上皮组织特异性表达N-LMP1和CR2转基因猪胚胎成纤维细胞的构建与鉴定

目的 构建上皮组织特异性表达鼻咽癌来源潜伏膜蛋白1（N-LMP1）和人补体受体2（CR2）真核表达载体,转染猪胚胎成纤维细胞并筛选整合有N-LMP1和CR2基因的细胞克隆,为构建与EBV感染相关的猪鼻咽癌模型奠定基础.方法 通过直接合成ED-L2启动子和N-LMP1,从人B淋巴细胞中的RNA经RT-PCR扩增出CR2,将上述3个片段逐个连接到真核表达载体pN1上,构建上皮组织特异性表达N-LMP1和CR2的载体pN1-ED-L2-N-LMP1-CR2;用脂质体转染猪胚胎成纤维细胞,经药物G418 筛选和PCR鉴定阳性克隆.结果 成功构建上皮组织特异性表达N-LMP1和CR2的真核表达载体pN1-ED-L2-N-LMP1-CR2,并成功整合到猪胚胎成纤维细胞的基因组中,获得了整合有目的 基因N-LMP1和CR2的猪胚胎成纤维细胞克隆.结论 获得了上皮组织特异性表达N-LMP1和CR2 的猪胚胎成纤维细胞克隆,为通过细胞核移植方法获得表达N-LMP1和CR2转基因猪提供了供体细胞.     

Image quality assessment of an iterative reconstruction algorithm applied to abdominal CT imaging

PurposeTo compare the noise and accuracy on images of the whole porcine liver acquired with iterative reconstruction (IMR, Philips Healthcare, Cleveland, OH, USA) and filtered back projection (FBP) methods.Materials and methodsWe used non-enhanced porcine liver to simulate the human liver and acquired it 100 times to obtain the average FBP value as the ground-truth. The mean and the standard deviation (“inter-scan SD”) of the pixel values on the 100 image acquisitions were calculated for FBP and for three levels of IMR (L1, L2, and L3). We also calculated the noise power spectrum (NPS) and the normalized NPS for the 100 image acquisitions.ResultsThe spatial SD for the porcine liver parenchyma on these slices was 9.92, 4.37, 3.63, and 2.30 Hounsfield units with FBP, IMR-L1, IMR-L2, and IMR-L3, respectively. The detectability of small faint features was better on single IMR than single FBP images. The inter-scan SD value for IMR-L3 images was 53% larger at the liver edges than at the liver parenchyma; it was only 10% larger on FBP images. Assessment of the normalized NPS showed that the noise on IMR images was comprised primarily of low-frequency components.ConclusionIMR images yield the same structure informations as FBP images and image accuracy is maintained. On level 3 IMR images the image noise is more strongly suppressed than on IMR images of the other levels and on FBP images.     

Viscoelastic shear properties of the corneal stroma

The cornea is a highly specialized transparent tissue which covers the front of the eye. It is a tough tissue responsible for refracting the light and protecting the sensitive internal contents of the eye. The biomechanical properties of the cornea are primarily derived from its extracellular matrix, the stroma. The majority of previous studies have used strip tensile and pressure inflation testing methods to determine material parameters of the corneal stroma. Since these techniques do not allow measurements of the shear properties, there is little information available on transverse shear modulus of the cornea. The primary objectives of the present study were to determine the viscoelastic behavior of the corneal stroma in shear and to investigate the effects of the compressive strain. A thorough knowledge of the shear properties is required for developing better material models for corneal biomechanics. In the present study, torsional shear experiments were conducted at different levels of compressive strain (0–30%) on porcine corneal buttons. First, the range of linear viscoelasticity was determined from strain sweep experiments. Then, frequency sweep experiments with a shear strain amplitude of 0.2% (which was within the region of linear viscoelasticity) were performed. The corneal stroma exhibited viscoelastic properties in shear. The shear storage modulus, G′, and shear loss modulus, G″, were reported as a function of tissue compression. It was found that although both of these parameters were dependent on frequency, shear strain amplitude, and compressive strain, the average shear storage and loss moduli varied from 2 to 8 kPa, and 0.3 to 1.2 kPa, respectively. Therefore, it can be concluded that the transverse shear modulus is of the same order of magnitude as the out-of-plane Young's modulus and is about three orders of magnitude lower than the in-plane Young's modulus.     

