2022-2023年山东省A(H1N1)pdm09流感病毒HA、NA基因变异特征分析

目的 了解山东省2022-2023流感监测年度分离的A(H1N1)pdm09亚型流感病毒血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)基因的变异特征,为流感的防控提供科学依据。方法 从流感监测网络实验室分离的流感毒株中按地市随机选取14株A(H1N1)pdm09亚型流感毒株,以WHO推荐的当季疫苗株为参考进行全基因组测序,并采用荧光法开展神经氨酸酶抑制(neuraminidase inhibition,NI)实验以评估药物敏感性。结果 山东省2022-2023年度分离的A(H1N1)pdm09流感病毒属于6B.1A分支中的5a.2a进化簇,核苷酸序列比对分析显示,HA和NA基因与2021-2023年度北半球疫苗株A/Victoria/2570/2019的亲缘关系较为接近,同源性分别为98.5%~98.7%和98.8%~99.1%。氨基酸序列分析显示,HA蛋白有20个位点发生了氨基酸序列变异,并发现1株病毒可能发生抗原漂移,有3株病毒发生了HA蛋白糖基化位点的缺失。NA酶相关重要位点未发生变异。NI实验显示,所测流感毒株均对抗流感病毒药物敏感。结论 所监测毒株与疫苗株整体同源性很高,但氨基酸存在一定程度变异,今后有必要持续开展流感病毒基因变异特征监测以了解流感流行的风险,以及评价基因变异对流感疫苗和治疗药物效果的影响。

Screening an Na^+/H^+ Antiporter Gene from the Halophiles Colonizing in the Dagong Ancient Brine Well of Zigong City,China

[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.

新型Na^(+)/H^(+)逆向转运蛋白UPF0118的Na^(+)结合鉴定

UPF0118蛋白是一种新型Na^(+)(Li^(+))/H^(+)逆向转运蛋白,属于AI-2E超家族,目前已被划分至Na^(+)/H^(+)逆向转运蛋白亚家族,但无直接证据表明该蛋白可与Na+产生相互作用。为鉴定UPF0118蛋白是否具有Na^(+)结合功能,利用PCR技术扩增upf0118基因编码序列,通过Eco RⅠ和PstⅠ酶切位点构建至原核表达载体p ETDuet-1,转化至E. coli BL21*(DE3),对诱导后大量表达的蛋白进行Ni^(2+)亲和层析和凝胶过滤层析,利用等温滴定量热技术对纯化蛋白进行滴定。结果表明,在pH 5.5条件下,Na^(+)滴入导致蛋白溶液产生明显放热现象。在pH 5.5条件下该蛋白具有Na^(+)结合功能,证明该蛋白Na+结合功能为其功能单元与分子机制的研究奠定基础。

渗透胁迫调节基因——Na^+/H^+ Antiporter基因与植物耐盐性

Na+/H+Antiporter基因与植物耐盐性密切相关,其编码产物Na+/H+逆向转运蛋白通过Na+外排和Na+区隔化来维持植物细胞内较低的Na+水平,降低Na+的毒害,从而对植物的耐盐性起重要作用。

同时鉴别诊断H7亚型和5种NA亚型禽流感病毒GeXP检测方法的建立

目的旨在建立同时鉴别诊断H7亚型及其5种NA亚型(N2、N3、N4、N7和N9)AIV的检测方法。方法针对H7亚型HA基因、5种NA亚型NA基因和所有亚型AIV M基因的保守序列,分别设计了6对亚型特异性引物和1对AIV亚型通用检测引物,利用GeXP多基因表达和毛细管电泳分析技术,建立同时检测H7亚型和5种NA亚型AIV的GeXP高通量分型鉴别诊断方法,对其进行特异性、敏感性和临床样品检测。结果特异性结果显示该法单管可同时检测H7、N2、N3、N4、N7和N9亚型AIV,均能检出对应的亚型AIV,通用检测引物检出所有亚型AIV,与其它常见禽病病原体不存在交叉反应。敏感性结果显示该法对7种AIV目的基因(含H7、N2、N3、N4、N7、N9基因)浓度均为100拷贝/μL时,仍可同时检测出。该法对150份临床样品的检测结果与病毒分离鉴定一致。结论本研究建立的同时鉴别诊断H7亚型和5种亚型AIV GeXP高通量检测方法具有特异性强、敏感性高、快速和简便等优点,为防控AIV提供新型检测方法。

