共查询到20条相似文献，搜索用时 31 毫秒
1.
本研究旨在分析热休克蛋白90(HSP90)基因的保守性,并为寻找新的HSP基因,探索HSP90的应用提供理论数据。利用CODEHOP设计简并引物,采用PCR方法克隆鲫鱼的HSP90序列,并将扩增的序列克隆到pMD18-T载体中构建克隆载体后进行测序,再将测序结果通过BLAST进行同源性比对并构建系统进化树。结果显示,鲫鱼与鲤鱼的同源性为99%;与斑马鱼的同源性为92%。本试验设计出的引物简并度较低,Tm值的修改较为灵活,产物特异性强,为研究鲫鱼的功能基因提供基础保障,并为热休克蛋白在进化上的保守性研究奠定理论基础。 相似文献
2.
为研究急性冷应激通过影响热休克蛋白含量的变化对阿勒泰羊抗寒性能、生产性能及抗病性能等的影响,试验设置常温组（15 ℃±2 ℃）与冷应激组（-25 ℃±2 ℃）,每组各5只羊,屠宰后分别采集急性冷应激组（冷应激24 h后）和常温组的心脏、肝脏、脾脏和肾脏组织。采用实时荧光定量PCR技术对HSP60、HSP70和HSP90 mRNA含量变化进行检测,并对HSP60、HSP70和HSP90基因进行同源性分析。结果显示,HSP70和HSP90基因与已有绵羊序列同源性高于99%,而HSP60基因与已有波斯金牛序列同源性高于99%;冷刺激后HSP60基因在心脏、肝脏、脾脏和肾脏组织中的表达较常温组均升高,且在肾脏中的表达量差异极显著（P<0.01）;冷刺激后HSP70基因在心脏、肝脏、脾脏和肾脏组织中的表达较常温组均升高,且在肝脏和肾脏组织中的表达量差异极显著（P<0.01）;冷刺激后HSP90基因在心脏、肝脏、脾脏和肾脏组织中的表达较常温组均升高,且在肝脏组织中表达量差异显著（P<0.05）,而在脾脏和肾脏组织中的表达量差异极显著（P<0.01）。表明急性冷应激极大程度地刺激了机体的产热机能,通过通路中的产热相关基因的相互调节使其能量代谢发生改变,从而提高细胞生存率,增强机体对环境胁迫的耐受力,能更好地适应环境温度的变化。试验结果为进一步深入研究急性冷应激对阿勒泰羊机体的影响提供一定理论依据。 相似文献
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为了表达牛环形泰勒虫（Theileria annulata）截短HSP70基因编码蛋白并研究该蛋白的结构与功能特性,本研究扩增牛环形泰勒虫HSP70目的基因并构建重组质粒pMD18-T-HSP70,选取其他种的同源HSP70蛋白序列构建系统进化树;利用生物信息学方法分析HSP70基因编码蛋白的氨基酸组成、基本理化性质、亲疏水性、跨膜区结构、信号肽、可能的磷酸化位点、亚细胞定位及蛋白的二级结构和三级结构;对重组蛋白HSP70进行蛋白互作网络分析;构建原核表达载体pET28a-HSP70,筛选诱导表达条件,镍柱纯化重组蛋白及检测反应原性。结果显示,牛环形泰勒虫HSP70蛋白序列与小泰勒虫的序列同源性较高,蛋白分子质量为42 ku,理论等电点（pI）为5.61,属于酸性亲水性蛋白,无跨膜区及信号肽;蛋白功能预测结果显示,HSP70包含32个可能的磷酸化位点,亚细胞定位分析显示该蛋白主要分布于细胞质。蛋白质二级结构中α-螺旋、β-转角、无规则卷曲、延伸链分别占39.18%、8.51%、30.41%和21.91%。蛋白互作网络构建结果显示,与HSP70相互作用的蛋白主要为HSP90家族成员,另外还有伴侣蛋白GrpE同系物,预示着HSP70可能在细胞内与HSP90形成复合体发挥作用。本试验成功构建原核表达载体,获得了大小约为48 ku的融合蛋白,以0.6 mmol/L IPTG于37 ℃诱导5 h,蛋白表达较好;点状印迹及Western blotting结果表明,表达产物可被自然感染的牛环形泰勒虫阳性血清识别,具有良好的反应原性。本试验结果为进一步探讨牛环形泰勒虫HSP70功能机制提供了理论依据。 相似文献
4.
