Molecular Characteristics and Phylogenetic Analysis of N Gene of Human Derived Rabies Virus

Objective To investigate the relationship between the molecular characteristics and phylogenetic evolution of rabies N gene.Methods Saliva samples were collected from rabies cases,and RT-PCR was used to amplify the N gene of rabies virus with the specific primers.The amplifying product of RT-PCR was cloned to pUCm-T vector and transformed into E.coli XL1-Blue and then the blue-white selection,PCR screening and gene sequencing were carried out to identify the positive clones.Finally,ExPASy and other bioinformatics softwares were used to analyze and predict the structure and biological characteristics of the N genome.Results The amplification product of RT-PCR was 1 353 bp,the recombinant plasmid pUCm-T/N was constructed,the whole length of the N gene open reading frame was composed of 1 353 nucleotide residues to code 450 amino acids （20 kinds）,the accession number submitted to the Genbank was HM756692,its sequence homology of nucleotides and amino acids compared with the vaccine strain CTN-1-V was 90% and 99% respectively.The evolutionary analysis showed that the isolated strain belonged to genotype I with certain geographic regionality.Conclusion The characteristics investigation and bioinformatics analysis of Hunan0806 N gene will provide fundament data to reveal the significance of the N gene characteristics for rabies epidemiology and its prevention control.

Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX

The structure genes spike(S) ,nucleocapsid(N) ,membrane(M) ,small membrane(sM) of a porcine epidemic diarrhea virus(PEDV) strain DX isolated in Gansu province,North-west of China,were cloned,sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S,sM,M and N genes open reading frame(ORF) of DX were 4 152,231,681 and 1 326 bases long respectively. There were transcription regulatory sequences(TRSs) upstream of the initiator ATG of the S,N and M genes. The amino acids sequences of S,M and N contained 30,3 and 7 potential asparagine(N) -linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06,JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China,and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.

Sequence Analyses on N Gene of Rabies Viruses Isolated from Guangdong Province

[ Objective] To find the dominant rabies virus strains that are epidemic in Guangdong Province and thus to better prevent and control rabies. [ Method] Canine brains and salivary glands collected from several regions of Guangdong Province were detected by direct imrnunofluorescence assay and nested RT-PCR. The isolates were further identified by intracranial inoculation in suckling mice. The N gene was amplified by RTPCR and the sequences were aligned with those of prevalent strains in Guangxi region and vaccine strains. [ Result] Two strains were isolated from specimens collected from Guangdong Province. Their N gene sequences had a high similarity of 97.9% -99.4% to those of Guangxi strains and also had a similarity of 87.7% -88.1% to those of vaccine strains of rabies virus. [ Conclusion] Apparently healthy dogs may carry rabies virus, and the prevalence of rabies virus shows obvious regional differences.

RIBOSOMAL PROTEIN S12 GENE MUTATION AFFECTING THE EXPRESSION OF BACTERIOPHAGE LAMBDA N GENE

Previous results in this laboratory indicated that in ribosomal protein S12 strepto-mycin-dependent mutants of Bacillus subtilis, the burst size of bacteriophage φ105 was decreased, and the protein synthesis was inhibited, while the DNA and

