家蚕转录因子AP-4基因cDNA的分子克隆与生物信息学分析

AP-4(activator protein 4)是一种转录因子,在生物的生长发育中有重要作用。根据家蚕表达序列标签(EST)并利用cDNA末端快速扩增(RACE)方法克隆了家蚕AP-4(Bombyx mori activator protein4)基因,结合生物信息学方法对所获的序列进行开放阅读框、序列同源性分析,预测了AP-4蛋白的理化性质。获得的家蚕AP-4基因cDNA的全长为1621bp,其开放阅读框为996bp,编码331个氨基酸,基因由3个外显子和2个内含子组成。同源性比对表明该基因推导的氨基酸序列与棉铃虫AP-4和谷蛀虫AP-4推测的蛋白同源性分别为76%和54%,第45-99位氨基酸序列是一个典型的保守结构域。实时定量RT-PCR显示该基因在所检测家蚕的各发育时期中,除幼虫1龄和蛹期第4天无表达外,其它时期均有表达,其中幼虫3龄和4龄期的表达量较高。     

野桑蚕CYP305B1V1基因的克隆与序列分析

对野桑蚕CYP305B1V1基因进行克隆和序列分析的结果显示,野桑蚕CYP305B1V1基因的ORF为1 464nt,编码487 aa;同源性分析表明,在DNA水平上野桑蚕CYP305B1V1基因与家蚕CYP305B1基因的同源性达99%,与推导的氨基酸序列完全一致。通过和NCB I中家蚕基因组数据比对和拼接,预测野桑蚕CYP305B1V1基因结构中至少含有6个内含子,且内含子与外显子之间的连接符合GT-AT法则。     

Molecular cloning and expression analysis of a CXC chemokine gene from large yellow croaker Pseudosciaena crocea

In the present study, we report the cloning of a CXCL12 chemokine gene homologue from the large yellow croaker Pseudosciaena crocea (LycCXCL12). The complete cDNA of LycCXCL12 is 678 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 11.1 kDa. The deduced LycCXCL12 contains a 22-aa signal peptide and a 75-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 57-68% and 32-36% aa sequence identities to known CXCL12 chemokines in fish species and other vertebrates, respectively. The LycCXCL12 gene was constitutively expressed in all tissues examined although at different levels. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL12 gene expression was significantly up-regulated in gills, liver, kidney, spleen and blood at 24 h after stimulation. Time course analysis using real-time PCR showed that LycCXCL12 gene expression reached peak level in spleen and kidney at 12 h or in gills at 24 h post-induction by poly(I:C), while its expression increased to the highest level in kidney at 24h or in gills and spleen at 48 h post-induction by bacterial vaccine, indicating that LycCXCL12 gene expression was differentially regulated by poly(I:C) and bacterial vaccine.     

家蚕真核翻译起始因子3f基因结构与表达研究

在对家蚕5龄丝腺cDNA文库测序的过程中,发现一个编码家蚕翻译起始因子基因(eIF3f)的EST序列。利用电子克隆、3′RACE(3′rapid amplification of cDNA ends)方法克隆了家蚕eIF3f基因cDNA序列全长,命名为eIF3f(GenBank登录号为DQ868530)。家蚕eIF3f基因cDNA全长为1 009 bp,由870 bp的开放阅读框序列(openreading frame,ORF)、55 bp的5′端非翻译区序列(5′-UTR)和65 bp的3′端非编码区序列(3′-UTR)组成,编码289个氨基酸。氨基酸序列比较分析显示,家蚕eIF3f与其他物种的eIF3f都具有翻译起始功能所必需的JAB_MPN结构域。家蚕eIF3f基因组结构分析:该基因由5个外显子和4个内含子组成;5′调控序列存在E74A、DFD、DRI等多个潜在的转录因子结合位点,但没有TATA盒启动子序列。家蚕eIF3f基因的表达在不同组织和不同发育时期存在差异,eIF3f是一种负调控翻译起始因子。研究结果有助于进一步研究真核翻译起始因子的相关基因调控规律。     