为了对蒙古绵羊Brachyury基因DNA序列进行研究,试验主要利用PCR技术对蒙古绵羊Brachyury基因序列进行扩增并克隆,首次获得了完整的Brachyury 基因序列。利用NCBI网站上的BLAST功能将测序结果同已发表绵羊Brachyury基因序列进行比对,结果显示同源性高达99%,大部分序列完全一致。将该序列与GenBank数据库中登录的牛、人、大鼠、小鼠、山羊、猴和蝾螈等10个不同物种的Brachyury分子序列进行了比对分析。结果显示,蒙古绵羊Brachyury氨基酸序列与牛Brachyury氨基酸序列同源性最高,为96.30%;最低的则是来源于蝾螈的Brachyury氨基酸序列,仅为58.45%。遗传进化分析进一步证实蒙古绵羊与牛的Brachyury亲缘关系较近,与其他物种则遗传距离较远。此外,序列分析发现蒙古绵羊Brachyury基因高度保守,仅个别碱基发生突变。研究结果表明,蒙古绵羊Brachyury基因序列与其他脊椎动物的Brachyury基因序列也具有高度的保守性。 相似文献
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In order to study Mongolian sheep Brachyury gene DNA sequence, main purpose of this article by using the method of PCR, DNA sequence of Mongolian sheep Brachyury gene cloning and the complete Brachyury gene sequences were obtained for the first time.The sequencing with NCBI BLAST function on the site, comparing the results with the published sequences of sheep Brachyury homology was as high as 99%.We compared these sequences with ten Brachyury genes of human, mouse and other animals in GenBank database.The results showed that the most sequences were identical.Mongolian sheep Brachyury shared a 96.30% amino acid homology with Bos taurus, the highest degree of homology indicated a close genetic relationship, and the Caudata had the lowest homology 58.45%.It could be further confirmed by phylogenetic analysis.Moreover, the results showed that Mongolian sheep Brachyury gene sequence of amino acids and other vertebrates Brachyury gene sequence of amino acids were highly conservative.The research results showed that Mongolian sheep Brachyury gene sequences and other vertebrates Brachyury gene sequences were highly conservative. 相似文献
6.
为了构建猪囊尾蚴（Cysticercuscellulosae）热休克蛋白的真核表达载体,试验利用PCR技术扩增HSP35．6的基因,将其与增强型绿色荧光蛋白的基因先后连接到pVAXI真核表达载体上,得到pVAXI—HSP35．6-EGFP的重组质粒,经酶切及测序鉴定正确。采用脂质体介导的DNA转染法,将阳性重组质粒转染Vero细胞。转染48h后荧光显微镜下观察到明亮的绿色荧光,说明融合基因可在Vero细胞中高效表达。本研究为进一步研究该蛋白生物学功能奠定了基础。 相似文献
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试验旨在分析牛支原体新疆分离株oppD/F基因序列,并探究其序列特征及编码蛋白的结构和功能。根据GenBank中PG45菌株oppD/F基因序列（登录号：AF130119.1）设计1对引物,应用PCR技术扩增获得分离株oppD/F基因,将oppD/F基因片段克隆至pMD19-T载体中进行测序,在采用生物信息学方法分析其核苷酸序列的基础上对其编码蛋白的基本理化性质、疏水性、可溶性、信号肽、跨膜域、亚细胞定位、磷酸化位点、糖基化位点、二级结构、三级结构和功能等进行分析和预测。结果显示,牛支原体新疆分离株oppD/F基因序列全长1 617 bp,与Mb PG45株和Mb JF4278株的同源性均为100%,处于同一分支;该基因编码蛋白是由539个氨基酸残基组成,不存在信号肽及跨膜结构,是一种稳定的亲水性蛋白质;oppD/F蛋白存在1个AAA家族结构域,50个潜在的磷酸化位点和3个糖基化位点,其二级结构是混合型,其中α-螺旋所占比例最高（61.78%）,无规则卷曲次之（20.78%）。功能预测结果显示,oppD/F蛋白在信号传导、受体、结构蛋白、离子通道、免疫应答和胁迫应答等方面的几率均较高。本研究结果为进一步分析牛支原体oppD/F基因的功能提供了一定的理论依据。 相似文献