猪传染性胃肠炎病毒S、N基因DNA疫苗载体构建及其免疫原性

【目的】构建猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)S、N基因的DNA疫苗载体,并进行免疫原性试验,为猪传染性胃肠炎(transmissible gastroenteritis,TGE)的防控和DNA疫苗研究提供技术支撑和基础数据。【方法】扩增S基因的A位点、D位点和N基因,并将N基因(单独)、A位点和D位点(融合)克隆至pCDNA3.1-His-C构建重组疫苗载体,运用生物信息学软件预测分析S(A-D)蛋白、N蛋白二级结构组成、三级构像、亚细胞定位和优势B细胞抗原表位。将构建成功的重组载体分别转染至PK-15细胞进行间接免疫荧光试验,运用共聚焦检测重组蛋白的表达分布情况。将重组疫苗载体单独或联合免疫小鼠,运用间接ELISA检测IgG抗体水平。【结果】扩增出S基因的A位点、D位点和N基因,大小分别为498、606、1149 bp。构建了A位点与D位点(融合)、N基因(单独)的DNA疫苗重组载体p-S(A-D)-His和p-N-His。生物信息学软件预测分析发现TGEV感染宿主细胞时N蛋白主要定位于细胞核和线粒体,S(A-D)蛋白主要定位于细胞质和线粒体,S(A-D)蛋白具有7个优势B细胞抗原表位,N蛋白具有8个优势B细胞抗原表位。重组载体p-S(A-D)-His和p-N-His均在PK-15细胞内成功表达,且S(A-D)-His和N-His在PK-15细胞核和细胞质中均有分布。重组疫苗载体免疫小鼠后,免疫效果由高至低依次为p-N-His>p-S(A-D)-His+p-N-His>p-S(A-D)-His。【结论】本研究构建了TGEV的S、N基因的DNA疫苗载体,免疫小鼠后均产生了较强的特异性抗体,为TGEV的核酸疫苗的研制提供了基础材料和依据。

Evolution of Influenza A H7N9 Virus with an Emphasis on Gene Constellation

The H7 subtype avian influenza viruses, including H7N2, HTN3 and HTN7, have posed a public health threat worldwide. Except one H7N7 fatal case in the Netherlands in 2003, the other H7 human cases have resulted in self-limiting conjunc- tivitis or mild upper respiratory illness.

大口黑鲈弹状病毒Taq Man荧光定量PCR检测方法的建立及初步应用

为建立快速、敏感的大口黑鲈弹状病毒(MSRV)流行株Taq Man荧光定量PCR (qPCR)方法,本研究采用NCBI分析MSRV流行株N基因的保守序列,设计特异性引物和Taq Man探针,以构建的并经PCR和测序鉴定正确的重组质粒标准品p MD18-MSRV-N为模板,通过优化各反应条件初步建立MSRV的Taq Man q PCR检测方法。结果显示,建立的Taq Man q PCR方法标准曲线的R2为0.9906,质粒标准品在6.79×10^(8)拷贝/μL~6.79×10^(2)拷贝/μL范围内与Ct值均存在较好的线性关系。采用该方法检测MSRV及相关病毒,评估该方法的特异性,结果显示,该方法可以特异性检测MSRV,而草鱼出血病病毒、神经坏死病毒、鲤春病毒血症病毒、大口黑鲈双RNA病毒、鳜蛙虹彩病毒、传染性脾肾坏死病毒、石斑鱼虹彩病毒、大口黑鲈蛙虹彩病毒等8种常见鱼类病毒检测结果均为阴性;采用建立的方法检测不同浓度的质粒标准品(6.79×10^(8)拷贝/μL~6.79×10^(1)拷贝/μL),评估该方法的敏感性,结果显示,该方法的检测限为6.79×10^(2)拷贝/μL,比文献中的常规PCR方法敏感1 000倍;以不同批次或同一批次提取的不同浓度的质粒标准品作为模板,分别利用该方法检测,评估该方法的重复性,结果显示,该方法批内和批间重复性试验的变异系数均小于2%。采用该方法和文献中的常规PCR同时检测71份临床发病的大口黑鲈、鳜鱼、鲮鱼组织样品,结果显示阳性率为16.9%(12/71),阴性率为83.1%(59/71),且阳性样品均是从发病大口黑鲈的组织样品中检测到,而发病的鳜鱼、鲮鱼组织样品中均未检测到该病毒;常规PCR方法的阳性率为9.9%(7/71),阴性率为90.1%(64/71),二者的总符合率为93.0%。本研究建立的MSRV Taq Man q PCR方法特异性强、敏感性高、重复性和准确性均较好,可用于大口黑鲈临床样品的检测,为MSRV的快速检测提供了可行的技术手段。

