羊源乙肝病毒与人乙肝病毒S基因序列同源性研究

为深入探讨动物乙肝与人乙肝之间的相关性，对用人乙肝诊断试剂盒检测出来的“三阳”（HBsAg、HBeAg、抗－HBc)羊源的乙肝病料进行了病毒分离和形态学鉴定，并以人乙肝病毒S基因两侧的保守区域作为模板，采用套式PCR方法扩增出羊源乙肝病毒部分基因。序列分析结果表明，羊源乙肝病毒与人乙肝病毒在S基因上核苷酸序列有一定的同源性，但差异是较大的。     

10株猪流行性腹泻病毒序列分析

为了分析2015-2018年北方地区猪流行性腹泻病毒(PEDV)遗传变异情况,通过RT-PCR分段扩增、测序,获得10株PEDV S基因序列。分析发现,10株S基因核苷酸序列同源性为96.8%～99.4%,推导编码的氨基酸序列同源性为96.5%～99.3%;与参考株核苷酸序列同源性为93.4%～99.5%,与参考株氨基酸序列同源性为92.0%～99.4%。其中,4株序列基因长度均为4 161 nt,编码1 386个氨基酸;6株序列基因长度均为4 158 nt,编码1 385个氨基酸,与目前流行的变异株和经典株CV777相比,这6株S基因推导的氨基酸序列已分别在131位、1 196位出现1个氨基酸缺失,在进化树中已形成多个小分支。由此表明,我国PEDV在流行过程中已出现较大变异。作者研究结果为监测和分析我国PEDV的变异和演化提供了有价值的基因信息数据。     

猪伪狂犬病病毒受体nectin-2基因的克隆与分子特征

为了进一步了解猪nectin-2基因的结构与功能,本研究采用生物信息学结合RT-PCR的方法从猪脑组织中克隆到了猪nectin-2基因,并对其核苷酸序列和推导的氨基酸序列进行了比较分析.猪nectin-2基因的编码区含有1440个核苷酸,编码479个氨基酸,其中信号肽由32个氨基酸组成,胞外域由330个氨基酸组成,含有2个潜在的N-糖基化位点和6个半胱氨酸残基,跨膜区由23个氨基酸组成,胞浆区由94个氨基酸组成,猪nectin-2基因与犬、马、家鼠、人、恒河猴、牛、黑猩猩的nectin-2基因核苷酸序列同源性分别为85.4%、85.7%、78.6%、82.1%、82.1%、81.9%和82.1%;推导氨基酸序列的同源性分别为84.5%、83.0%、74.7%、75.7%、76.4%、78.4%和75.5%.本试验为进一步深入研究猪伪狂犬病病毒与宿主之间的关系奠定了基础.     

猪瘟病毒石门株不同代次E2基因主要抗原编码区序列差异分析

利用RT－PCR及序列测定对猪瘟病毒石门株不同代次毒株E2基因主要抗原编码区及同一代次不同年代主要抗原编码区序列进行了分析，结果所有毒株E2基因主要抗原编码区序列呈现较高的同源性，石门株不同代次毒株E2基因主要抗原编码区核苷酸及氨基酸同源性分别为97．7％－100％，97．3％－100％；同一代次不同年代主要抗原编码区序列核苷酸及氨基酸同源性均为98．6％－100％。只有3个代次毒株发生较小的变异，核苷酸及氨基酸呈现1－3个碱基或氨基酸的变异，其他代次的毒株序列完全一致。初步证实了猪瘟病毒分子结构的遗传稳定性，说明猪瘟病毒的变异可能更多的与种群病毒的优势选择有关。     

2011—2012年四川省猪流行性腹泻病毒的遗传变异分析

为分析猪流行性腹泻病毒(PEDV)的遗传变异情况,本试验从四川省11个市19个不同地区发生仔猪腹泻疾病的猪场采集的病料中分别扩增到8个PEDV(SC-8)ORF3和部分S基因序列,将其进行比对和遗传演化分析。结果显示,SC-8的ORF3基因包含674~676个碱基,编码224个氨基酸,与GenBank中登录的国内外地方流行株的核苷酸序列同源性为95.7%~99.9%,氨基酸序列同源性为98.2%~100%,与标准毒株CV777相比,核苷酸序列和氨基酸序列除存在点突变现象外,部分序列还存在核苷酸的插入和缺失性突变;部分S基因扩增长度为981bp,编码327个氨基酸,与国内外参考毒株的核苷酸序列同源性为93.2%~99.5%,氨基酸序列同源性为93.5%~100%,与标准毒株CV777相比,四川-绵阳(SCMY)分离株在92~93nt之间存在1个核苷酸插入,在2~29aa之间也有18个氨基酸发生了突变;系统进化树分析表明,8株PEDV ORF3基因均与野毒株亲缘关系较近,而与弱毒疫苗株亲缘关系较远;SC-8的部分S基因均与国内地方流行毒株亲缘关系较近,而与国外分离株的亲缘关系较远。     