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试验旨在对新疆绵羊脑炎临床分离株LM90SB2单增李斯特菌llsB基因进行克隆及生物信息学分析，以期进一步完善单增李斯特菌溶血素S的功能研究。根据GenBank中单增李斯特菌F2365基因全长序列（登录号为：AE017262）设计其特异性引物，利用PCR方法对新疆分离株LM90SB2的llsB基因进行扩增，回收目的基因与pMD19-T载体连接，采用PCR、双酶切鉴定筛选阳性菌并进行测序，对所获序列进行同源性比对及遗传变异分析。结果显示，新疆分离株LM90SB2的llsB基因序列全长为876 bp，共编码291个氨基酸；LM90SB2分离株llsB基因核苷酸序列与10-0809、81-0592、81-0558、02-1792、NTSN、02-1289不同分离株同源性均为100%，与CⅡMS-PH-1、NRRLB-57603株的同源性均为99.9%，与J1816、R2-502株的同源性为44.7%~45.0%。分子进化树显示，LM90SB2菌株llsB基因与血清型为4b的菌株亲缘关系较近，聚类为同一分支。蛋白质二级结构预测表明，LM90SB2 llsB蛋白为亲水性蛋白，无信号肽，不形成跨膜结构。本试验成功克隆了LM90SB2株llsB基因，为深入探讨该基因功能提供全面的理论依据。 相似文献
9.
The study was aimed to obtain carp iNOS cDNA full-length sequence, and investigate the changes in peripheral blood leukocyte expression of iNOS in the stimulation of different mitogens.Based on EST sequences of iNOS that obtained from carp normal peripheral blood leukocytes cDNA library, full-length cDNA sequence of carp iNOS was successfully amplified by using gene library screening and rapid-amplification of cDNA 5'ends (5'-RACE) method.Carp peripheral blood leukocytes were divided into control and experimental groups and cultured.The experimental groups were stimulated by LPS (1.0 μg/mL) and ConA (1.0 μg/mL) for 4 and 12 h, respectively.And control groups were in the same cultured conditon time without mitogen stimulated.Real-time PCR was used to detect the expression of iNOS in peripheral blood leukocytes of each group.The results showed that the cDNA fragment was total 3 704 bp containing 74 bp 5'untranslated region (UTR), 246 bp 3'UTR and a 3 384 bp ORF encoding 1 127 amino acids.Sequence homology analysis showed that the amino acid sequence of carp iNOS shared 100% identity with Carassius auratus.After LPS and ConA stimulation, the iNOS expression of peripheral blood leukocytes in the experimental groups were increased, and the expression in the short time stimulation condition (4 h) was higher than the long time (LPS 12 h; ConA 24 h).In conclusion, iNOS expression of peripheral blood leukocytes was raised after LPS and ConA stimulation, suggesting that there were dynamic changes in the inflammation. 相似文献
10.
为明确沙葱萤叶甲Galeruca daurica热激蛋白70 (Heat shock protein 70,Hsp70)基因的序列结构及其系统进化关系,本研究通过PCR技术克隆沙葱萤叶甲Hsp70基因cDNA全长序列与基因组序列,并进行生物信息学与表达谱分析。结果显示,克隆获得了沙葱萤叶甲2条Hsp70基因GdHsp70-2(GenBank登录号：MZ853083)和GdHsp70-3(Genbank登录号：OK585088),基因全长分别为2 410 bp和2 242 bp,各自编码657和646个氨基酸,均含有3个保守的HSP70家族特征序列,预测蛋白三维结构均由N-端ATPase功能域和C-端底物结合功能域所组成;系统发育分析表明GdHSP70-2,GdHSP70-3分别与松墨天牛(Monochamus alternatus)MaltHSC70-1、玉米根萤叶甲(Diabrotica virgifera virgifera) DvirHSP70-2的亲缘关系最近;基因组DNA克隆获得GdHsp70-2的两段内含子序列,GdHsp70-3则不含有内含子;表达谱分析结果表明GdHsp... 相似文献
11.