猪流行性腹泻病毒SYBR Green I实时荧光定量PCR检测方法的建立

为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。经过一系列试验表明,该检测方法线性关系良好,R^(2)值为0.99;特异性强,敏感性高,最低可检测至2.23 copies/μL,比普通PCR灵敏约100倍;重复性好,组内变异系数为0.25%~0.43%,组间变异系数为0.67%~0.97%;对于各地区96份临床样品检测出PEDV阳性率为25%。本研究建立的实时荧光定量PCR检测方法为PEDV的临床诊断、流行病学调查以及定量研究提供了有效的检测工具。

猪流行性腹泻病毒nsp1、nsp5和N蛋白的重组腺病毒载体的构建及免疫原性比较

为研究猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)nsp1、nsp5和N蛋白的免疫原性,试验以复制缺陷型人腺病毒5型(AdMax系统)为载体,利用PEDV-GDgh毒株(登录号为MG983755),通过PCR方法扩增出GDgh毒株nsp1、nsp5、N基因,并将其连接到腺病毒穿梭载体上,构建重组穿梭质粒。将3个重组穿梭质粒和腺病毒骨架载体共转染至HEK-293A细胞中获得第1代重组腺病毒,分别命名为rAd-nsp1、rAd-nsp5、rAd-N。用第1代3株重组腺病毒接种HEK-293A细胞,连续传代,通过RT-PCR和Western-blot方法鉴定第3代重组腺病毒,并采用Reed-Muench法计算病毒半数组织培养感染剂量(TCID_(50))。以3株重组腺病毒的第3代病毒液免疫小鼠,收集血清并检测特异性IgG抗体水平。结果表明:试验成功构建出带有nsp1、nsp5、N基因的重组穿梭质粒;分别用重组腺病毒rAd-nsp1、rAd-nsp5、rAd-N感染正常HEK-293A细胞,可产生典型的细胞病变并观察到荧光信号;目的基因正常转录后得到的目的蛋白可正确表达;3株重组腺病毒rAd-nsp1、rAd-nsp5、rAd-N的TCID_(50)依次为1×10^(-8.35)/mL、1×10^(-8.50)/mL、1×10^(-7.66)/mL;免疫小鼠后均可产生针对目的蛋白的特异性抗体。说明试验成功构建的3株正常表达目的蛋白的重组腺病毒均具有免疫原性。

结直肠癌组织中lncRNA CCAT2和NDRG1的表达及意义

目的探讨结直肠癌(CRC)患者组织中长链非编码RNA(lncRNA)结肠癌相关转录物2(CCAT2)、N-myc下游调节基因(NDRG)1的表达及与临床病理特征及预后的关系。方法选取2018年2月至2020年2月在南通市中医院行CRC根治性手术治疗的96例CRC患者作为研究对象。采用实时荧光定量PCR检测组织中lncRNA CCAT2、NDRG1 mRNA表达,采用免疫组织化学检测组织中NDRG1蛋白表达,采用Kaplan-Meier曲线分析不同lncRNA CCAT2、NDRG1 mRNA表达组患者的预后差异,采用多因素Cox回归分析CRC预后影响因素。结果与癌旁组织比较,癌组织中lncRNA CCAT2表达较高,NDRG1 mRNA表达及蛋白阳性率较低,差异有统计学意义(P<0.05)。与TNM分期Ⅰ~Ⅱ期、无淋巴结转移比较,TNM分期Ⅲ期、有淋巴结转移的CRC患者癌组织中lncRNA CCAT2表达较高,NDRG1 mRNA表达较低(P<0.05)。lncRNA CCAT2高表达组3年累积生存率低于lncRNA CCAT2低表达组,而NDRG1 mRNA高表达组3年累积生存率高于NDRG1 mRNA低表达组,差异有统计学意义(P<0.05)。TNM分期、淋巴结转移,lncRNA CCAT2、NDRG1 mRNA是CRC预后影响因素(P<0.05)。结论CRC组织中lncRNA CCAT2表达升高,NDRG1表达降低,二者均参与CRC肿瘤的进展,可作为评估CRC患者生存预后的新指标。

