虾流行病的病原学、流行病学及诊断和防治方法的研究,部分学者曾在中国对虾(P.chinensis)中观察到呼肠孤样病毒颗粒[3],但未报道较详细的电子显微镜观察资料.     

中国对虾继饥饿后的补偿生长研

1999年7至8月份,在25.0±0.5℃条件下对中国对虾（湿重,1.454±0.150g）进行了不同时间的饥饿处理后再供食的恢复生长实验。对照组C连续饱食投喂32d;处理组S4、S8和S12分别饥饿4、8和12d后再饱食投喂28、24和20d。主要结果如下饥饿结束时各处理组的干重和湿重显著低于对照组（P<0.05）;实验结束时S4组和对照组间的干重和湿重差异不显著（P>0.05）,而S8和S12两组的干重和湿重仍显著低于对照组（P<0.05）;恢复生长后各处理组的湿重摄食率显著高于对照组（P<0.05）。实验结果表明,中国对虾继饥饿后再恢复喂食出现完全或部分补偿生长效应,且这种补偿生长效应主要是通过恢复生长阶段食欲增大,摄食水平提高实现的。     

微生态调节剂对提高中国对虾抗病力的研究

应用从健康中国对虾肠道中分离筛选出的无毒、无害菌群（ＪＸ５、ＪＸ６）制成微生态调节剂,将其作为饲料添加剂饲养中国对虾,对感染暴发性流行病的抗病力进行试验研究。结果表明：（１）微生态调节剂能够明显地提高中国对虾感染后的成活率,使死亡高峰时间延迟;（２）感染后的血液病理指标显示,病虾的血清总蛋白、白蛋白、球蛋白等指标下降,甘油三酯、碱性磷酸酶、乳酸脱氢酶等指标上升;（３）投喂微生态调节剂的试验组与对照组经感染致病后,其血液指标与健康虾比较变化幅度不同,试验组虾血液指标变化幅度小于对照组。本文同时对微生态调节剂抗病力的作用机制进行了初步探讨。     

中国对虾两种球状病毒的免疫检测

以江苏、河北某虾场病虾组织中提取的对虾的两种球状病毒作为抗原,制备出特异性强、效价较高的抗血清,用对流免疫电泳,酶联免疫吸附,免疫电镜技术对五个不同地区的病虾材料进行检测,结果表明：江苏、青岛、河北地区的病虾组织中均含有相应的球状病毒病原。     

Detection of Vibrio parahaemolyticus in Penaeus chinensis using an indirect fluorescent antibody staining procedure

An indirect fluorescent antibody technique (iFAT) incorporating fluorescein-isothiocyanate conjugated anti-rabbit globulin goat serum, and rhodamine-isothiocyanate conjugated bovine serum albumin as background stain has been developed for the detection of Vibrio parahaemolyticus.     

温度和体重对中国对虾碳收支的影响

于 1996年 5～ 9月 ,测定了 3种规格 ( 0 .2 71± 0 .0 41g、3 .5 0 9± 0 .3 0 1g、11.0 62± 1.0 2 7g)池塘养殖中国对虾 (Penaeuschinensis)在 2 0℃、2 5℃和 3 0℃下 ,摄食日本刺沙蚕 (Neanthesjaponica)的C收支 .结果表明 ,温度和体重对摄食C有显著影响 ,随温度升高和体重的下降 ,C的摄食量显著增大 ,在 2 0℃、2 5℃和 3 0℃下的平均C摄食量分别为 12 .41、19.12和 2 6.0 8mg·g-1·d-1,在 3个规格下的平均C摄食量分别为 3 6.0 6、12 .17和 9.3 8mg·g-1·d-1.不同规格中国对虾对摄食C的分配比例无显著影响 ,温度是影响中国对虾摄食C分配的主要因素 .在 3个温度下摄食沙蚕的生长C、排粪C、蜕壳C和代谢C占摄食C的平均分配比例分别为 3 1.2 3 %、4.3 8%、7.94%、5 6.45 % ;2 6.83 %、2 .92 %、6.69%、65 .79% ;16.86%、2 .3 8%、5 .99%和 74.76% .