杂交牛(大额牛×云南黄牛)朊蛋白基因 的分子克隆及其序列分析

朊蛋白(prion protein,PRNP)基因编码朊蛋白,是引起疯牛病的主效基因。本研究利用PCR方法首次从杂交牛(大额牛×云南黄牛)基因组中扩增了PRNP基因,GenBank登录号为HQ875337。PCR产物直接双向测序表明,该序列包含杂交牛PRNP基因795 bp的开放阅读框(ORF),编码264个氨基酸前体蛋白。生物信息学分析结果发现,该蛋白包含1个信号肽、3个α螺旋、2个β折叠、6个八肽重复序列、1个疏水区域、1个二硫键和1个糖基磷脂酰肌醇锚定位点。与已报道的其他牛PRNP基因进行序列比对分析,核苷酸和氨基酸的同源性均在97%以上。     

非洲狮朊蛋白基因的克隆与序列分析

根据已报道的哺乳动物朊蛋白基因序列设计了1对引物，采用PCR方法扩增了16只非洲狮的朊蛋白基因，序列分析结果表明，所得到的非洲狮朊蛋白基因片段长678bp、编码226个氨基酸的前体蛋白，其核苷酸序列的同源性为99．79％以上，发现了4个核苷酸多态性位点，无氨基酸变异。经与已报道的猫、貂、绵羊、牛、鼠和犬等哺乳动物的氨基酸序列进行比较，与猫（AF003087，96．7％）和绵羊（96．2％）的氨基酸同源性最高。     

牛朊蛋白(bPrPc)基因的克隆和序列分析

分别从9头牛(3头牦牛、3头荷斯坦牛和3头秦川黄牛)全血中提取基因组总DNA，用所设计引物以聚合酶链式反应扩增出细胞型朊蛋白(PrP^c)基因，并克隆到pMD18-T载体。序列分析表明所克隆的牛PrP。的片段大小为795bp，包含了牛朊蛋白基因的完整编码区序列，为含单一外显子的完整开放阅读框，与国内外报道的已知序列基本相同。本次所报道的牛PrP(bPrP^c)基因含6个短而富含G-C的元件，可编码八肽Pro-His-Gly-Gly-Gly-Trp-Gly-Gln／Arg或九肽Pro-Gin／His-Gly-Gly-Gly-Gly-Trp／Arg-Gly-Gin。这些bPrPC基因序列相比较，其核苷酸和氨基酸同源性分别在99．0％～100．0％和99．2％～100．0％之间。在整个18个碱基替换中，多数替换是保守的，为同义突变，仅有5个替换引起氨基酸突变，分别为HN200302的W60R、HN200303的S154N、MN200301的A129V、NN200302的Q234R和NN200303的Q94R突变。发现牦牛朊粒基因在126(A-G)、234(G-A)和678(T-C)位的特征性核苷酸替换，但均为同义码替换。     

微小牛蜱铁蛋白编码基因的克隆和分析

从微小牛蜱克隆到1个新的铁蛋白编码基因，cDNA全长642bp，编码区为123-639bp，编码172个氨基酸残基，该蛋白预测的分子量为19．9ku，等电点为4．24。经过分析，其预测氨基酸序列与已报道的变异革蜱、非洲钝缘蜱和蓖子硬蜱铁蛋白同源性分别为93．60％、88．37％和83．72％。且核苷酸序列在mRNA 5’未翻译区(5’UTR)的茎环结构存在铁应答元件(IRE)，其氨基酸序列上带有典型的亚铁氧化酶中心结构的保守序列。RT-PCR分析表明，该基因在微小牛蜱卵、幼蜱、半饱血雌蜱、饱血雌蜱和雄蜱这几个阶段均有表达。     

2种家养动物朊蛋白基因的扩增与分析

采用PCR方法扩增了云南矮马和北京油鸡的PrP^C基因，并进行序列测定，结合已发表种属朊病毒基因序列，运用分子生物学软件进行同源性分析。结果表明，云南矮马PrP^C基因与已报道的马PrP^C基因比较，其同源性达99％，氨基酸同源性达100％。与牛PrP^C基因比较，同源性达89％，氨基酸同源性达91％。北京油鸡PrP^C基因与鸡的已知序列比较，同源性达99％；与鸭、鸽、鹌鹑的已知序列比较，同源性94％～97％。其翻译的氨基酸序列与鸡的已知序列比较，同源性达99％，与鸭、鸽、鹌鹑比较，氨基酸同源性88％～99％。从本试验结果来看，云南矮马PrP^C基因与牛的PrP^C基因同源性较远，因此感染牛源朊病毒的风险较小，禽类与哺乳动物PrP^C氨基酸属于2个不同的进化分支，因此哺乳动物海绵状脑病不易传染给禽类或引起朊蛋白构型上的改变。这些结果可为海绵状脑病种间屏障补充详细的数据，也为制定相关预防控制措施提供了一些理论依据。     