Mining rice new germplasm containing S_5~n gene by functional molecular marker and sequencing

Inter-subspecific hybrids between indica and japonica varieties yield strong biological heterosis, but it is difficult to utilize the hybrids directly in commercial production due to sterility of F1. A special gene S5n may overcome the hybrids sterility, which is caused by the interaction between S5 loci. Recently, S5n had been cloned, and it was revealed containing a large DNA deletion sequence that made the gene nonfunctional, compared to S5i or S5j. We designed a pair of primers flanking the deletion sequence of S5n, and then applied to distinguish the varieties with S5n or non-S5n, convincing result suggested that the primers could be served as functional molecular marker to efficiently identify the new germplasm with S5n. Using the functional marker, we surveyed 197 varieties from China National Micro-core Rice Collection, and found ten of which represented S5n including following varieties: Haobuka, Sanbangqishiluo, Mubanggu, Xiaohonggu, Mowanggu’neiza, Laozaogu, Fanhaopi, Feie’nuo2, Baoxie-7B, Teqingxuanhui. Among them, two varieties Sanbangqishiluo and Laozaogu was previously reported to contain S5n gene. Further sequence analysis on the DNA sequence covering both sides of deletion in S5n of the 10 varieties confirmed that the detected sequences in above varieties was identical with those of varieties containing S5n, such as 02428 and Linglun. These results suggested that the gene in S5 locus of the ten varieties was also nonfunctional and it proved the presence of S5n gene.

BmNPV侵染对家蚕β-N-乙酰葡萄糖苷酶活性及其相关基因转录水平的影响

为了深入解析β-N-乙酰葡萄糖苷酶(GlcNAcases)在病毒感染过程中的作用机制,将家蚕核型多角体病毒(BmNPV)经口感染5龄起家蚕,在感染后3、6、9、12、24 h提取家蚕血淋巴、脂肪体和中肠,采用荧光定量PCR方法检测GlcNAcases基因的表达水平,同时利用GlcNAcases测试盒测定GlcNAcases活性变化情况。结果显示,经口感染BmNPV后,家蚕血淋巴、脂肪体和中肠组织中GlcNAcases活性整体呈现先逐渐升高后急剧降低的趋势;而家蚕机体4个GlcNAcases编码基因的转录水平在不同组织中存在一定差异。在感染BmNPV后3~12 h,血淋巴中BmGlcNAcase1和BmGlcNAcase2的表达量均明显上调,BmGlcNAcase3和BmGlcNAcase4表达量与对照组(未感染)无差异;在感染后24 h,血淋巴中4个GlcNAcases基因表达量均开始下调,且表达量明显低于对照组。脂肪体组织中BmGlcNAcase2和BmGlcNAcase3在感染BmNPV后其表达量呈现先上调后下调的趋势,其中BmGlcNAcase2的表达量在感染BmNPV后3 h就开始上调,BmGlcNAcase3的表达量在9 h才开始上调,BmGlcNAcase1和BmGlcNAcase4的表达量无明显变化。感染BmNPV后,中肠BmGlcNAcase2和BmGlcNAcase4的表达量整体先逐渐上调,增高至一个峰值,随后急剧减弱,表达受到明显抑制;而BmGlcNAcase1和BmGlcNAcase3在感染BmNPV后其表达量呈现下调趋势,表达量逐渐降低。综上,BmGlcNAcase2编码的GlcNAcases在家蚕抵御BmNPV的侵染过程中发挥了一定的免疫防御功能。