Muscovy duck retinoic acid-induced gene I (MdRIG-I) functions in innate immunity against H9N2 avian influenza viruses (AIV) infections

Retinoic acid inducible gene I (RIG-I) is a cytosolic pattern recognition receptor that senses pathogen-associated molecular patterns (PAMPs). Muscovy duck (Cairina moschata) is a large duck different from other species of ducks, and is more susceptible to some microbial pathogens. In this study, the Muscovy duck RIG-I gene (MdRIG-I) was identified. Quantitative RT-PCR showed that MdRIG-I mRNA was widely expressed in different tissues, especially in those with mucosa. RIG-I null DF-1 cells transfected with DNA constructs encoding MdRIG-I or CARDs domain can activate IRF-3 and NF-κB to up-regulated activity of IFN-β promoter. The components of the signaling pathway downstream of RIG-I in mammalian cells including IRF-3, NF-κB, IFN-β and the IFN-stimulated genes Mx-1, PKR and MDA5 were significantly up-regulated in CARDs-overexpressing-DF-1 cells. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression in brain, spleen, lung and bursa were up-regulated in ducks challenged with H9N2 avian influenza virus (AIV), whose six internal genes were closely related to the H7N9 and H10N8 AIV. In vitro, DF-1 cells transfected with MdRIG-I plasmid can respond significantly to H9N2 AIV, evident through enhancement of IFN-β promoter activity and decreased virus titer. Altogether, these results indicated that MdRIG-I is a novel member of RLR gene family, engaging in the early stage of antiviral innate immunity.     

野桑蚕细胞色素P450基因CYP9A20V1的克隆与序列分析

研究和比较家蚕与野桑蚕细胞色素P450基因的结构和功能,可从分子生化机制上探索家蚕对农药的抗性。基于此,利用RT-PCR方法从野桑蚕中肠组织中克隆了一个P450家族基因CYP9A20V1(GenBank登录号:FJ378716)。序列分析表明,该基因的开放阅读框(ORF)为l 596 bp,编码531个氨基酸,预测分子质量约61.4 kD,等电点8.1。在线Blast分析结果表明:野桑蚕CYP9A20V1基因与家蚕CYP9A20的同源关系最近,同源性达到98.7%;与棉铃虫CYP9A12基因的同源性也达到57.1%。根据已知的P450蛋白序列结构进行比较分析,野桑蚕CYP9A20V1与家蚕CYP9A20编码氨基酸序列在底物识别位点SRS1、SRS2、SRS4、SRS6区域完全相同,在SRS3和SRS5区域的同源性分别是88.9%和94.1%,推测这2个基因具有相同的底物识别能力;野桑蚕CYP9A20V1与棉铃虫CYP9A12在SRS1、SRS4、SRS5、SRS6区域的同源性分别为47.1%、88.2%、76.5%和60%,在底物识别区域的同源性较高。初步推测野桑蚕CYP9A20V1基因可能与抗除虫菊脂类农药有关。     

cDNA cloning, genomic structure, expression analysis and biological activity of porcine A Proliferation-Inducing Ligand (APRIL)

A Proliferation-Inducing Ligand (APRIL) is a novel member of the tumor necrosis factor (TNF) superfamily. In this study, a novel cDNA has been isolated from pig spleen by homology cloning and 3'- and 5'-rapid amplification of cDNA ends (RACE) strategies and designates porcine APRIL (pAPRIL). The open reading frame (ORF) of this cDNA covers 756 bases, encoding 251 amino acids. The soluble part of pAPRIL shows 89% identity with its human counterpart at the level of the primary protein structure. The pAPRIL gene is approximately 2.1kb in size and comprises six exons and five introns. Southern blotting analysis indicated that the pAPRIL gene is a single copy gene. Real-time PCR analysis revealed that pAPRIL is constitutively expressed in various tissues. Recombinant His(6)-tagged psAPRIL protein was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. In vitro, purified recombinant psAPRIL protein co-stimulated the proliferation of porcine splenic B-cells in response to formalin-fixed Staphylococcus aureus Cowan 1 (SAC).     

