Molecular Cloning and Expression of IL2 cDNA from the Tibet Pig

Interleukin-2 is a vital cytokine secreted by activated T lymphocytes, and plays important role in the regulation of cellular and humoral immunity of animals. In our experiment, IL2 cDNA of the Tibet Pig was first cloned by RT-PCR from ConA-stimulated lymphocytes in the blood and subcloned into pMD-18 T vector, which then was identified with endonuclease restriction. The sequencing result showed that Tibet pig IL-2 (TPIL-2) cDNA was 503 bp long (ORF was 465 bp) (Genbank accession number: AY 294018). The recombinant prokaryotic and eukaryotic expression plasmids of the cDNA were then constructed to analyse the ability to stimulate the proliferation of porcine lymphocytes in vitro. The recombinant porcine IL-2 expressed in the prokaryotic cells was found to be of 43 kDa molecular mass, which was consistent with a 17.4 kDa protein deduced from the IL-2 cDNA sequence (glutathione S-transferase molecular mass is 26 kDa); the recombinant protein in eukaryotic cells was confirmed by use of specific rabbit anti-porcine IL-2 serum in an ELISA. The bioactivity of TPIL-2 was detected through MTT colorimetry by stimulating the proliferation of pig ConA-stimulated blasts in vitro. The results indicate that the TPIL-2 significantly promoted the proliferation of ConA-stimulated blasts of pig. This confirms that IL-2 cDNA of the Tibet pig was successfully cloned and expressed in prokaryotic and eukaryotic cells, which lays the foundation for the the preparation of specific recombinant IL-2 protein and development of novel immune adjuvants to raise the immunity of pigs against various infectious pathogens and increase the immunoprotective efficacy of vaccines.     

Effect of dipeptide on intestinal peptide transporter 1 gene expression: An evaluation using primary cultured chicken intestinal epithelial cells

Peptide transporter 1 (PepT1) is a transporter responsible for absorbing dipeptide and tripeptide in enterocytes and is upregulated by dipeptide in mammals. It has not been certain whether intestinal PepT1 expression is responsive to dipeptides in chickens because of the lack of in vitro study using the cultured enterocytes. This study established a primary culture model of chicken intestinal epithelial cells (IECs) in two-dimensional monolayer culture using collagen gel by which the response of chicken PepT1 gene expression to dipeptide stimuli was evaluated. The cultured chicken IECs showed the epithelial-like morphology attached in a patch-manner and exhibited positive expression of cytokeratin and epithelial cadherin, specific marker proteins of epithelial cells. Moreover, the chicken IECs exhibited the gene expression of intestinal cell type-specific marker, villin1, mucin 2, and chromogranin A, suggesting that the cultured IECs were composed of enterocytes as well as goblet and enteroendocrine cells. PepT1 gene expression was significantly upregulated by synthetic dipeptide, glycyl-l-glutamine, in the cultured IECs. From the results, we herein suggested that dipeptide is a factor upregulating PepT1 gene expression in chicken IECs.     

Effect of dexmedetomidine and xylazine followed by MK-467 on gastrointestinal microperfusion in anaesthetized horses

Objective

To compare the effects of MK-467 during isoflurane anaesthesia combined with xylazine or dexmedetomidine on global and gastrointestinal perfusion parameters.

中国美利奴羊TLR2基因多态性及其与布鲁氏菌病的相关性分析

试验旨在研究Toll样受体2（Toll-like receptor,TLR2）基因的多态性及其与中国美利奴羊布鲁氏菌病易感性的相关性。利用生物信息学方法对NCBI上公布的绵羊TLR2基因序列进行比对,选出多态位点丰富的片段进行扩增,运用PCR-SSCP的方法对206个中国美利奴布鲁氏菌病阴性样本和80个中国美利奴羊布鲁氏菌病阳性样本进行TLR2基因的多态性检测,然后对不同等位基因的PCR产物进行测序,确定该基因的多态性位点,经卡方检验分析每个SNP位点的等位基因频率、基因型频率及其多态性与布鲁氏菌病易感性的相关性,利用生物信息学软件分析RNA二级结构及蛋白质的二级结构。结果表明,在279 bp的序列中共检测到3个SNPs,分别为：C1731T、G1737C和G1749T,均未引起对应氨基酸的改变,属于无义突变。这些位点在病例组和对照组之间的等位基因频率及基因型频率均不存在显著差异（P>0.05）。各突变位点均能引起RNA二级结构和最小自由能的改变,而蛋白质的二级结构均未改变。由此得出,中国美利奴羊TLR2基因的3个SNPs位点（C1731T、G1737C和G1749T）与中国美利奴羊布鲁氏菌病易感性无相关性。     

猪δ冠状病毒胶体金试纸条检测方法的建立

为建立一种方便、快捷、实用的检测猪δ 冠状病毒(PDCoV)方法,本研究利用原核表达猪δ 冠状病毒N 蛋白,以纯化的N 蛋白免疫BALB/C小鼠,利用杂交瘤细胞技术研制2株分泌PDCoV N 蛋白单克隆抗体的杂交瘤细胞株,分别命名为E8、A10。2株杂交瘤细胞分泌单克隆抗体敏感性及特异性良好,E8亚类为IgG2a,A10亚类为IgG1。以E8作为金标抗体,A10作为检测抗体,兔抗鼠作为质控线抗体,制备猪δ 冠状病毒胶体金检测试纸条,检测猪δ 冠状病毒敏感性为100TCID50,检测猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪瘟病毒、猪圆环病毒、猪δ 冠状病毒阴性对照均为阴性。本研究建立的胶体金试纸条检测方法敏感性及特异性良好。该检测方法的建立,为猪δ 冠状病毒(PDCoV)流行病学调查提供一种方法,同时为规模化养殖集团及基层兽医组织提供一种检测猪δ 冠状病毒产品。     

