Cloning and Characterization of UL34 Gene of Pseudorabies Virus HB Isolate

In this study, China isolate HB of pseudorabies virus(PRV) was confirmed and genotypically characterized by amplifying and sequencing of partial UL34, a conservative gene involved in the egress of nucleocapsids from the nucleus, for phylogenetic analysis. The open reading frame(orf) of UL34 of PRV HB isolate is composed of 786 nucleotides, which encoded 262 amino acids. In addition, a potential transmembrane domain(241-260 aa) and 11 potential phosphorylation sites were also found in the UL34 of PRV HB isolate. Multiple amino acids alignment indicated that UL34 proteins of PRV strains derived from different geographic origins were highly conservative, but some mutations were also found. Phylogenetic analysis based on UL34 protein indicated that PRV HB strain was evolutionarily distinct from other recent China strains sequenced so far, forming a single clade within the phylogeny. Moreover, PRV HB isolate had close evolutionary relationship with Bo HV-1 and Bo HV-5 within the Alphaherpesvirinae. Taken together, these results indicated that PRV strains were in the progress of evolution. This study has expanded the knowledge of genetic profiles of PRV strains.     

Thymidine kinase gene mutation leads to reduced virulence of pseudorabies virus

To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK^― mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK^― mutant were sequenced. Analysis of the tk gene sequence of TK^― mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All other nucleotides of TK^―mutant were identical to that of wt virus. Deletion and insertion of the nucleotide sequence resulted in a frameshift and a premature chain termination, and the resultant TK protein was not active. Analysis of the amino acid sequence revealed that TK protein of PRV HB contained the conserved consensus sequence of herpesviral TKs and an additional conserved-DHR-motif. The results of this work also indicated that TK^― mutant was genetically stable. Compared to PRV HB, virulence of TK^― mutant was greatly decreased. Mice vaccinated with TK^― mutant were completely protected against a lethal challenge with virulent PRV (HB).     

Pseudo Rabies Virus Protein UL34 Interacted with UL31 to Disrupt the Human Lamin A

Pseudo rabies virus (PRV) egresses from the nucleus by budding from the inner nuclear membrane (INM). The nuclear lamina forms a rigid meshwork of intermediate filaments underlying the INM. It remains unknown whether PRV infection induces the disruption of lamina. In this paper, it can be observed that nuclear Lamin A became fractured during PRV infection. UL34 was localized at the nuclear rim, but UL31 was accumulated in the nucleus as distinct patches. Interestingly, a part of UL31 was localized at the INM in the presence of UL34. Immunoprecipitation (IP) assay confirmed that PRV UL31 and UL34 interacted in the transfected cells. Importantly, the co-expression of UL31 and UL34 directly disrupted Lamin A, resembling that observed during PRV infection. In conclusion, PRV infection induces the disruption of Lamin A, and UL34 and UL31 play a critical role in the disruption of Lamin A.     

黄瓜花叶病毒小青菜分离物RNA3全长序列分析

对侵染十字花科小青菜的黄瓜花叶病毒YN分离物（CMV-YN）RNA3进行全长克隆和序列分析．CMV-YNRNA3全长2220nt，分别编码279个氨基酸的3a蛋白和218个氨基酸的CP．序列同源性比较结果如下；CMV-YNRNA3核苷酸及其编码蛋白的氨基酸序列与亚组IA株系CMV-Fny、亚组IB株系CMV-Nt9、亚组Ⅱ株系CMV-Q的同源性，RNA3序列分别为92．7％、96．7％、74．2％，3a蛋白氨基酸序列分别为96．4％、98．6％、83．2％，CP氨基酸序列分别为97．7％、98．2％、83．1％．该结果表明CMV-YN与亚组IB株系CMV-Nt9的同源关系更密切．对CP核苷酸序列的系统进化树分析表明：CMV-YN归属于亚组IB，本研究为首次报道侵染我国十字花科植物的CMV基因组序列．     

燕麦分离蛋白消化特性和消化产物对STC-1细胞分泌胆囊收缩素的影响

为研究燕麦分离蛋白的消化特性及其消化产物对肠内分泌细胞分泌胆囊收缩素(CCK)的影响,以燕麦为原料,采用碱提酸沉法得到燕麦分离蛋白,分析燕麦分离蛋白在模拟胃肠道消化过程中的分子质量变化、氮释放规律、消化产物的氨基酸组成,计算氨基酸评分、化学评分和必需氨基酸指数,并以STC-1细胞为肠内分泌细胞模型评价消化产物对STC-1细胞分泌CCK的影响。实验结果表明:燕麦分离蛋白经胃肠消化后,消化产物的分子质量小于10kDa,释放的可溶性氮的比例为90.11%,消化产物可溶性部分中游离氨基酸的比例为22.8%,肽的比例为67.31%,并且肽的分子质量主要在1000Da以下(约74%);燕麦分离蛋白消化产物中必需氨基酸总量占38.75%,必需氨基酸与非必需氨基酸的比值为0.63,且必需氨基酸指数(0.95)大于0.90。燕麦分离蛋白消化产物对STC-1细胞影响的实验结果表明,燕麦分离蛋白消化产物对STC-1细胞生长具有显著的促进作用,且能够增加CCK的合成和分泌。研究结果表明,燕麦分离蛋白作为一种优质的蛋白质原料,不仅具有优良的营养功能,而且具有促进肠内分泌细胞分泌CCK的生物活性,在食品领域具有较大的应用潜力。     

