黏病毒抗性蛋白A(myxovirus resistance protein A,MxA)是由干扰素诱导的具有重要抗乙肝病毒(hepatitis B virus,HBV)功能的蛋白质,我们前期工作发现,MxA主要依赖其中心互作结构域(central interactive domain,CID)与病毒直接相互作用...黏病毒抗性蛋白A(myxovirus resistance protein A,MxA)是由干扰素诱导的具有重要抗乙肝病毒(hepatitis B virus,HBV)功能的蛋白质,我们前期工作发现,MxA主要依赖其中心互作结构域(central interactive domain,CID)与病毒直接相互作用发挥功能,但其具体的抗病毒功能区以及功能区是否具有独立的抗病毒活性仍不清楚。本研究拟鉴定MxA蛋白上的抗乙肝病毒活性肽。首先从全长MxA构建缺失突变体ΔCID和截短体CID,以HepG2.2.15细胞为病毒模型,分别转染空载质粒、MxA、ΔCID和CID,免疫荧光法检测转染效率,Western印迹法检测质粒表达,酶联免疫法测定细胞培养液中HBsAg、HBeAg的量及荧光定量PCR法测定乙肝病毒DNA的量,评估CID段的抗乙肝病毒活性。根据CID段的晶体结构,缩短肽段长度,构建α1、α2、α3等9段肽段质粒,鉴定各段的抗乙肝病毒活性和细胞毒性(MTT法)。运用计算生物学手段——分子对接法预测MxA蛋白与病毒相互作用的模式和位点。结果显示,ΔCID、CID和9段肽段质粒的序列及表达正确,9段肽段的表达量未见显著性差异,无显著的细胞毒性。CID组和黏病毒抗性蛋白A组较对照组乙肝病毒的复制水平显著降低,CID组细胞上清中HBsAg、HBeAg及乙肝病毒DNA的量分别减少了55.57%±8.48%、68.37%±6.24%、66.67%±6.40%,P<0.01;MxA组的3个指标分别减少了61.63%±3.11%、70.77%±7.25%、73.73%±6.18%,P<0.01;ΔCID组较对照组无明显变化。9段肽段中α1组较对照组HBsAg、HBeAg及乙肝病毒DNA的量有显著下降,分别减少了48.33%±1.70%、70.67%±3.30%、68.95%±2.55%,P<0.001,表明α1对乙肝病毒具有强抑制活性。分子对接的结果显示,384~408位氨基酸是MxA蛋白与病毒互作的关键位点,该区域落在α1肽段上,验证了α1是MxA蛋白抗乙肝病毒及与乙肝病毒相互作用中的关键区段。本研究筛选并鉴定出人干扰素诱导蛋白MxA上最有效的抗乙肝病毒活性肽α1,研究结果为抗乙肝病毒多肽类新药的研发奠定了基础。展开更多
Natural resistance associated macrophage protein (Nramp) is an innate resistance protein to intracellular parasites, which is expressed plentifully in macrophage cells. Nramp has been studied in mouse, human, cattle, ...Natural resistance associated macrophage protein (Nramp) is an innate resistance protein to intracellular parasites, which is expressed plentifully in macrophage cells. Nramp has been studied in mouse, human, cattle, rainbow trout and channel catfish. However, little was known about the structure of Pagrus major Nramp. In order to get the complete sequence of Pagrus major Nramp, a pair of primer is designed according to a 200bp known sequence of Pagrus major Nramp cDNA. By the use of SMART RACE, the full Nramp of Pagrus major cDNA about 5 000 bp was obtained, including about 200 bp 5′ terminal region (UTR), complete encoding region and 3′ terminal region. There were 3 ployA signals, which showed many possibilities of cutting at 3′ terminal region. The character of Pagrus major Nramp nucleotide sequence and deduced amino acid sequence are analyzed. 12 putative transmembrane(TM) regions, a consensus transport motif (CTM), a predicted protein kinase C phosphroylation site and three predicted N-link glycosylation sites are indicated in its deduced amino acid sequence. The ’consense transport motif’ CTM is located between TM8 and TM9. Furthermore, a protein kinase C phosphroylation site and three N-link glycosylation sites were predicted. The alignment of amino acid sequences between Pagrus major Nramp cDNA and several animals is analyzed and the deduced amino acid sequence of Pagrus major Nramp had 77.8%, 83.0%, 82.3%, 80.0%, 81.1%, 60.4%, 70.3%, 58.5%, 69.5% identity with rainbow trout α(AAD20721), rainbow trout β (AAD20722), channel catfish(AF400108), fathead minnow (AAF01778), common carp (CAB60196), mouse 1(AAA39838), mouse 2(AAC42051), human 1(D50403), human 2(NP-000608), respectively. The alignment reveals high conservation in TM and CTM regions. Analysis result makes us get familiar with the structure and character of fish Nramp, furthermore, offers some information for the enhancement of immunity of fish and genetic amelioration on fish breeding.展开更多