口蹄疫病毒受体牛源β1亚基基因的分子特征

从口蹄疫病毒试验感染康复牛肺组织中克隆了β1亚基基因,并对其核苷酸和推导氨基酸序列进行了比较分析.结果显示,牛β1亚基基因的编码区含有2 397个核苷酸,编码798个氨基酸,含有10个潜在的糖基化位点,其中信号肽由20个氨基酸组成,胞外域由708个氨基酸组成,跨膜区由29个氨基酸组成,胞浆域由41个氨基酸组成.与GenBank中的牛β1基因的同源性达99.5%,从起始密码子开始共有12个碱基发生了变化,引起第217、247、268、281、321、691、709位的氨基酸改变,分别由F、S、W、V、H、Y、V变为S、P、R、G、Y、C、G.牛β1基因与猪、猩猩、猫、犬、人、小鼠和鸡的β1基因核苷酸序列同源性分别为93.8%、89.3%、91.6%、90.2%、89.6%、85.4%、75.6%,推导氨基酸序列同源性分别为98.2%、93.7%、97.5%、96.7%、94.2%、93.2%、84.1%.牛与猪β1亚基同源性最高.     

猪伪狂犬病病毒受体nectin-2基因的克隆与分子特征

为了进一步了解猪nectin-2基因的结构与功能,本研究采用生物信息学结合RT-PCR的方法从猪脑组织中克隆到了猪nectin-2基因,并对其核苷酸序列和推导的氨基酸序列进行了比较分析。猪nectin-2基因的编码区含有1440个核苷酸,编码479个氨基酸,其中信号肽由32个氨基酸组成,胞外域由330个氨基酸组成,含有2个潜在的N-糖基化位点和6个半胱氨酸残基,跨膜区由23个氨基酸组成,胞浆区由94个氨基酸组成,猪nectin-2基因与犬、马、家鼠、人、恒河猴、牛、黑猩猩的nectin-2基因核苷酸序列同源性分别为85.4%、85.7%、78.6%、82.1%、82.1%、81.9%和82.1%;推导氨基酸序列的同源性分别为84.5%、83.0%、74.7%、75.7%、76.4%、78.4%和75.5%。本试验为进一步深入研究猪伪狂犬病病毒与宿主之间的关系奠定了基础。     

11株禽腺病毒遗传变异分析

为了分析2019年禽腺病毒(FAdV)遗传变异情况,通过PCR分段扩增、测序,获得11株FAdV Hexon基因序列。经分析发现,11株腺病毒Hexon基因高变区核苷酸序列同源性为66.1%～100.0%,推导编码的氨基酸序列同源性为56.5%～100.0%;与参考株核苷酸序列同源性为65.4%～100%,与参考株氨基酸序列同源性为56.5%～100.0%。其中9株序列基因长度均为738 nt,编码246个氨基酸;GX-1901001毒株序列基因长度均为750 nt,编码250个氨基酸;HB-1911039毒株序列基因长度均为756 nt,编码252个氨基酸。与目前流行株相比,本试验获得的11株腺病毒Hexon基因存在不同程度的变异或缺失,在进化树中已形成多个小分支。表明我国FAdV在流行过程中已出现变异。本试验结果为监测和分析我国FAdV的变异和演化提供了有价值的基因信息数据。     

新型鸭呼肠孤病毒S组基因克隆及序列分析

采用RT-PCR方法对NDRV-QY分离株的S组基因编码区进行RT-PCR扩增、克隆及序列测定。结果表明,QY株S组基因包含的ORF大小分别为1 517 bp、1 251 bp、1 104 bp和1 104 bp。将QY株的S组基因各节段编码区核苷酸序列及其推导氨基酸序列与正呼肠孤病毒属的呼肠孤病毒的相似性比较和遗传进化分析结果表明,QY株与ARV、TRV、MDRV、NDRV均有不同程度的同源性,其中与NDRV的同源性最高,核苷酸序列和氨基酸序列同源性均达90%以上。QY株与NDRV、MDRV、ARV同在正呼肠孤病毒的第Ⅱ亚群,与NDRV处在同一个分支,与MDRV和ARV处在不同分支,建议将新型鸭呼肠孤病毒分类在正呼肠孤病毒属的第Ⅱ亚群当中。     