5种β受体拮抗剂类药物中的N-亚硝基类杂质的含量研究

目的测定普萘洛尔、美托洛尔、阿替洛尔、艾司洛尔、比索洛尔原料药/制剂中N-亚硝基类杂质含量,明确其含量的关注阈值。方法采用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱技术。以ACE Excel 3 C18-AR为色谱柱,以含0.01 mol/L乙酸铵的0.2%甲酸溶液-甲醇为流动相进行梯度洗脱,流速为0.60 mL/min,柱温为40℃,进样量为5μL;以可加热的电喷雾离子源为离子源,以全扫描-选择离子监测模式进行正离子扫描。采用该法对10家企业生产的15批β受体拮抗剂类药物原料药/制剂中N-亚硝基类杂质含量进行测定,并采用Discovery Studio软件对待测杂质进行毒性预测和关注阈值估算。结果5种β受体拮抗剂类药物中,N-亚硝基普萘洛尔、N-亚硝基美托洛尔、N-亚硝基阿替洛尔、N-亚硝基艾司洛尔、N-亚硝基比索洛尔检测质量浓度的线性范围分别为1.01~503.38、1.02~508.38、0.97~483.63、1.11~554.27、1.05~523.92 ng/mL(r>0.999),定量限分别为1.04、0.25、0.05、0.55、1.05 ng/mL,检测限分别为0.52、0.08、0.02、0.17、0.52 ng/mL,精密度、重复性、加样回收率、稳定性、耐用性试验的RSD均小于7.5%(n=6或n=5)。15批样品中,除1批样品外,其余批次均检出了N-亚硝基普萘洛尔(1.07~8.91 ng/mg)、N-亚硝基美托洛尔(1.43~3.37 ng/mg)、N-亚硝基阿替洛尔(1.33 ng/mg)、N-亚硝基艾司洛尔(0.19 ng/mg)、N-亚硝基比索洛尔(1.27 ng/mg)。经预测,上述5种杂质有不同程度的生育毒性、致突变性、致癌性,关注阈值分别为1.0、0.4、4.3、0.2、46.7 ng/mg。结论所建方法简单快捷、灵敏度高、专属性强,估算的关注阈值明确,可用于多种β受体拮抗剂类药物中N-亚硝基类杂质的含量控制。

猪流行性腹泻病毒贵州流行株N基因的克隆、测序与生物信息学分析

为了了解贵州省猪流行性腹泻(porcine epidemic diarrhea,PED)的流行情况,试验对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)贵州流行株(GZ2022)N基因序列进行克隆、测序,并利用DNAStar、ProtParam、ProtScale等生物信息学分析软件对PEDV-GZ2022株N基因的同源性和遗传进化地位,以及N蛋白的分子特征和潜在功能进行分析预测。结果表明:PEDV-GZ2022株N基因大小约为1364 bp,编码441个氨基酸;PEDV-GZ2022株N基因核苷酸序列与GenBank中的PEDV参考毒株N基因的相似性为95.5%~99.8%,与PEDV经典毒株CV777 N基因核苷酸序列的相似性为95.6%,与SDLY2020毒株N基因核苷酸序列的相似性高达99.8%,PEDV-GZ2022毒株与经典毒株CV777关系相隔较远,与PEDV变异株GⅡ-b位于同一分支;PEDV-GZ2022株N蛋白中含有较多的疏水性氨基酸,为疏水性蛋白;该蛋白质存在4个N糖基化位点和37个O糖基化位点,有31个丝氨酸、14个苏氨酸及3个酪氨酸可能被磷酸化,并且抗原指数较好。说明PEDV-GZ2022株为GⅡ-b变异株,PEDV-GZ2022 N蛋白可能具有良好的抗原性,可以作为潜在的诊断抗原。

Synthesis of N-methylene phosphonic chitosan(NMPCS)and its potential as gene carder

N-Methylene phosphonic chitosan （NMPCS）, an amphiphilic macromolecule with powerful chelating ability of Ca^2＋ ions, was synthesized and characterized. The physicochernical properties of NMPCS and the interactions between NMPCS and plasmid DNA were investigated by FTIR, ^13C NMR, X-ray, agarose gel electrophoresis retardation assay, atomic force microscopy （AFM） and circular dichroism （CD）. The results suggest that at charge ratio 2：1 or above, DNA could be completely entrapped and spherical complexes with mean size of 80-210 nm were formed. Taking HeLa as host cell, luciferase expression mediated by NMPCS improved about 100 times compared to the expression mediated by chitosan.