H9N2亚型禽流感病毒M1和NA基因在昆虫细胞中的表达

为通过昆虫细胞表达出H9N2亚型禽流感病毒M1和NA蛋白,并鉴定其免疫活性,利用PCR技术扩增H9N2亚型禽流感病毒M1和NA基因,以pFastBacDual为转移载体构建重组转移载体pFastBacDual-M1和pFastBacDual-NA;将阳性重组转移载体分别转化至DH10Bac感受态细胞,得到重组杆粒rBacmid-M1和rBacmid-NA;将重组杆粒分别转染Sf9昆虫细胞,获得含M1和NA基因的重组杆状病毒rBV-M1和rBV-NA;运用IFA和Western-blot鉴定M1和NA蛋白的表达情况,同时以NA蛋白为包被抗原,运用间接ELISA方法鉴定NA蛋白的反应活性。结果显示,IFA鉴定均出现特异性绿色荧光,Western-blot检测M1和NA蛋白大小分别约为28和52 ku,ELISA检测NA蛋白对抗H9N2阳性血清有很高的反应值。结果表明,M1和NA蛋白可在昆虫细胞中特异性表达且具有反应原性。本试验为进一步研发H9N2亚型禽流感诊断技术和疫苗奠定了基础。

Cloning and Identification of A New Na^+/H^+ Antiporter Gene ZmSOS1 in Maize(Zea mays L.)

[ Objective] The study aimed to clone and identify Na^＋/H^＋ antiporter genes in maize, and provided the information for characterizing the function of such genes in abiotic stress tolerance of maize. Method The in silico cloning, RT-PCR, and bioinformatics analysis were used in this study. Result By in sifico cloning, a plasma membrane Na^＋/H^＋ antiporter gene, named as ZmSOS1 （EMBL accession No. BN001309）, was cloned from maize （ Zea mays L. ）. ZmSOS1 has an open reading frame （ORF） of 3 411 bp which encoded a protein of 1 136 amino acids. By multiple sequence alignment analysis, it showed the predicated peptide of ZmSOS1 were 61% and 82% identities in amino acids to the plasma membrane Na^＋/H^＋ antiporter AtSOS1 and OsSOS1, respectively. The RT-PCR analysis revealed that ZmSOS1 could be significantly up-regulated by salt stress, which indicated ZmSOS1 might play a role in salt tolerance of maize. Conclusion ZmSOS1 is a putative plasma membrane Na^＋/H^＋ antiporter gene and may play a role in abiotic stress tolerance of maize.

Whole-genome identification and expression analysis of K^+ efflux antiporter(KEA) and Na^+/H^+ antiporter(NHX) families under abiotic stress in soybean

Sodium toxicity and potassium insufficient are important factors affecting the growth and development of soybean in saline soil. As the capacity of plants to maintain a high cytosolic, K^＋/Na^＋ ratio is the key determinant of tolerance under salt stress. The aims of the present study were to identify and analyse expression patterns of the soybean K^＋ efflux antiporter（KEA） gene and Na^＋/H^＋ antiporter（NHX） gene family, and to explore their roles under abiotic stress. As a result, 12 soybean Gm KEAs genes and 10 soybean Gm NHXs genes were identified and analyzed from soybean genome. Interestingly, the novel soybean KEA gene Glyma16g32821 which encodes 11 transmembrane domains were extremely up-regulated and remained high level until 48 h in root after the excessive potassium treatment and lack of potassium treatment, respectively. The novel soybean NHX gene Glyma09g02130 which encodes 10 transmembrane domains were extremely up-regulated and remained high level until 48 h in root with Na Cl stress. Imaging of subcellular locations of the two new Glyma16g32821-GFP and Glyma09g02130-GFP fusion proteins indicated all plasma membrane localizations of the two novel soybean genes. The 3D structures indicated that the two soybean novel proteins Glyma09g02130（NHX） and Glyma16g32821（KEA） all belong to the cation/hydrogen antiporter family.