DING Xuedong WANG Guohua PAN Xiangchen LIU Dongdong LUO Xuedong MA Yuan LI Maolin CUI Qi ZHANG Qijin 《中国畜牧兽医》2007,47(12):3815-3824
The purpose of this study was to investigate the variation of Orf virus (ORFV) immune related genes after infection with different species.The ORFV genomes of sheep and camel were extracted and named ORFV-Y and ORFV-LT,respectively.Based on ORFV genome sequence published in GenBank (accession No.:KF234407.1),three pairs of specific primers were designed and synthesized to amplify the B2L,F1L and VIR gene fragments of ORFV-Y and ORFV-LT,respectively,and the amplified fragments were cloned into pMD19-T vector,transformed into E.coli DH5α competent cells.The recombinant plasmid was identified,and positive clones were selected for sequencing,DNAStar software was used to analyze the homology,amino acid sequence and phylogenetic tree of 13 ORFV genome sequences published on NCBI.The results showed that the nucleotide homology of B2L,F1L and VIR genes were 92.8% to 99.2%,95.7% to 99.5% and 77.6% to 100%,respectively.After comparing the amino acid sequence between the two genomes and the reference sequence,it was found that there were obvious differences in the immune related genes between the two genomes,and F1L gene had some rules to follow.The phylogenetic analysis of B2L,F1L and VIR genes showed that ORFV-Y was closely related to the Chinese Fujian goat strain,while ORFV-LT was far from the reference strains,and was a separate branch.The results showed that ORFV had obvious difference in immune related genes between sheep and camels,it provided a reference basis for further research on the changes of ORFV gene sequences in different species and the development of vaccines for different species in the future. 相似文献
12.
试验旨在探究羊口疮病毒（Orf virus，ORFV）在感染不同物种后其免疫相关基因所表现出的差异变化。对采集到的羊和骆驼ORFV病料进行病毒基因组提取，分别命名为ORFV-Y和ORFV-LT。根据GenBank中ORFV全基因组序列（登录号：KF234407.1）设计并合成3对特异性引物，分别扩增ORFV-Y和ORFV-LT的B2L、F1L和VIR基因片段，并将扩增得到的基因片段分别克隆到pMD19-T载体上，转化大肠杆菌DH5α感受态细胞，对重组质粒进行鉴定，选取阳性克隆质粒进行测序，用DNAStar软件对所获序列进行拼接后，与NCBI上已公布的13组ORFV全基因组相应基因序列进行同源性比对、氨基酸序列分析和系统进化树构建。结果显示，两组基因组与参考序列的B2L、F1L和VIR基因核苷酸同源性分别为92.8%~99.2%、95.7%~99.5%和77.6%~100%。将两组基因组与参考序列进行氨基酸序列比对分析后发现，两组基因组之间的免疫相关基因均表现出较为明显的差异，且F1L基因有一定的规律可循。对B2L、F1L和VIR基因进行系统进化树构建分析后发现，ORFV-Y与中国福建山羊株亲缘关系较近，而ORFV-LT与参考毒株进化关系均较远，且单独成为一个分支。综上，ORFV在感染羊和骆驼时其免疫相关基因出现了较为明显的差异，为深入探究ORFV感染不同物种其基因序列发生的变化及未来针对不同物种的疫苗研制提供参考。 相似文献