四川猪2型链球菌病的PCR检测

2005年7月中下旬,四川贵阳等地人、猪陆续发生一种发病惠、死亡快、高度散发的疾病,从病死猪组织中分离出19株链球菌,设计合成3对引物,分别对分离菌株进行了猪链球菌16SrRNA、CPS2J、MRP基因片段扩增,有15株菌3个基因的扩增结果均为阳性,表明此次疲情的病原为猪2型链球菌。     

Immunopathology of <Emphasis Type="Italic">Dirofilaria immitis</Emphasis> Infection

Heartworm disease caused by Dirofilaria immitis affects canine and feline hosts, with infections occasionally being reported in humans. Studies have shown that both dirofilarial antigens and those derived from its bacterial endosymbiont Wolbachia, interact with the host organism during canine, feline and human infections and participate in the development of the pathology and in the regulation of the host’s immune response. Both innate and acquired immune responses are observed and the development of the acquired response may depend on the host and, or on its parasitological status. This review aims at illustrating current research on the role of both D. immitis and Wolbachia, in the immunology and immunopathology of dirofilariosis.     

猪细小病毒检测技术研究进展

近年来,随着猪细小病毒分子生物学的研究进一步深入,各种新型疫苗的成功研制,为猪细小病毒病的防制带来希望。但目前为止猪细小病毒的检测技术以传统方法为主,大量研究发现猪细小病毒常与其他病原微生物混合感染,为了根除本病,必须提高诊断和防疫水平。文章就该病诊断方法的研究进展做一综述。     

山羊ACSS2基因的克隆、序列分析及组织表达研究

本研究旨在对山羊乙酰辅酶A合成酶2（acetyl-CoA synthetase 2,ACSS2）基因进行克隆和生物信息学分析,并检测其在山羊不同泌乳时期乳腺组织中的表达量变化。以山羊乳腺组织RNA为模板,采用RT-PCR方法扩增并克隆山羊ACSS2基因完整CDS区序列,对测序结果进行生物信息学分析,并对ACSS2基因在山羊不同泌乳时期乳腺组织中的表达量进行分析。结果显示,山羊ACSS2基因CDS区序列长2 106 bp,编码701个氨基酸;山羊ACSS2基因与牛、马、人、犬、猪、小鼠和鸡的同源性分别为97.8%、92.0%、91.3%、91.3%、91.1%、88.1%和73.3%。蛋白理化性质分析结果表明,ACSS2蛋白分子质量为78.72 ku,理论等电点为6.03,属于酸性蛋白;跨膜结构和信号肽分析表明,ACSS2蛋白不含跨膜结构和信号肽;结构域分析表明,该蛋白含有1个乙酰辅酶A合成酶N端结构域。亚细胞定位分析结果表明,该蛋白主要分布在内质网（44.4%）、线粒体（33.3%）、细胞质（11.1%）和细胞核（11.1%）中。蛋白质结构预测发现ACSS2蛋白含有α-螺旋（29.10%）、延伸链（21.54%）、β-转角（9.84%）及无规则卷曲（39.52%）。实时荧光定量PCR分析结果表明,ACSS2基因在不同泌乳时期均有表达,其中在泌乳中期表达量最高,在干奶期表达量最低。本试验结果为进一步研究山羊ACSS2基因在脂质代谢过程中的功能及转录调控机制提供了参考。     

The use of quantitative PCR to detect Felis catus papillomavirus type 2 DNA from a high proportion of queens and their kittens

Squamous cell carcinomas are common feline skin cancers that have been associated with infection with Felis catus papillomavirus type 2 (FcaPV-2). Currently, little is known about the epidemiology of FcaPV-2 infection. The aim of this study was to develop a real-time PCR assay to quantify FcaPV-2 DNA in plucked hairs and skin swabs from 11 healthy breeding queens and their kittens. Samples were taken prior to kittening and then 2, 7 and 28 days after kittening to determine the age at which the kittens were first exposed to the virus. FcaPV-2 DNA was amplified from all of the queens and from 91% of the kittens at 2 days of age. There was a wide range in the quantity of FcaPV-2 DNA detected, from 1 to 92,520 copies per swab, and from 0.01 to 234 copies per copy of reference gene DNA in the hair plucks. The quantity of FcaPV-2 DNA detected in samples collected from the kittens was strongly correlated to that of their respective queens and the mean viral DNA load was similar for cats within a household but varied significantly between households. This is the first time that quantitative PCR has been used to detect FcaPV-2 DNA and the results suggest that the virus is ubiquitous but there is a wide variation of viral DNA loads. Kittens appear to be exposed to FcaPV-2 early in life, presumably from direct contact with their queen. These results are important when determining if FcaPV-2 infection of cats is preventable.