Expression analysis of a Na^+/H^+ antiporter gene PeNHX1 from Populus euphratica

Na+/H+ antiporters play an important role in the salt tolerance of a wide variety of plants.Using the rapid amplification of cDNA ends method,a Na+/H+ antiporter gene (PeNHX1) was isolated from Populus euphratica.The deduced amino acid sequence contained 528 amino acid residues with a conserved amiloride-binding domain (77LFFIYLLPPI86) and shared more than 68% identity with that of AtNHX1 from Arabidopsis thaliana.PeNHX1 can confer resistance to Na+,as well as Li+,to (EP432) an Escherichia coli strain deficient in both nhaA and nhaB,thus proving that it is a functional Na+/H+ antiporter.PeNHX1 expression profile in EP432 reflected pH independent manner.PeNHX1 expression was regulated by salt at the transcriptional level.Meanwhile,results demonstrated that transcripts of PeNHX1 in P.euphratica calli showed a salt dependent response,and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response in P.euphratica.

Effect of salt stress on the expression of NHX-type Na^+/H^+ antiporters in Populus euphratica and P.pruinosa calli

Populus euphratica and Populuspruinosa, sister species in the Turanga Section （Salicaceae）, growing in semi-arid saline areas are known for their high salinity tolerance. In this study, by combining growth level with Na＋ and K＋ contents, the expression level of vacuolar Na＋/H＋ antiporters was investigated for NaCl-induced changes in P. euphratica and t3. pru- inosa calli. Compared to R euphratica, P. pruinosa calli grew well in 200 mM NaC1 stress from 14. to 21 days. Increasing the stressed time caused an increase in Na＋ content concomitant with a decrease in K＋ content in P. euphratica calli, whereas, with the presence of 200 mM NaCI, K＋ content has a less increase in 14 and 21 days than in 7 days which was detected in R pruinosa calli. The transcript levels of six genes coding for NHX-type Na＋/H＋ antiporters suggest that vacuolar NHX1-NHX6 antiporters play important roles in responding to salt stress in R pruinosa. Our data suggest that there exists a higher salt tolerance for P. pruinosa than P. euphratica at the cellular level, Na＋ avoidance or accumulation is observed in cellular compartments, and that expression of NHX antiporters is linked to the accumulator phenotype.

Cloning and Characterization of the Na^+/H^+ Antiport Genes from Triticum aestivum

The Na+/H+ antiport genes namedTaNHX1andTaNHX2were cloned by screening a salt_stressed wheat cDNA library using rice Na+/H+ antiport cDNA fragment as the probe. Sequencing analysis showed thatTaNHX1was 2 029 bp in length and contained a complete ORF of 1 638 bp. TheTaNHX1encodes a polypeptide of 546 amino acids with a transmembrane domain DIFFIYLLPPI.TaNHX2was 1 693 bp in length consisting of a partial ORF followed by a 3′_UTR of 808 bp. The amino acid sequence of these two genes were about 70% identical to the known NHX genes from rice, Arabidopsis and Atriplex. A RT_PCR assay showed that the level ofTaNHX1transcripts was increased and reached a steady higher level in the seedlings after 3 h treatment with 400 mmol/L NaCl.