番鸭呼肠孤病毒ZJ99株σC蛋白基因的克隆与序列分析

参考已发表的番鸭呼肠孤病毒(mDRV)的S4序列设计了S4对扩增σC蛋白基N的引物，对mDRV ZJ99株的RNA抽提物进行RT—PCR扩增，获得了特异性的扩增片段。将PCR产物克隆测序，序列经同源性比较，发现mDRV ZJ99株σC蛋白基N序列与福建MW9710株对应基N的同源性为99．5％，与法国89026株对应基因的同源性为94．2％，与法国89330株对应基因的同源性为95．0％；相应的氨基酸序列同源性分别为98．9％、94．1％、93．7％。基于σC基因及其推导氨基酸序列的蛋白质结构及物理化学性质预测结果，与国外学者对mDRV σC蛋白的报道相似。     

口蹄疫病毒诱导的牛α－干扰素基因cDNA的克隆及序列分析

参照已发表的牛α-干扰素基因cDNA序列设计了1对引物，用感染口蹄疫病毒的牛外周血为材料，提取总RNA。经RT-PCR，获得与预期大小相符的DNA片段。所获得的PCR产物长度是604bp，与参考序列的同源性是99．7％。第23位～91位的69个碱基为信号肽序列，编码23个氨基酸。与参考序列比较，第43位的碱基由C变为A，即由TTC→TTA，对应的氨基酸由苯丙氨酸(F)变为亮氨酸(L)。第45位的碱基由G变为C，即由CGA→CCA，对应的氨基酸由精氨酸(R)变为脯氨酸(P)。第92～589位的498个碱基为牛α-干扰素成熟蛋白基因序列，编码166个氨基酸，与参考序列完全相同，即同源性为100％。第590～592位的碱基为TGA终止子。整个牛α-干扰素前体蛋白基因共570个碱基。编码189个氨基酸。     

东北虎朊病毒基因的克隆与序列分析

根据已报道的哺乳动物朊病毒基因序列设计引物，采用PCR方法扩增了25只东北虎的朊病毒基因，克隆、测序及序列分析表明，所得到的东北虎朊病毒基因片段为402bp，编码134个氨基酸的前体蛋白，核苷酸序列同源性为99．67％。共发现了4个核苷酸多态性（T423C，A501G，C511A，A610G），其中C511A和A610G的碱基突变导致K171Q和A204T氨基酸的变异。与已报道的猫、貂、绵羊、鼠和犬等哺乳动物的氨基酸序列比较，结果与猫（AF003087，97．3％）和绵羊（97．3％）的氨基酸同源性最高。     

堆型艾美耳球虫子孢子表面抗原基因cSZ1的克隆及序列分析

根据已报道的堆型艾美耳球虫(Eimeria acervulina)子孢子表面抗原基因cSZ1的cDNA序列设计特异性引物,以孢子化12h的E.acervulina广东株卵囊的总RNA为模板,用RT-PCR方法成功克隆了其子孢子表面抗原基因cSZ1(cSZ1(Gd))的cDNA。所克隆cSZ1(Gd)的cDNA全长940bp,其中第3～512位是其阅读框架,共编码170个氨基酸,两端为非编码区。与已报道的cSZ1基因的cDNA序列相比,cSZ1(Gd)有4个位置的核苷酸发生了变异,即原序列中第294、443、569、586位的G、G、G、A在cSZ1(Gd)分别为A、A、A、G,二者核苷酸序列的同源性为99.57％(936/940),其中阅读框架的核苷酸同源性为99.61%(508/510)。比较根据核苷酸序列推导的氨基酸序列,第294位的变异导致了所编码氨基酸由缬氨酸(V)变为异亮氨酸(Ⅰ),而第443位的变异则为无义突变,氨基酸序列的同源性为99.41％(169/170)。     

小球隐孢子虫子孢子表面蛋白CP15/60基因的克隆和序列分析

将小球隐孢子虫子孢子表面蛋白（CP15／60）编码区基因克隆及测序，并对其抗原决定簇进行分析。抽提牛源小球隐孢子虫孵囊基因组DNA，用聚合酶链反应（PCR）扩增编码CP15／60的基因，然后将其克隆到p MD18-T载体中，用双脱氧链末端终止法测DNA序列。结果，用PCR扩增获得了CP15／60的编码区基因，并测定了该基因编码区序列。与GebBank比较，核苷酸序列同源性为：98．9％；推导出的氨基酸序列同源性为：99．3％。     

Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene

ABSTRACT: Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.     