13.
试验旨在获得鲤鱼诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)cDNA全长序列,并以此为基础探讨在丝裂原刺激下外周血白细胞中iNOS的表达变化。以从鲤鱼正常外周血白细胞cDNA文库中获得的iNOS的EST序列为基础,采用基因文库筛选和cDNA5'末端快速扩增技术(5'-RACE)相结合的方法,成功扩增出鲤鱼iNOScDNA全长序列,然后进行鲤鱼外周血白细胞原代培养,分成对照组和试验组,其中试验组分别为脂多糖(LPS,1.0μg/mL)刺激4、12h,刀豆蛋白A(ConA,1.0μg/mL)刺激4、24h,对照组为相同培养时间无丝裂原刺激的外周血白细胞,根据得到的iNOScDNA全长序列和鲤鱼β-actin序列分别设计特异性引物,应用实时荧光定量PCR方法检测各组外周血白细胞中iNOS在mRNA水平上的表达情况并进行分析。序列分析结果显示,最后获得的cDNA片段共3704bp,包含74bp的5'端非编码区,246bp的3'端非编码区,一个3384bp的完整的开放阅读框(ORF),共编码1127个氨基酸。序列同源性分析结果显示,该序列与鲫鱼iNOS基因同源性高达100%;实时荧光定量PCR结果显示,经LPS、ConA刺激的试验组外周血白细胞中iNOS表达量均升高,且短时间(4h)刺激的表达量高于长时间(LPS12h;ConA24h)刺激的表达量。综上所述,在LPS、ConA刺激过程中,iNOS在外周血白细胞中表达量均有上调,结果提示存在炎症反应的动态变化。 相似文献
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研究旨在对努比亚山羊脂肪和肥胖相关蛋白（fat mass and obesityassociated protein,FTO）基因进行克隆和分析,并构建其真核表达载体。取努比亚山羊背最长肌组织作为试验材料,采用RT-PCR扩增出FTO基因的编码区,测序鉴定后对得到的序列用相应的分析软件进行生物信息学分析,然后将FTO基因片段与pMD19-T载体连接后转化大肠杆菌DH5α感受态细胞,构建pMD19-T-FTO载体,测序正确的重组质粒双酶切后,连接pEGFP-N1载体构建pEGFP-N1-FTO真核表达载体,然后转染3T3-L1细胞,培养48 h后,在荧光显微镜下观察细胞表达荧光的情况。结果表明,试验成功克隆了努比亚山羊FTO基因的编码区,序列长度为1 518 bp,编码505个氨基酸,分子质量为57 142.24 u。努比亚山羊FTO基因编码区序列与NCBI上公布的山羊、牛、绵羊、猪、原鸡、小鼠的相似性分别为98.7%、96.4%、98.3%、87.2%、64.3%、81.7%,该基因系统进化树分析显示,努比亚山羊与山羊遗传距离最近,与原鸡遗传距离最远,在不同物种中具有高度保守性。努比亚山羊FTO蛋白二级和三级结构以α-螺旋和无规则卷曲为主。构建的真核表达载体pEGFP-N1-FTO转染3T3-L1细胞48 h后,在显微镜下观察到绿色荧光的表达,说明FTO基因真核表达载体构建成功。本试验构建努比亚山羊FTO基因真核表达载体,其在3T3-L1细胞中成功表达,为以后研究FTO基因与山羊脂肪代谢的相关性奠定基础。 相似文献
15.
本研究旨在克隆绵羊SUN2（Sad1 and UNC84 domain containing 2）基因并进行生物信息学分析,构建真核表达载体并检测其在A549细胞中的表达。根据绵羊SUN2基因序列（GenBank登录号：XM_015095026.2）设计特异性引物,应用RT-PCR方法扩增并克隆绵羊SUN2基因CDS序列,测序鉴定后对其进行生物信息学分析,并构建pEGFP-C1-SUN2重组质粒。利用脂质体转染方法将pEGFP-C1-SUN2重组质粒转染至A549细胞并检测其表达。结果显示,绵羊SUN2基因CDS区序列全长2 187 bp,编码728个氨基酸,理论分子质量为81.06 ku,分子式为C3593H5639N1031O1110S14,等电点（pI）为6.20,总原子数为11 387,不稳定系数为56.88,属于不稳定蛋白。绵羊SUN2基因序列与山羊、牛、猪、马、家犬、大鼠、小鼠及人的相似性分别为98.9%、97.2%、91.6%、90.6%、87.1%、82.4%、82.1%和86.1%,不同物种间基因相似性较高,进化过程中相对保守。系统进化树分析表明,绵羊SUN2基因与山羊、牛、猪、马和家犬等哺乳动物的遗传距离相对较近,与鸡的遗传距离较远。结构域及跨膜结构域预测显示,绵羊SUN2蛋白含有1个高度保守的SUN结构域,且存在跨膜结构域。信号肽预测显示其不存在信号肽位点,疏水性分析为亲水性蛋白,氨基酸序列二级结构主要为α-螺旋（51.65%）,其余为无规则卷曲（31.46%）、延伸链（12.36%）和β-转角（4.53%）。试验成功构建了绵羊SUN2基因真核表达载体pEGFP-C1-SUN2,将其转染至A549细胞并检测到蛋白表达。本试验结果为绵羊SUN2基因功能的研究提供了参考依据,为后续探究SUN2基因在绵羊肺腺瘤病中的作用奠定了基础。 相似文献
16.