猪圆环病毒2型－细小病毒－伪狂犬重组病毒免疫小鼠试验研究

为了研究表达猪2型圆环病毒ORF2基因和猪细小病毒VP2基因的重组伪狂犬病毒疫苗株SA215(D)的疫苗价值,对该病毒株安全性、免疫原性、保护力及对PRV Fa株定值进行了研究。研究结果表明重组病毒SA215（D）毒力显著低于PRV Fa强毒株,可以抵御伪狂犬病强毒Fa株的攻击,并能抵御其在小鼠体内的定植;抗体检测显示SA215（D）免疫小鼠后第2周抗PRV、PCV2和PPV的抗体水平均很低,加强免疫后第4周抗体水平大幅上升,且显著高于阴性对照组,而与阳性对照组无明显差异,表明SA215（D）可诱导小鼠产生体液免疫反应。淋巴细胞增殖试验显示SA215（D）可诱导小鼠产生较强的淋巴细胞增殖反应,与亲本株SA215组相比,差异显著(0.01     

检测PCV2、PPV、PRV疫苗株与野毒株的多重PCR方法

本文建立了一种同时检测猪圆环病毒2型(PCV2)、细小病毒(PPV)、及伪狂犬病毒(PRV)疫苗株与野毒株的多重PCR方法.根据GenBank上发表的PCV2、PPV和PRV gB、gE基因序列,针对各自保守区各设计一对特异性引物,用这四对引物对同一样品中的PCV2、PPV和PRV gB、gE进行检测,结果可同时扩增出269bp(PCV2)、581bp(PPV)、372bP(PRV gB)及147bp(PRV gE)四条特异性片段.对JEV、PRRSRV、大肠杆菌和双蒸水的PCR扩增结果均为阴性;敏感性测定结果表明,该多重PCR能检出10pg PCV2、PPV和PRV gB、gE检测敏感度分别为10-6.2、10-3.8、10-5.8TCID50的模板.该方法的建立对临床上进行这三种疾病的鉴别诊断和混合感染的检测具有重要意义.     

猪轮状病毒JL94中国分离株衣壳蛋白基因的分析

根据GenBank发表的猪轮状病毒衣壳蛋白vp4基因、vp6基因和vp7基因保守序列,设计合成3对引物,分别扩增猪轮状病毒中国分离株JL94株的此3段基因并进行序列分析。结果表明,JL94株vp4基因与国外分离株CRW-8株、OSU株、Gottfried株、4F株和4S株的VP4氨基酸同源性分别为97.16％、95％、71.88％、73.45％和70.88％;JL94株vp6与CRW-8株、OSU株、Gottfried株、4F株和4S株的VP6氨基酸同源性分别为96.98％、97.48％、93.20％、97.48％和93％;JL94株vp7基因与CRW-8株、OSU株、Gottfried株、4F株和4S株的VP7氨基酸同源性分别为98％、99.90％、75.40％、84.66％和80％。可见JL94株同CRW-8株、OSU株同源性更高些;vp4和vp7基因在同型间高度保守而不同型间差距较大,vp6基因在同群和非同群间差别不是很明显。同时可以判定JL94属于A群Ⅰ亚群G5P7血清型PRV。研究猪轮状病毒中国分离株JL94株衣壳蛋白基因的特征,为研制高效抗病毒疫苗奠定基础。     

猪繁殖与呼吸综合征病毒N基因的克隆及高效表达*

本试验参照GenBank公布的Porcine reproductive and respiratory syndrome virus(PRRSV)VR2332株的核苷酸序列,设计并合成了一对引物,应用RT-PCR方法扩增出了PRRSV的核衣壳蛋白基因(N基因).在对N基因及pET32a载体双酶切后进行连接,构建了高效原核表达载体pETN.将pETN重组质粒转化BL21(DE3)宿主菌后,对培养条件及诱导表达条件(IPTG最佳浓度、作用时间)等影响表达的因素进行优化,实现了PRRSV核衣壳蛋白基因的高效表达.     