文摘黏病毒抗性蛋白A(myxovirus resistance protein A,MxA)是由干扰素诱导的具有重要抗乙肝病毒(hepatitis B virus,HBV)功能的蛋白质,我们前期工作发现,MxA主要依赖其中心互作结构域(central interactive domain,CID)与病毒直接相互作用发挥功能,但其具体的抗病毒功能区以及功能区是否具有独立的抗病毒活性仍不清楚。本研究拟鉴定MxA蛋白上的抗乙肝病毒活性肽。首先从全长MxA构建缺失突变体ΔCID和截短体CID,以HepG2.2.15细胞为病毒模型,分别转染空载质粒、MxA、ΔCID和CID,免疫荧光法检测转染效率,Western印迹法检测质粒表达,酶联免疫法测定细胞培养液中HBsAg、HBeAg的量及荧光定量PCR法测定乙肝病毒DNA的量,评估CID段的抗乙肝病毒活性。根据CID段的晶体结构,缩短肽段长度,构建α1、α2、α3等9段肽段质粒,鉴定各段的抗乙肝病毒活性和细胞毒性(MTT法)。运用计算生物学手段——分子对接法预测MxA蛋白与病毒相互作用的模式和位点。结果显示,ΔCID、CID和9段肽段质粒的序列及表达正确,9段肽段的表达量未见显著性差异,无显著的细胞毒性。CID组和黏病毒抗性蛋白A组较对照组乙肝病毒的复制水平显著降低,CID组细胞上清中HBsAg、HBeAg及乙肝病毒DNA的量分别减少了55.57%±8.48%、68.37%±6.24%、66.67%±6.40%,P<0.01;MxA组的3个指标分别减少了61.63%±3.11%、70.77%±7.25%、73.73%±6.18%,P<0.01;ΔCID组较对照组无明显变化。9段肽段中α1组较对照组HBsAg、HBeAg及乙肝病毒DNA的量有显著下降,分别减少了48.33%±1.70%、70.67%±3.30%、68.95%±2.55%,P<0.001,表明α1对乙肝病毒具有强抑制活性。分子对接的结果显示,384~408位氨基酸是MxA蛋白与病毒互作的关键位点,该区域落在α1肽段上,验证了α1是MxA蛋白抗乙肝病毒及与乙肝病毒相互作用中的关键区段。本研究筛选并鉴定出人干扰素诱导蛋白MxA上最有效的抗乙肝病毒活性肽α1,研究结果为抗乙肝病毒多肽类新药的研发奠定了基础。
文摘Natural resistance associated macrophage protein (Nramp) is an innate resistance protein to intracellular parasites, which is expressed plentifully in macrophage cells. Nramp has been studied in mouse, human, cattle, rainbow trout and channel catfish. However, little was known about the structure of Pagrus major Nramp. In order to get the complete sequence of Pagrus major Nramp, a pair of primer is designed according to a 200bp known sequence of Pagrus major Nramp cDNA. By the use of SMART RACE, the full Nramp of Pagrus major cDNA about 5 000 bp was obtained, including about 200 bp 5′ terminal region (UTR), complete encoding region and 3′ terminal region. There were 3 ployA signals, which showed many possibilities of cutting at 3′ terminal region. The character of Pagrus major Nramp nucleotide sequence and deduced amino acid sequence are analyzed. 12 putative transmembrane(TM) regions, a consensus transport motif (CTM), a predicted protein kinase C phosphroylation site and three predicted N-link glycosylation sites are indicated in its deduced amino acid sequence. The ’consense transport motif’ CTM is located between TM8 and TM9. Furthermore, a protein kinase C phosphroylation site and three N-link glycosylation sites were predicted. The alignment of amino acid sequences between Pagrus major Nramp cDNA and several animals is analyzed and the deduced amino acid sequence of Pagrus major Nramp had 77.8%, 83.0%, 82.3%, 80.0%, 81.1%, 60.4%, 70.3%, 58.5%, 69.5% identity with rainbow trout α(AAD20721), rainbow trout β (AAD20722), channel catfish(AF400108), fathead minnow (AAF01778), common carp (CAB60196), mouse 1(AAA39838), mouse 2(AAC42051), human 1(D50403), human 2(NP-000608), respectively. The alignment reveals high conservation in TM and CTM regions. Analysis result makes us get familiar with the structure and character of fish Nramp, furthermore, offers some information for the enhancement of immunity of fish and genetic amelioration on fish breeding.