This study was conducted to construct zinc finger and BTB domain containing 38 (Zbtb38) gene lentiviral overexpression plasmid vector.Firstly,the CDS sequence of mouse Zbtb38 gene was amplified by overlap extension PCR method using mouse spinal cord tissue,and the Zbtb38 CDS sequence front amplification primer 1 and the latter amplification primer 2 were designed respectively.Using the amplification products of the above two primers as template,primer 3 was designed and subjected to fusion PCR amplification,thereby obtaining the full CDS sequence of Zbtb38.Then,the obtained Zbtb38 gene CDS full sequence was ligated to the plasmid pGM-T,and the recombinant plasmid was transferred into E.coli DH5α competent cells,the positive clones were identified and verified by direct PCR and sequencing.Finally,the Zbtb38 CDS sequence was ligated into the lentiviral vector Plvx-puro by XhoⅠ and BamHⅠ double digestion to obtain the shuttle plasmid Plvx-puro-Zbtb38,which was co-transfected with lentiviral packaging plasmids to produce lentiviral particles in 293T cells.The results showed that the primer 1 amplification product contained the first 1-1 794 bp of the Zbtb38 CDS sequence,and the actual sequencing length of the amplified product was 1 772 bp.The primer 2 amplification product contained the 1 762-3 594 bp of the entire sequence of Zbtb38 CDS,and the actual sequencing length of the amplified product was 1 818 bp,indicating that the fragmented amplification target fragment was successfully obtained.The expected fusion products amplified by primer 3 and the PCR amplification products of the colonies both contained the complete sequence of Zbtb38 CDS (3 594 bp),which was compared with the CDS sequence of Zbtb38 gene in NCBI database,and the coincidence rate was 99.99%,indicating that the Zbtb38 CDS sequence was successfully amplified.The Real-time PCR results showed that the virus titer reached 3.25×108 Tu/mL,which met the experimental animal injection standard requirement,indicating that the lentiviral vector was successfully constructed.In summary,the mouse Zbtb38 gene lentiviral overexpression plasmid vector was successfully constructed in this experiment. 相似文献
17.
本研究旨在构建Zbtb38（zinc finger and BTB domain containing 38）基因慢病毒过表达质粒载体。以小鼠脊髓组织为材料,采用重叠延伸PCR方法扩增小鼠Zbtb38基因的CDS序列,分别设计Zbtb38 CDS序列前段扩增引物1和后段扩增引物2,再以上述两对引物的扩增产物作为模板,设计引物3并进行融合PCR扩增,由此得到Zbtb38 CDS全序列。将获得的Zbtb38基因CDS全序列连接到质粒pGM-T,转化大肠杆菌DH5α感受态细胞,采用直接PCR鉴定阳性克隆并进行测序验证。利用XhoⅠ和BamHⅠ双酶切方法将Zbtb38 CDS序列连接至慢病毒载体Plvx-puro,获得穿梭质粒Plvx-puro-Zbtb38,再与慢病毒包装质粒共转染293T细胞生产慢病毒颗粒。结果显示,引物1扩增产物包含Zbtb38 CDS全序列的第1-1 794 bp,扩增产物实际测序长度为1 772 bp,引物2扩增产物包含Zbtb38 CDS全序列的第1 762-3 594 bp,扩增产物实际测序长度为1 818 bp,说明分段扩增目的片段已成功。引物3扩增的预期融合产物及菌落PCR扩增产物均包含Zbtb38 CDS全序列（3 594 bp）,与NCBI数据库Zbtb38基因的CDS序列比对后的重合率达99.99%,表明Zbtb38 CDS序列扩增成功。实时荧光定量PCR检测结果显示,病毒滴度达3.25×108 Tu/mL,达到普通动物注射标准要求,提示慢病毒载体构建成功。综上所述,本试验成功构建了小鼠Zbtb38基因慢病毒过表达质粒载体。 相似文献
18.