梅花鹿朊蛋白基因(PRNP)的克隆及序列分析

根据GenBank中鹿朊蛋白基因序列设计特异性扩增引物，采用PCR方法从中国梅花鹿基因组中扩增得到梅花鹿的朊蛋白基因，采用PCR产物直接测序，并将其克隆到pGEM—TEasy载体中测序进行进一步确认，通过分析表明所克隆的梅花鹿朊蛋白基因的ORF基因片段包含771bp，编码256个氨基酸的前体蛋白，相对分子质量约为28200。与已报道的其他品种鹿朊蛋白基因序列进行对比分析，核苷酸及氨基酸序列的同源性均在98％以上。本试验为进一步研究中国梅花鹿朊蛋白的多态性提供了数据。     

Prion protein gene polymorphism and blood lymphocyte profile in cows naturally infected with bovine leukemia virus

The polymorphic loci of the bovine prion protein (PRNP) gene, comprising 23-bp insertion/deletion (23-bp indel) within the promoter sequence and 12-bp insertion/deletion (12-bp indel) within the intron 1 sequence, are located in regions which play a key role in gene expression. The objective of this study was to determine whether the 23-bp and 12-bp insertion/deletion polymorphism within the PRNP gene leads to significant differences in the blood lymphocyte profile and to investigate changes in the composition of these cells in cattle naturally infected with Bovine Leukemia Virus. An analysis of the effect of the bovine PRNP gene polymorphism on the blood lymphocyte profile revealed considerable differences between animals with the 23-bp indel genotypes, and small and statistically non-significant differences between those with the 12-bp indel genotypes. 23-bp del/del homozygotes had a significantly lower percentage of T lymphocytes with the phenotypes CD2 (P < 0.01), CD8 (P < 0.01) and WC1-N2 (P < 0.05), and a higher ratio of CD4 to CD8 T lymphocytes, compared to animals with the 23-bp ins/ins genotype. The obtained results indicate that the 23-bp indel polymorphism, in contrast to the 12-bp indel polymorphism, has a significant effect on changes in the blood lymphocyte profile. The size of blood lymphocyte subpopulations was also found to change under the influence of enzootic bovine leukosis. The direction of those changes in EBL-positive animals is consistent with that observed in 23-bp del/del homozygotes, which may testify to the adverse effect of this genotype on immunological efficiency.     

新疆地方绵羊品种PRNP基因多态性研究

从新疆采取了8个地方绵羊品种的血液样品171份,提取绵羊基因组DNA,用PCR方法扩增绵羊PRNP基因,通过序列测定,对它们的PRNP基因型进行研究,确定了PRNP基因136、154、171位密码子的多态性为136(A/A),154(H/R)和171(Q/R/H/K),结果发现所检测的新疆地方绵羊品种PRNP基因136位密码子均为A,其基因型均为A型痒病抵抗性基因型。     

Technical note: determination of alleles of the ovine PRNP gene using PCR-single-strand conformational polymorphism analysis

Susceptibility to scrapie in sheep is linked to variation at codons 136, 154, and 171 in the host prion protein gene (PRNP). A number of techniques are available for detecting these polymorphisms, but none allow for a rapid and accurate determination of genotype. Here we describe PCR coupled with single-strand conformational polymorphism (SSCP) analysis, which allows for the accurate identification of ovine PRNP alleles. A gene region including codons 136 to 171 was amplified by PCR, and the amplimers were then denatured and subjected to electrophoresis in a nondenaturing polyacrylamide gel. Nine unique SSCP patterns, representing nine different alleles of the ovine PRNP gene, could be resolved. A new polymorphism (I/T) at codon 142 also was detected. The profiles produced by SSCP allowed for the accurate differentiation of PRNP alleles and could be employed to genotype PRNP in sheep.     