贵阳地区犬细小病毒NS1基因的克隆及序列分析

为了解贵阳地区犬细小病毒(CPV)非结构蛋白NS1的遗传变异进化状况,试验采用PCR检测、分子克隆、测序及生物信息学软件对分离自贵阳的8株CPV的NS1基因进行序列分析。结果表明:成功扩增并克隆测序得到8株CPV NS1基因,全长为2 007 bp,共编码668个氨基酸;分离株间NS1基因核苷酸序列的同源性为98.4%～99.8%,与10株犬类参考毒株CPV NS1基因序列的同源性为98.3%～99.6%,与其他11株非犬类参考毒株NS1基因核苷酸的同源性为39.4%～99.1%。说明CPV NS1基因具有一定的种属特异性,其亲缘关系越近同源性越高。     

鸡源类人乙肝病毒与人乙肝病毒S基因序列分析

为揭示动物乙肝病毒与人乙肝病毒之间的相关性研究打下基础,以人乙肝病毒Ｓ基因两侧的保守区域作为模板,采用套式ＰＣＲ方法,成功地扩增出鸡源类人乙肝病毒部分基因（１．２ｋｂ）。序列分析结果表明,鸡源类人乙肝病毒与人乙肝病毒Ｓ基因似有一定的同源性,但在保守区域（Ｓ区）内存在较大的核苷酸序列差异。     

鸡传染性支气管炎病毒KIBVXJ株S1基因的克隆及序列分析

根据已发表的鸡传染性支气管炎病毒S1基因,设计并合成了一对引物,经RT-PCR扩增后获得全长约为1700bp的核苷酸序列。序列分析表明,S1基因的最大开放阅读框位于12位～1685位碱基之间,编码557个氨基酸;KIBVXJ株S1基因序列在19位～292位点的氨基酸区域内有较多的氨基酸置换、插入和与缺失现象;S1的裂解位点的序列为HRRRR,具有我国地方流行株的特征。KIBVXJ株的S1基因与国内外疫苗株的同源性分析表明,核苷酸同源率为73.8%～74.2%,氨基酸同源率为74.9%～76.0%。遗传进化分析表明,KIBVXJ株与国内外的主要疫苗株亲缘关系最远,与国内近年来分离的A2株、LX4株和QXIBV株的亲缘关系最近。     

Characterization of canine caspase-3

The canine caspase-3 gene was cloned and sequenced. The canine caspase-3 cDNA clone was 1473 base pairs in length and encoded 277 amino acids. The predicted amino acid sequence showed 88.4%, 88.0%, 85.9%, 65.9% and 60.6% homology with that of human, pig, mouse, chicken and zebra fish caspase-3, respectively. The caspase-3 mRNA was confirmed to express in skin, lymph node, bone marrow, small intestine and lung from a healthy dog by RT-PCR analysis.     

Canine CD20 gene

The human CD20 antigen, a 35kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.     

cDNA and protein sequence for canine arachidonate 5‐lipoxygenase: stuctural modelling and species differences

5‐lipoxygense is an important enzyme in the genesis of leukotrienes through arachidonic acid metabolism to 5‐hydroxyecosatetranoic acid (5)‐HETE. Not only is this enzyme important in producing the chemotactic leukotrienes, but it may also be important in producing autocrine lipid signaling molecules (5‐oxo‐ETE, LTB4) for cellular proliferation. mRNA was isolated in our laboratory from canine leukocytes, and transitional carcinoma cells using the Qiagen whole blood mRNA extraction kit. An internal cDNA fragment was cloned using primers designed according to homologous regions in human and rat 5‐LOX. Once an internal sequence was determined both 5′ and 3’ RACE reactions were performed according to the manufacturer's suggestions using the Ambion RACE kit. The 5′ region just upstream of the ATG start site to 300 bp beyond the terminal codon was sequenced. Protein structural modeling was performed through threading of the presumed sequence onto the known rabbit 15‐LOX using Swiss PDB viewer. Based on the known human sequence and structural modeling there are three areas of interest in the canine structure. (i) In the catalytic domain there are a number of charged amino acid differences in an external alpha helix at amino acids 645‐660, which may confer structural differences between human and canine 5‐LOX. (ii) An important phosphorylation site at ser 532 is actually a threonine in canine 5‐LOX. (iii) All mammalian 5‐lipoxygense enzymes are between 673–674 amino acids; however, the canine 5‐LOX sequence contains two additional amino acids, a leucine and valine at residues 379 and 380 respectively, making canine 5‐LOX 676 amino acids rather than the 674 identified in human, mouse and bovine.     