HA和NA匹配度对H5亚型高致病性禽流感假病毒影响的研究

为研究不同H5亚型高致病性禽流感病毒(H5 HPAIV)的HA和NA匹配度对流感假病毒组装和功能的影响,本研究将5株H5 HPAIV的HA和NA基因节段分别克隆于pCMV/R载体,构建3个HA和5个NA重组质粒,两两随机组合后将HA、NA质粒与pCMV/RΔ8.9质粒、p HR-CMV-Luc质粒构成4质粒系统,利用磷酸钙法将4个质粒转染HEK293T细胞,检测各细胞培养上清液血凝效价,结果显示:有3个上清液无血凝效价,其余12个上清液的血凝效价在100 HAU/m L~400 HAU/m L,表明获得12株具有血凝效价的流感假病毒。超速离心后获得浓缩假病毒,通过western blot检测假病毒HA蛋白的表达,结果显示各假病毒均在75 ku、50 ku和25 ku有特异性条带。利用各假病毒分别感染MDCK.1细胞60 h后裂解细胞,采用荧光素酶试验检测假病毒的相对荧光素酶活性(RLA),结果显示各假病毒RLA在5.0×10~1~4.5×10~5不等。上述结果首次表明不同H5 HPAIV的HA和NA相互匹配会对假病毒的血凝效价和相对荧光酶活性(RLA)造成影响。本研究为后续流感病毒灭活疫苗制备及抗病毒药物的研发奠定基础。

14株H9N2亚型禽流感病毒分离株的HA和NA基因序列分析

为了解浙江省H9N2亚型禽流感病毒(AIV)血凝素(HA)和神经氨酸酶(NA)基因的分子特征和遗传变异情况,本试验对2012—2021年分离到的14株H9N2亚型AIV分离株进行HA和NA基因序列测定和分析。结果显示:14株分离株的HA和NA基因均属于Y280分支,HA核苷酸同源性为91.5%~99.4%,NA核苷酸同源性为89.0%~98.7%,其与疫苗株CK/SS/94、CK/F/98、CK/6/96和CK/1/00的HA核苷酸同源性为86.0%~91.1%。14株分离株HA蛋白裂解位点氨基酸组成均为RSSR↓GLF;受体结合位点主要表现为K141N、A142T、T155N、H183N和V190T突变,其中第226位和第228位全部为L和G,可与α-2,6唾液酸受体结合,具有感染人的特性;蛋白潜在糖基化位点均为6个,主要变异表现在第200~202位1个位点缺失和第295~297位1个位点增加;抗原相关位点主要表现为G82E、S137D、D145G、N159G、A160D、T192R和N193D突变,此外,有5株出现D160N突变。14株分离株NA蛋白存在6个潜在糖基化位点,其中1株发生N86H突变,缺失1个潜在糖基化位点;均出现第61~63位颈部氨基酸缺失。结果表明,浙江省H9N2亚型AIV流行毒株不断变异,与疫苗株存在抗原性差异,需加强病毒变异监控和新疫苗研发。

广东地区H9N2亚型禽流感病毒HA和NA基因生物学特征分析

【目的】监测广东地区H9N2亚型禽流感病毒(Avian Influenza Virus,AIV)的遗传变异和分子进化趋势,为我国H9N2亚型AIV的防控提供参考。【方法】对2022年广东地区3株H9N2亚型AIV的HA和NA基因进行扩增、克隆、测序,使用DNAStar、PhyloSuite软件进行序列拼接和比对,再运用MEGA、Megalign、NetNGlyc 1.0Server等软件,对其核苷酸和氨基酸序列进行遗传进化、核苷酸同源性分析,以及受体结合、蛋白活性、耐药性和糖基化等关键位点分析。【结果】系统发育树和核苷酸同源性分析结果显示,3株分离株的HA和NA基因分别属于h9.4.2.5分支和1分支,与早期毒株的核苷酸同源性分别为81.6%~91.7%和88.2%~91.2%,分别命名为h9.4.2.5c分支和1.2分支;核苷酸和氨基酸序列分析发现,HA裂解位点为PSRSSR↓GLF,受体结合位点产生I155T、H183N、A190T/V、T212I、Q226L、Q227M突变,糖基化位点产生218N非糖基化和新增313N糖基化突变;NA茎部缺失187~195位9个核苷酸(ACAGAGATA),导致缺失63~65位3个氨基酸(TEI);NA红细胞结合位点产生K/E/S368N、D369N突变,与NA蛋白活性,耐药性的相关位点E119、D151、R152、R224、E276、R292、R371均未发生突变。【结论】2022年广东地区分离的3株H9N2亚型AIV已经进化成新的亚群,部分突变可能增强了对哺乳动物的适应性,且其抗原性发生改变,但尚未获得对奥司他韦和扎那米韦等抗流感病毒药物的耐药性。