Polymorphism of human cytomegalovirus （HCMV） UL144 gene in low passage clinical isolates

To explore the impact of gene polymorphism of human cytomegalovirus （HCMV） on virus virulence, the full-length UL144 gene and partial sequence of glycoprotein B （UL55） gene were sequenced and analyzed for 23 clinical strains of HCMV isolated from urine samples of pediatric patients with congenital or postnatal HCMV infection. Among the 23 isolates, 13 （57 %） were UL144 genotype 1A, 3（13 %） were UL144 genotypes 2 and 7（30 %） were UL144 genotype 3; geographic differences in geno- type distribution were found for both UL144 and gB gene. No UL144 genotypes 1B and 1C were found in this study while these two genotypes were common in HCMV strains isolated in the US. Our results also demonstrated that for all clinical strains of gB genotypes I, III and UL144 genotypes 1A, 2, and 3 found in this study, mother-to-fetus vertical transmission was possible.     

感染番茄抗病品种的番茄黄化曲叶病毒的基因变异分析

近几年来,生产中发现一些番茄抗病品种在不同环境条件下或应用几年后出现感病现象。为了解不同抗病品种中番茄黄化曲叶病毒(tomato yellow leafcurl virus,TYLCV)基因变异的情况,对5个感染TYLCV的番茄抗病品种进行了TYLCV全长基因克隆和序列测定。扩增结果显示,5个样品携带的TYLCV基因组长度均为2 781 bp,且均编码6个功能蛋白。基因组序列比较发现,这5个分离物与TYLCV-Israel株系同源性达到99%以上;通过功能蛋白比对发现,复制增强因子AC3蛋白存在变异,同世界各地报道的TYLCV-Israel株系典型分离物的AC3蛋白存在7处氨基酸差异的位点。分析结果表明,这五个病毒分离物均属于TYLCV-Israel株系,其AC3蛋白的氨基酸序列变异程度均不显著,并没有产生新的病毒株系。     

中国多年生野生大豆Glycine亚属rbcS基因的结构和系统发生的研究

用PCR法从我国多年生野生大豆Glycine亚属的烟豆 (GlycinetabacinaBenth .)和多毛豆 (短绒野大豆GlycinetomentellaHayata)的总DNA中扩增到Rubisco小亚基基因 (rbcS) ,2个基因的顺序分析结果表明它们包含了 5 37bp长的编码区 ,其编码的 178肽的Rubisco小亚基前体由 5 5个氨基酸组成的前导肽和 12 3个氨基酸组成的成熟肽组成 .基因内有 2个内含子 .比较Glycine亚属内烟豆和多毛豆之间的rbcS间碱基同源性为94 5 % ,差别主要存在于内含子中 ;Soja亚属的G .soja和G .max之间该基因同源性高达 99 7% ,而Glycine亚属和Soja两亚属之间rbcS同源性则为 84 8% .从这 2个亚属内 4个种的rbcS成熟肽 12 3个氨基酸顺序可知 ,同一亚属内 2个种之间仅有 2～ 3个氨基酸不同 ,而Glycine亚属和Soja亚属之间差异增大至 5～ 6个氨基酸 .系统进化树分析也表明Glycine亚属与Soja亚属在进化过程中分离较早 ,这从分子水平上为大豆属的分类研究提供了科学的依据 .     

人巨细胞病毒临床低传代病毒株UL97 基因的克隆与分析

分离人巨细胞病毒（HCMV）临床低传代GZ02病毒株,并根据GenBank提供的实验室标准病毒株HCMV AD169磷酸转移酶基因UL97 DNA序列及有关文献设计引物,从HCMV GZ02病毒株基因组DNA中通过PCR扩增UL97基因,并克隆至pGEM3Z质粒载体．重组质粒经测序鉴定,发现HCM VGZ02病毒株UL97基因序列保守区域与HCMV AD169 UL97长度完全一致,但发生G205A、G761A、T823C、T1173C、T1282C、T1334C、T2106C等7个位点的碱基突变,涉及到编码氨基酸E69K,S275P,C428R和F445S位点发生改变。     