Molecular cloning of canine Mcl-1 gene and its expression in tumor cell lines

The canine Mcl-1 gene was cloned and sequenced. Canine Mcl-1 clone was 2694 base pairs in length and encoded 350 amino acids. The predicted amino acid sequence was 87.7%, 77.1% and 75.7% homologous to predicted human, mouse and rat Mcl-1, respectively. RT-PCR analysis revealed that canine Mcl-1 mRNA was expressed in PBMCs (peripheral blood mononuclear cells), bone marrow cells, MDCK (Madin-Darby canine kidney) and GL-1 (canine B cell leukemia) whereas undetectable in CL-1 (canine T cell lymphoma) cell line.     

9株鸡毒支原体29 Ku多肽基因的克隆与序列分析

根据已发表的鸡毒支原体(MG)S6株29Ku多肽基因序列设计了1对引物,以9株(广西分离株5株、标准株4株)DNA为模板进行PCR扩增,均得到802bp的特异性片段,将9株MG PCR产物纯化后克隆到pMD18-T载体上,得到重组质粒.重组质粒经PCR法和EcorⅠ、SalⅠ双酶切等方法鉴定后,测定了9株29 Ku多肽基因序列,并在基因库中S6标准株的29 Ku多肽基因序列进行分析比较.结果表明,5株分离株与5株标准株29Ku多肽基因核苷酸序列同源性分别为94.4%～99.9%,推导的氨基酸同源性分别为89.7%～99.2%.从各毒株的进化分析表明,5个分离株与标准强毒株S6、A5969、K1501和PG31强毒株间遗传距离较近,而5个分离株与标准株F疫苗株间遗传距离则较远.     

细毛羊黑素皮质激素受体1(MC1R)基因与毛色表型的研究

为了研究黑素皮质激素受体1(MC1R)基因与黑色素生成的关系及该基因突变导致毛色发生改变的机理,研究采用RT-PCR和PCR等技术克隆得到了白色东北细毛羊MC1R基因长1 093 bp的cDNA片段,编码364个氨基酸,其中包括70个氨基酸信号肽和294个氨基酸的成熟肽,具有7个跨膜结构域。结果表明:细毛羊cDNA与绵羊、牛、马、人、小鼠、犬等同源性均大于91%,氨基酸序列与其他动物的同源性高于89%;除绵羊外与其他动物相比有5处发生突变,且这5处突变均位于MC1R蛋白跨膜结构区;而细毛羊和绵羊之间只有1处发生突变,由K→M,同源性达98.97%。说明细毛羊与绵羊的亲缘关系最近。     

Prokaryotic Expression and Bioinformatics Analysis of VP2 Gene of Infectious Bursal Disease Virus C4 Strain

This study was aimed to investigate the relationship between the virulence characteristics of infectious bursal disease virus(IBDV) C4 strain and its VP2 amino acid sequence. The RNA of IBDV C4 strain was extracted,and its VP2 gene was amplified by RT-PCR.VP2 nucleotide sequences and deduced amino acids of different virulent IBDV strains were compared. At the same time, prokaryotic expression vector pET-32a(+) was used to express the VP2 gene. The expression of recombinant VP2 protein was detected by SDS-PAGE and Western blotting. The results showed that the VP2 gene of IBDV C4 strain belonged to the very virulent infectious bursal disease virus (vvIBDV) in evolutionary relationship, the VP2 nucleotides homology between IBDV C4 strain and other vvIBDV strains were 98.1% to 98.7%, and there were no mutations in S-W-S-A-S-G-S (326-332 amino acids) and 222(A), 256(I), 294(I) and 299(S). The VP2 amino acid sequence of IBDV C4 strain was consistent with the characteristics of other vvIBDV strains. However, there were three differences amino acids sites at 201(D/G), 281(G/R) and 313(V/A) between the amino acids of the C4 strain and the very virulent strain UK661. And the change of 281(R) was in the small hydrophilic region of 279 to 290, which was related to the antigenicity of the virus; The recombinant VP2 protein molecular weight expressed in Escherichia coli BL21 was about 67 ku. This study provided a basis for further research on antigenic changes resulting from amino acid variation of 201(G), 281 (R) and 313(A). These results indicated that the VP2 gene of the IBDV C4 strain was consistent with the major characteristics of the vvIBDV strain VP2 gene. The difference of three amino acid sites in the vvIBDV strain C4 might be related to the evolution of virulence of IBDV strain in China.