BAO Shijun ZHU Caihong XING Xiaoyong DING Xiaoqin WEN Fengqin WU Xiaochun XUE Huiwen 《畜牧兽医学报》1956,51(11):2895-2902
To explore the biological function of NADH oxidase NOX2 from Mycoplasma bovis (Mb), according to the nox2 gene sequence of Mb strain Hubei (GenBank.CP002513.1), the primers were designed and the nox2 gene of Mb strain Lintao was amplified by PCR. Based on sequencing and gene optimization, the prokaryotic expression vector pET-nox2 was constructed, and was expressed in Escherichia coli Rosetta (DE3). Subsequently, the analysis of the enzymatic activity and the immunogenicity of recombinant proteins rMbNOX2 were completed. And then the subcellular localization of NOX2, complement-dependent bactericidal activity of anti-rMbNOX2 serum, and as well as inhibition effect of anti-rMbNOX2 serum to Mb adhering to host cells was determined. The results showed that the CDS sequence of the nox2 gene of Mb Lintao strain was 1 350 bp, and showed 99.93% homology with nox2 gene of all Mb except Mb JF4278 strain in GenBank. The result of SDS-PAGE displayed the optimized nox2 was successfully expressed in E. coli. The recombinant protein rMbNOX2 was about 67 ku. Enzyme activity analysis showed that the purified rMbNOX2 had good enzymatic activity. The results of ELISA and Western blot showed that the rMbNOX2 has excellent immunogenicity, and the Mb NOX2 distribute both in the cell membrane and cytoplasm, but it's more distributed in the cytoplasm. Complement-dependent mycoplasmacidal assay and adherence inhibition assay confirmed anti-rMbNOX2 serum has distinct complement-dependent mycoplasmacidal activity and can also effectively inhibit the adherence of Mb to host cells. The results of this study lay a foundation for further study on the biological function of Mb NOX2. 相似文献
19.
为了表达牛环形泰勒虫(Theileria annulata)截短HSP70基因编码蛋白并研究该蛋白的结构与功能特性,本研究扩增牛环形泰勒虫HSP70目的基因并构建重组质粒pMD18-T-HSP70,选取其他种的同源HSP70蛋白序列构建系统进化树;利用生物信息学方法分析HSP70基因编码蛋白的氨基酸组成、基本理化性质、亲疏水性、跨膜区结构、信号肽、可能的磷酸化位点、亚细胞定位及蛋白的二级结构和三级结构;对重组蛋白HSP70进行蛋白互作网络分析;构建原核表达载体pET28a-HSP70,筛选诱导表达条件,镍柱纯化重组蛋白及检测反应原性。结果显示,牛环形泰勒虫HSP70蛋白序列与小泰勒虫的序列同源性较高,蛋白分子质量为42 ku,理论等电点(pI)为5.61,属于酸性亲水性蛋白,无跨膜区及信号肽;蛋白功能预测结果显示,HSP70包含32个可能的磷酸化位点,亚细胞定位分析显示该蛋白主要分布于细胞质。蛋白质二级结构中α-螺旋、β-转角、无规则卷曲、延伸链分别占39.18%、8.51%、30.41%和21.91%。蛋白互作网络构建结果显示,与HSP70相互作用的蛋白主要为HSP90家族成员,另外还有伴侣蛋白GrpE同系物,预示着HSP70可能在细胞内与HSP90形成复合体发挥作用。本试验成功构建原核表达载体,获得了大小约为48 ku的融合蛋白,以0.6 mmol/L IPTG于37℃诱导5 h,蛋白表达较好;点状印迹及Western blotting结果表明,表达产物可被自然感染的牛环形泰勒虫阳性血清识别,具有良好的反应原性。本试验结果为进一步探讨牛环形泰勒虫HSP70功能机制提供了理论依据。 相似文献
20.
根据绵羊DRA基因序列和酿酒酵母表面展示载体pYD1上的多克隆位点设计特异性引物,从绵羊组织中提取总RNA并逆转录,利用PCR技术扩增得到DRA基因,克隆获得762 bp目的片段,并提交GenBank,登录号:KR422362。将该基因通过双酶切连接到表面展示载体pYD1 上,成功构建了酿酒酵母表面展示重组质粒pYD1-DRA。将DRA基因外显子2两端进行基因点突变,形成新的酶切位点,继而对外显子2设计特异性引物,对绵羊大样本进行DNA池化,并将外显子2扩增产物测序,分析其多态性获得多态位点。重组质粒双酶切得到246 bp具有多态性的外显子2,将其连接到经同样酶双酶切的表面展示重组突变载体pYD1-DRA-TB上,成功构建了酿酒酵母表面展示库。将其转化用于表面展示的酿酒酵母EBY100感受态细胞中,得到酵母转化子,挑取酵母菌的单克隆通过PCR 扩增及序列测定证实了DRA基因库已成功整合到酿酒酵母基因组中。经半乳糖诱导后,通过免疫荧光法在荧光显微镜下检测得到DRA基因库已成功展示在酵母细胞表面。 相似文献