蚕豆VfNHX1基因克隆及初步功能验证

【目的】探究蚕豆(Vicia faba L.)VfNHX1基因在响应盐胁迫过程中的作用。【方法】通过3′和5′RACE方法,从蚕豆中克隆了1个Na^(+)/H^(+)逆向转运蛋白编码基因VfNHX1,并对其进行了生物信息学分析、亚细胞定位、盐胁迫下的表达分析和初步功能验证。【结果】(1)该基因全长2255 bp,CDS编码区长1629 bp,编码542个氨基酸;(2)生物信息学分析显示,该蛋白有10个跨膜区,不具有信号肽,是一个结构稳定的膜蛋白,且包含1个NHX蛋白家族特有的Na-H Exchanger结构域;亚细胞定位分析显示VfNHX1定位在液泡膜上;(3)实时荧光定量PCR(qRT-PCR)分析显示,在NaCl处理后,叶片中VfNHX1表达量呈现先降低后升高,随即又下降的变化趋势,且在12 h时达最高值;根中VfNHX1表达量先降低后升高,在48 h时表达量显著升高(P<0.01);(4)酵母生长试验结果表明,VfNHX1可以提高盐敏感酵母突变体AXT4K对高盐的耐受能力。【结论】VfNHX1基因能够响应盐胁迫,是蚕豆潜在抗盐功能基因。

Na^(+)/H^(+)交换体1在肾脏疾病中的作用

细胞内稳态的维持是细胞正常生命活动的前提。分布广泛的钠氢交换体1(Na^(+)/H^(+)exchanger 1,NHE1)是调节细胞内pH和细胞体积的重要机制,还能进一步影响细胞周期、细胞增殖和物质代谢等一系列生理过程。作为NHE家族中发现最早,研究甚广的亚型,NHE1还是一种重要的结构蛋白,锚定于细胞骨架并定位在特定质膜区域,通过独立于离子转运的机制影响细胞粘附、迁移,生存和凋亡,成为众多信号通路的交汇点。NHE1相关的研究主要集中在各种肿瘤和心血管疾病,在肾脏疾病中的作用尚不明确。目前肾脏疾病大多缺乏有效的治疗药物,尽管众多研究试图从现有治疗药物中探索促进再生或抑制凋亡等肾脏保护的新机制,但鲜有新药能登上KIDGO指南的推荐药物之列。

Involvement of Plasma Membrane Ca^(2+)/H^+ Antiporter in Cd^(2+) Tolerance

Cation exchangers （CAXs） belong to the cation/Ca2＋exchanger superfamily which have been extensively investigated in plant tonoplasts over the last decade. Recently, the roles of CAXs involved in heavy metal accumulation and tolerance in plants have been studied for phytoremediation and food security. In this mini review, we summarize the roles of the Ca2＋/H＋ antiporter in Ca2＋ signal transduction, maintaining ion homeostasis and sequestering heavy metals into the vacuole. Moreover, we present a possible role of the plasma membrane Ca2＋/H＋ antiporter in heavy metal detoxification.

Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na^+/H^+Antiport Activity of Three Malus species with Different Salt Tolerance