PRNP haplotype and genotype frequencies in Brazilian sheep: issues for conservation and breeding programs

Polymorphisms of PRNP gene have been strongly correlated to the susceptibility/resistance to scrapie in sheep. Variants at the coding positions 136, 154 and 171 have been the most frequently associated to susceptibility to classical scrapie. The aim of this study was to estimate PRNP haplotype and genotype frequencies in a sample of 1400 sheep from 13 different breeds that are representative of the main production regions in Brazil. A total of four different alleles (ARR, ARQ, AHQ and VRQ) and nine genotypes were observed at different frequencies among the investigated breeds. There were distinct patterns of allelic distribution between naturalized and commercial/specialized breeds and different geographic regions. These results will influence the development and management of breeding and conservation programs and will help to develop Brazilian efforts to avoid scrapie epidemics.     

Evaluation of associations between prion haplotypes and growth, carcass, and meat quality traits in a Dorset x Romanov sheep population

There is concern about potential antagonistic correlated responses due to intensive selection for scrapie-resistant haplotypes of the prion (PRNP) gene in sheep. The objective of the present research was to test for associations of PRNP haplotypes for codons 136, 154, and 171 with growth, carcass, and meat quality traits in an F2 Dorset x Romanov population (n = 415) segregating the 2 callipyge alleles. Haplotypes of the 3 PRNP codons were determined for each sheep, and breed of origin of each gamete was predicted by genotyping 6 microsatellite markers flanking the PRNP locus. Twenty-five growth, carcass, and meat quality traits were evaluated. Data were analyzed using a basic model consisting of fixed effects of year, sex, and callipyge genotype, the random effect of sire, and 7 covariates corresponding to the probability that a lamb inherited a specific PRNP haplotype of either Dorset or Romanov origin. A fixed effect of litter size was added to the model for growth traits. The model for carcass traits contained the linear and quadratic effects of chilled carcass weight and the interactions among callipyge genotype and linear and quadratic terms. For meat quality traits, the model contained chilled carcass weight as a covariate and the interaction between callipyge genotype and chilled carcass weight. A contrast between the resistant ARR haplotype and the average effect of other PRNP haplotypes was tested to investigate the effects of potential selection for ARR within each breed of origin (Dorset, ARR vs. ARQ, VRQ, and AHQ; Romanov, ARR vs. ARQ and VRQ). There was limited evidence that selecting for scrapie resistance would cause correlated responses due to linkage disequilibrium. Associations of only 3 traits with PRNP haplotypes were detected in either breed of origin. In Romanov, the ARR haplotype was associated with longer carcasses (P < 0.013), narrower rumps (P = 0.038), and less marbling (P = 0.022) than the average of ARQ and VRQ haplotypes. No significant contrasts were detected for Dorset. This study is the first to account for breed of origin while investigating haplotype associations in an F2 population. This study provided limited evidence of associations between PRNP haplotypes and growth, carcass, and meat quality traits.     

PRNP gene polymorphisms in main indigenous Turkish goat breeds

Tropical Animal Health and Production - The polymorphisms of the PRNP gene influence the susceptibility to scrapie in goats. In this study, caprine PRNP gene was analysed in a total of 249...     

利用RNAi抑制牛骨髓间充质干细胞中朊蛋白基因（PRNP）的表达

利用RNAi技术，根据牛源PRNP基因eDNA设计3段siRNA序列和1个阴性对照序列，分别将其连接到RNA干扰载体pRNAT-U6．1／Neo上构建成shRNA载体，并将shRNA载体转染牛骨髓间充质干细胞（BMSC）；通过Real-timePCR和WesternBlotting筛选抑制效果最佳的载体；并用800mg／LG418对转染最佳载体的细胞进行药物筛选。结果显示：成功构建了3个靶向shRNA载体和1个阴性对照shRNA载体；转染后48h在荧光镜下检测各组均可观察到绿色荧光的表达；Real-timePCR和WesternBlotting结果显示，3个靶向shRNA载体在不同时间段均在一定程度上下调了PRNPmRNA的表达，抑制了朊蛋白PrP^c 的生成，得到了1个最佳干扰载体sh3；通过药物筛选出了稳定转染的细胞单克隆。本研究获得了1个有效抑制朊蛋白基因表达的shRNA载体，并筛选出稳定转染的细胞单克隆，上述结果可为抗疯牛病体细胞核移植提供供体细胞。