PRV广东株gE基因去信号肽片段的克隆与序列测定

根据伪狂犬病病毒（PRV）Rice株gE基因的序列设计并合成了1对引物,以我国PRV地方毒株广东株的基因组DNA为模板,通过PCR方法获得了一大小约1.6kb的DNA片段,并将其克隆到pMD18-T载体上进行测序,序列测定结果显示,该片段长1665bp,编码555个氨基酸,与PRV Rice株gE基因的核苷酸序列同源性为97.7%,氨基酸序列同源性为95.9%。     

猪繁殖和呼吸综合征病毒ORF7基因的克隆与序列分析

根据猪繁殖和呼吸综合征病毒（PRRSV）已发表的核苷酸序列，设计了1对特异性引物P1／P2，用RT-PCR扩增了PRRSV福建毒株FJ-1和FJ-2的全长核衣壳蛋白基因（ORF7），将扩增产物连接到pUCm-T载体并转化大肠杆菌DH10B，提取阳性重组质粒进行序列测定与分析．结果表明：FJ-1和FJ-2毒株ORF7基因均为372bp，编码123个氨基酸，与美洲型参考毒株VR-2332及欧洲型参考毒株LV的核苷酸同源性分别为95．16％，94．35％和61．40％，60．65％，其推导的氨基酸同源性分别为95．93％，95．12％和56．49％，55．73％．研究表明，福建流行的FJ-1和FJ-2属于美洲型PRRSV毒株．     

红鳌鳌虾胚胎发育过程中蛋白质含量及氨基酸组成的分析

采用生物化学方法对红整鳌虾胚胎发育过程中蛋白质与氨基酸的组成进行了测定和分析。总蛋白与总氨基酸的含量均呈下降趋势。总蛋白由受精卵期的65.32%(占干重)降低到准备孵化期的40.28%;总氨基酸含量由受精卵期的58. 05%(占干重)减少至准备孵化期的47.49%.必需氨基酸(除Met外)的变化与总氨基酸含量呈显著性相关，且变异系数在8.25-13.34%之间;非必需氮基酸中Glu,Asp的含量较高，Cys的含量较低。蛋白质和氨基酸不仅是红鳌鳌虾胚胎发育的构建物质而且在胚胎发育过程中提供能量。     

JWA, a novel microtubule－associated protein, regulates homeostasis of intracellular amino acids in PC12 cells

Our previous studies demonstrated that JWA,a novel retinoic acids responsive and cytoskeleton related gene, is associated with cell differentiation and apoptosis. In the present study, to elucidate if the JWA is a novel kind of microtubnle-associated proteins (MAPs) and functiona llylink to microtubule, we first successfully identified JWA from the physically purified MAPs complex of rat braint issues. The results of co-immunoprecipitation, gene transfection and immunofluorescence microscopy assays from HBE and NIH3T3 cells provide strong evidence for a linkage between JWA and β-tubulin. In general, JWA is stably binding to β-tubulin whenever microtubule is polymerized or not,and it may be critical to the mitosis process. In addition, by use of the antisense oligonucleotides technique, we also showed that JWA is a negative modulator on intracellular amino acids in PC12 cells. Further analysis indicated that JWA selectively regulates both taurine, an inhibitory aminoacid, and glutamate, an excitatory amino acid. In conclusion,JWA is not only structurally associated, but also a novel functional MAP.     

六品系猪SLA-2多肽结合区分子结构差异研究

为研究我国饲养的不同品系猪sLA_2抗原多肽结合区分子结构差异,设计引物,克隆了六品系猪SLA-2基因cDNA全长序列,用分子生物学软件分析其基因特征,比较各品系猪SLA-2分子结构差异,并通过同源模建显示变异住点的空间位置．结果表明,六品系猪SLA-2cDNA全长为1119bp,其中3-1097为编码区,共编码364个氨基酸,分别在第125,188,227,283位置出现半胱氨酸残基,含有2对链内二硫键．六品系猪SLA-2胞外区氨基酸差异主要集中在α1区的62-70及77-82和口2区的143-156．同源模建显示,这些变异位点均位于SLA-2多肽结合区的口螺旋结构上,推测可能与动物的抗病毒免疫特性有关．可为猪品系的改良及抗病毒免疫研究提供参考．     

杂色鲍肠道益生菌的分离和鉴定

从杂色鲍(Haliotis diversicolor)肠道内,共分离出84株细菌,经16S rRNA基因序列比对,共产生了35种类型(OTU),分属于17个属,利用纸片扩散法进行了细菌消化酶活性及抑菌活性检测.有75株菌在不同程度上表现出淀粉酶活性、蛋白酶活性、脂肪酶活性、琼胶酶活性、纤维素酶活性或褐藻酸钠酶活性;有24株菌不同程度地抑制2～8株致病菌的生长.结合实验结果,对9株潜在益生菌进行溶血实验检测及动物安全性实验.其中AP12和AP34不产生溶血素,并且在菌株浓度108 CFU/g时对鲍无明显的毒害作用,表明此2株细菌可以作为潜在的益生菌应用于杂色鲍的养殖.AP34具有5种消化酶活性,与fu同温层芽孢杆菌(Bacillus stratosphericus)16S rRNA基因序列同源性最高,为99.87％;AP12对8株致病菌均表现出较强的抑菌活性,该菌16S rRNA基因序列与济州岛褐色杆菌(Phaeobacterdaeponensis)相似性最高,为98.63％.     