猪瘟和猪繁殖与呼吸综合征混合感染的快速检测及地域流行性分析

呼吸道和繁殖障碍疾病是猪场最常见的两种疾病,严重影响猪群的健康和正常生产,已经给世界养猪业造成了很大的经济损失[1].而且这两种疾病的病原复杂多样,不同的猪场病因可能完全不同,从而给疾病的诊断与防治带来了极大的困难.     

PMWS病猪猪圆环病毒2型全基因组序列分析

根据GenBank中发表的猪圆环病毒2型(PCV2)全基因序列,设计一对特异性引物,用PCR方法直接从5省病料中分别扩增出5个PCV2毒株的全基因组,分别命名为FujianPCV、GuangxiPCV、HainanPCV、HunanPCV和ShandongPCV.将扩增片段克隆于pMD 18-T载体,进行序列测定,结果表明,除FujianPCV株核苷酸长度为1768bp,其余四株均为1767 bp.应用DNAstar序列分析软件分析表明,本实验的5个PCV2分离株与世界上其它地区的PCV2分离株密切相关,核苷酸序列同源性达93%～98.8%,与PCV1毒株的序列同源性只有68.4%～70%.其中ORF1和ORF2所编码的氨基酸序列与国内外PCV2毒株比较,同源性也很高,分别为98.1%～99.4%和88%～99.1%.     

猪圆环病毒2型流行株的分离及其感染性克隆的构建

【目的】通过分离一株猪圆环病毒2型(PCV2)流行毒株,并构建其感染性克隆,为研究PCV2基因功能提供操作平台。【方法】通过PCR方法,从疑似患断奶仔猪多系统衰竭综合症(PMWS)的仔猪淋巴结中鉴定为猪圆环病毒(Porcine circovirus,PCV)2型阳性。把阳性病料接种PK-15细胞传代培养,在培养物中扩增出PCV2的全基因序列。对扩增出的全序列进行序列测定,并与GenBank中公布的5株广东PCV2分离株(GD-pz、GD-gj、GD-jm、GD-ss和GD-sz)进行同源性分析。通过EcoRⅠ和SalⅠ将PCV2全基因组序列克隆进pUC18载体中,获得含PCV2 GD-zq株全基因组单拷贝的重组质粒pPCV2-GD-zq,再通过SalⅠ和HindⅢ把另一个全长拷贝克隆进pPCV2-GD-zq质粒中,使PCV2 GD-zq株基因组DNA以头尾相接的双重复方式克隆进pUC18载体中,获得重组质粒pPCV2-2GD-zq。将pPCV2-2GD-zq DNA纯化和定量后转染PK-15细胞,拯救PCV2 GD-zq病毒。【结果】从PMWS感染的猪淋巴结中分离到了一株PCV2,命名为GD-zq株;序列分析结果显示,GD-zq株全基因组为1 767 bp,与GenBank中公布的5株广东PCV2分离株ORF1核苷酸一致性为97.1%-99.7%,编码氨基酸一致性为98.7%-100%;ORF2核苷酸一致性为93.2%-99.6%,编码氨基酸一致性为92.3%-99.1%;全基因一致性为96.0%-99.6%。pPCV2-2GD-zq质粒转染PK-15细胞后,其通过间接免疫荧光实验(IFA)能从转染细胞及其传代细胞中,检测到拯救出的病毒。【结论】分离了一株PCV2广东株GD-zq,成功构建了PCV2 GD-zq株的感染性克隆。