Salt stress contains osmotic and ionic stress, while iso-osmotic polyethylene glycol （PEG） has only osmotic stress. This study aimed to compare the different effects on the activity of H＋-ATPase, proton pump and Na＋/H＋antiport in Malus seedlings between osmotic and ionic stress. Species of salt tolerant Malus zumi, middle salt tolerant Malus xiaojinensis and salt sensitive Malus baccata were used as experimental materials. Malus seedlings were treated with NaCl and iso-osmotic PEG stress. The activity of H＋-ATPase, proton pump and Na＋/H＋antiport of plasmolemma and tonoplast in Malus seedlings were obviously increased under salt stress, and those in salt-tolerant species increased more. Under the same NaCl concentration, the activity of H＋-ATPase, proton pump and Na＋/H＋antiport of plasmolemma and tonoplast in salt-tolerant species were all obviously higher than those in salt-sensitive one. Higher Na＋/H＋antiport activity of plasmolemma and tonoplast in salt-tolerant species could help to extrude and compartmentalize sodium in roots under salt stress. The ascent rate of activity of H＋-ATPase, proton pump and Na＋/H＋antiport in Malus seedlings under the three salt concentration stress was all obviously higher than that under the iso-osmotic PEG stress. It indicated that the sodium ion effect had more stimulation on the activity of H＋-ATPase, proton pump and Na＋/H＋antiport in salt-tolerant species, and salt-tolerant species has higher capability of sodium extrusion and compartmentalization in roots and is therefore more salt tolerant.

枯草芽孢杆菌YvdSR蛋白转运Na^(+)关键残基的研究

对枯草芽孢杆菌(Bacillus subtilis strain 168)双组分基因YvdSR表达的双组分蛋白YvdSR进行功能鉴定,得出YvdSR具有转运Na^(+)/Li^(+)功能,为探究YvdSR离子转运机制,分别对两种组分作同源序列比对,从每种组分中筛选出完全保守的酸性氨基酸残基并进行氨基酸残基定点突变。结果表明,YvdS和YvdR共有的E13在同源物中完全保守且突变为丙氨酸后均失去Na^(+)(Li^(+))/H^(+)逆向转运活性,说明两种组分的E13均是蛋白质行使功能不可或缺的,YvdS的E13A经回复突变后可恢复高水平Na^(+)(Li^(+))/H^(+)逆向转运活性,YvdR的E13A经回复突变后仍失去Na^(+)(Li^(+))/H^(+)逆向转运活性,表明YvdS和YvdR在Na^(+)(Li^(+))/H^(+)逆向转运中行使不同功能。

Severe acute respiratory syndrome coronavirus 2 may cause liver injury via Na^(+)/H^(+) exchanger

The liver has many significant functions,such as detoxification,the urea cycle,gluconeogenesis,and protein synthesis.Systemic diseases,hypoxia,infections,drugs,and toxins can easily affect the liver,which is extremely sensitive to injury.Systemic infection of severe acute respiratory syndrome coronavirus 2 can cause liver damage.The primary regulator of intracellular pH in the liver is the Na+/H+exchanger(NHE).Physiologically,NHE protects hepatocytes from apoptosis by making the intracellular pH alkaline.Severe acute respiratory syndrome coronavirus 2 increases local angiotensin II levels by binding to angiotensinconverting enzyme 2.In severe cases of coronavirus disease 2019,high angiotensin II levels may cause NHE overstimulation and lipid accumulation in the liver.NHE overstimulation can lead to hepatocyte death.NHE overstimulation may trigger a cytokine storm by increasing proinflammatory cytokines in the liver.Since the release of proinflammatory cytokines such as interleukin-6 increases with NHE activation,the virus may indirectly cause an increase in fibrinogen and D-dimer levels.NHE overstimulation may cause thrombotic events and systemic damage by increasing fibrinogen levels and cytokine release.Also,NHE overstimulation causes an increase in the urea cycle while inhibiting vitamin D synthesis and gluconeogenesis in the liver.Increasing NHE3 activity leads to Na+loading,which impairs the containment and fluidity of bile acid.NHE overstimulation can change the gut microbiota composition by disrupting the structure and fluidity of bile acid,thus triggering systemic damage.Unlike other tissues,tumor necrosis factor-alpha and angiotensin II decrease NHE3 activity in the intestine.Thus,increased luminal Na+leads to diarrhea and cytokine release.Severe acute respiratory syndrome coronavirus 2-induced local and systemic damage can be improved by preventing virus-induced NHE overstimulation in the liver.