家蚕类胰凝乳蛋白酶基因的发掘与表达特征分析

类胰凝乳蛋白酶(CTLP)是鳞翅目昆虫幼虫中肠中的主要蛋白酶。基于构建的家蚕中肠等组织差异表达基因的SSH文库,发掘和克隆了一条新的家蚕类胰凝乳蛋白酶基因Ctlp(GenBank登录号:JQ081296)。该基因定位于18号染色体nscaf2901,靠近端粒,有5个外显子和4个内含子,全长cDNA序列976 bp,ORF为840 bp,编码279个氨基酸残基,信号肽序列1～18 aa(分值0.985),1～20 aa和50～100 aa区域为2个由内到外的跨膜螺旋,推测蛋白质相对分子质量为29 780.10,pI为8.75,蛋白质功能域(保守区)和分子进化分析显示其为丝氨酸蛋白酶家族的类胰凝乳蛋白酶。家蚕Ctlp基因具有在5龄幼虫中肠特异性高表达和伴随进食量增加而上调表达的特征,同时也具有性别差异性和熟蚕期特异性上调表达的现象,暗示CTLP可能具有促进消化以外的功能。构建pET32a-ctlp重组表达载体,SDS-PAGE分析和Western blot鉴定结果显示,重组蛋白在原核表达系统中的表达量较低。     

猪Mx1基因真核表达载体的构建、鉴定及其表达

为获得转Mx1基因的阳性陆川猪成纤维细胞,本研究以干扰素诱导猪成纤维细胞Mx1基因表达,提取细胞总RNA,RT-PCR获得编码猪Mx1蛋白的cDNA;以pMSCV-IRES-GFP为骨架构建猪Mx1基因表达载体pMSCV-IRES-GFP-Mx1,并利用脂质体2000介导重组质粒转染陆川猪胎儿成纤维细胞,通过荧光观察和PCR检测分析结果表明Mx1蛋白基因整合进入陆川猪胎儿成纤维细胞。     

PRRSV-SCQ株结构蛋白基因核酸序列分析及其氨基酸序列推导

以重庆分离病毒株PRRSV-SCQ构建的包含SCQ株ORF2、ORF3、ORF4、ORF5、ORF6和ORF7 6个结构蛋白基因的cDNA文库质粒pRSF2、pRSF3、pRSF4、pRSF5、pRSF6和pRSF7为基础材料,通过Sanger′s双脱氧末端终止法进行核酸序列分析。测序结果表明,文库质粒DNA中分别含有PRRSV-SCQ株中为结构蛋白编码的阅读框ORF2、ORF3、ORF4、ORF5、ORF6和ORF7的完全序列,其起始密码子均为ATG,终止密码子分别为TGA、TAG、TGA、TAG、TAA和TGA。阅读框ORF2的cDNA片段长度为721 bp,ORF3的cDNA片段长度为764 bp,ORF4的cDNA片段长度为547 bp,ORF5的cDNA片段长度为621 bp,ORF6的cDNA片段长度为509 bp,ORF7的cDNA片段长度为436 bp。推导的蛋白质序列中,GP2、GP3、GP4、GP5、M和N蛋白的氨基酸数量分别为258,254,178,200,174 aa和123aa。每条多肽链都以甲硫氨酸为起始氨基酸。     

猪Mx1基因真核表达载体的构建及表达

根据GenBank中猪Mx1基因序列设计并合成引物,对Mx1基因ORF区全长1 992个碱基进行扩增。将扩增片段与pVax1载体连接,构建pVa-Mx1-ORF真核表达载体。将构建的表达载体导入BHK-21和HEK-293细胞进行表达,用RT-PCR和间接免疫荧光对表达产物进行鉴定。结果表明,所构建的pVa-Mx1-ORF表达载体在BHK-21和HEK-293细胞中成功表达。