处理厨余垃圾的高温菌剂研制及其降解性质研究

利用高温菌耐热嗜热的特性和有机物好氧分解的基本原理,从厨余垃圾处理系统中分离筛选到6株在65 ℃能产生淀粉酶、脂肪酶、蛋白质酶及纤维素酶的高温高效菌种.经鉴定,所分离得到的高温菌株都有芽孢,属于兼性细菌.经安全性检测6株高温菌中未检测到沙门氏菌和志贺氏菌等致病菌,说明分离到的高温菌不存在致病性因素.通过最佳菌株组合后对厨余垃圾进行降解试验,最终确定4株（HB1,HB2,HB4和HB6）制成高温菌剂.将该菌剂在100 kg厨余垃圾处理机上进行降解试验,结果表明：一次性投入5%菌剂后升温至65℃,24 h内对厨余垃圾中粗脂肪和粗纤维有明显的降解效果,降解效率分别为30.7%和11.3%,粗蛋白含量增加了9.5%.继续对厨余垃圾处理48 h,其有机物分解率几乎没有得到提高,说明大部分有机物在24 h内即可被分离出的高温菌剂所降解.     

番木瓜环斑病毒复制酶基因的克隆和序列分析

用ＲＴ－ＰＣＲ技术从ＰＲＶ－ＡＬ中分离到复制酶（ＲＰ）基因，将基因克隆进载体ｐＵＣ１８，用双脱氧链终止法测定了基因序列，表明其全长为１６０２ｂｐ，与国内外报道的ＨＡ５－１、ＹＫ和Ｓｍ的ＲＰ基因相比，同源性分别达８２．８０％，９５．０７％和９１．８３％．     

用于乙肝疫苗效力检定的小鼠种群的建立

目的在进行乙肝疫苗效力检测时应用统一的动物种群。方法目前评价乙肝疫苗免疫效力的主要方法是用小鼠测定乙肝疫苗的ED50值(接种疫苗后使50%的动物抗体阳转的疫苗稀释度的倒数),而小鼠对HBsAg免疫应答的水平与H-2单倍型密切相关。因此我们通过微量细胞毒法和PCR方法检测小鼠的H-2单倍型的特点,得出乙肝疫苗与不同动物种群的相关性,解决实际工作中的问题。结果通过多方面的实验验证,NIH鼠可以对乙肝疫苗产生高应答效应。结论NIH鼠的H-2单倍型中有q型基因,同时NIH鼠保持了原封闭群动物繁殖力强生长速度快等优点,所以便于进行乙肝疫苗效力检测时应用。     

南海圆鮀鲣肌肉组织酶解产物抗疲劳活性的初步研究

圆鮀鲣是我国南海高产低值的一类金枪鱼。对其肌肉组织的普通肉和暗色肉进行了酶解及氨基酸组成分析,并进行了抗疲劳活性动物实验评价。圆〖XCTUO-K.tif,+3.8mm。3.8mm;%110%110〗鲣的普通肉酶解产物冻干粉和暗色肉酶解产物冻干粉的氨基酸质量分数分别为57.06%和58.90%,其中支链氨基酸含量占总氨基酸的约18%。普通肉酶解产物冻干粉和暗色肉酶解产物冻干粉中分子质量5000Da以下肽的质量分数均达到70%以上,700Da以下的肽占20%以上。与空白对照组相比,圆鮀鲣的普通肉酶解产物冻干粉和暗色肉酶解产物冻干粉的各剂量组均具备显著延长小鼠负重游泳时间,增加小鼠肝糖原储备和降低运动后小鼠血清尿素氮含量的作用,表明2种酶解产物均有显著的抗疲劳活性,且二者无显著差异。研究结果表明,圆鮀鲣的普通肉和暗色肉组织均可作为抗疲劳保健产品潜在的开发对象。     

高产聚-3-羟基丁酸重组大肠杆菌的筛选

将能以葡萄糖为唯一碳源积累P(3HB)的工程菌进行优化筛选,获得6个较高产的菌株,PHB含量最高为69.1%.经丙酸驯化,虽然没有PHBV的积累,但PHB的量均有增加,最高可达71.13%.另外,抗生素对菌体生长及PHB积累有重要影响.