TaARR1, a cytokinin response regulator gene in Triticum aestivum, is essential in plant N starvation tolerance via regulating the N acquisition and N assimilation

Plant N starvation response is closely associated with the N signaling components that involve transduction of the low-N cues. In this study, we functionally characterized Ta ARR1, a cytokinin(CK) response regulator gene in Triticum aestivum, in mediating the N starvation adaptation in plants. Ta ARR1 harbors two conserved domains specified by plant ARR family members;subcellular localization analysis indicated its target onto nucleus after endoplasmic reticulum assortment. Ta ARR1 displayed modified expression upon the N starvation stressor, showing upregulated expression in roots and leaves over a 27-h N starvation treatment and whose induced transcripts were gradually recovered along with progression of the N recovery treatment. The tobacco lines overexpressing Ta ARR1 displayed improved low-N stress tolerance, displaying enlarged phenotype, increased biomass and N accumulation, and enhanced glutamine synthetase(GS) activities compared with wild type(WT) following the N starvation treatment. Expression analysis on genes encoding the nitrate transporter(NRT) and GS proteins in Nicotiana tabacum revealed that Nt NRT2.2 and Nt GS3 are upregulated in expression in the N-deprived transgenic lines, whose expression patterns were contrasted to other above family genes that were unaltered on transcripts between the transgenic lines and WT. Transgene analysis validated the function of Nt NRT2.2 and Nt GS3 in regulating N accumulation, GS activity, growth traits, and N use efficiency in plants. These results suggested the internal connection between the Ta ARR1-mediated N starvation tolerance and the modified transcription of distinct N acquisitionand assimilation-associated genes. Our investigation together indicates that Ta ARR1 is essential in plant N starvation adaptation due to the gene function in transcriptionally regulating distinct NRT and GS genes that affect plant N uptake and assimilation under the N starvation condition.

Expression and Biological Function of N-myc Down-regulated Gene 1 in Human Cervical Cancer

The expression of N-myc down-regulated gene 1 (NDRG1) has previously been reported to be involved in the proliferation,differentiation,invasion and metastasis of cancer cells,but its role in cervical cancer is still unclear.This study aimed to investigate the expression of NDRG1gene in human cervical cancer and its effect on aggressive tumor behaviors.The NDRG1 expression in cervical tissues and cells was detected by RT-PCR.Specific expression plasmid pEGFP-N1-NDRG1-GFP was used to enhance the expression of NDRG1 in human cervical cancer cell lines.The mRNA and protein level of NDRG1 was assessed by RT-PCR and Western blotting,respectively.Its effects on cell proliferation,migration,invasion,cell cycle and apoptosis were detected by MTT,transwell migration assay and flow cytometry (FCM),respectively.The results showed that the expression of NDRG1 in cervical cancer tissues and cells was significantly lower than in normal cervical tissues (P【0.001).After transfection with pEGFP-N1-NDRG1-GFP,the mRNA and protein expression of NDRG1 was up-regulated in Siha cells,which suppressed cell proliferation (P【0.001),induced cell cycle arrest (P【0.05),reduced invasion and migration of Siha cells (P【0.05),but caused no cell apoptosis.Moreover,vascular endothelial growth factor (VEGF),a tumor-induced angiogenesis factor,was markedly reduced and E-cadherin,a cell adhesion molecule,was increased in the cells transfected with pEGFP-N1-NDRG1-GFP.It was concluded that up-regulated NDRG1 may play a role in the suppression of malignant cell growth,invasion and metastasis of human cervical cancer.

猫传染性腹膜炎病毒N蛋白表达及其多克隆抗体制备

本研究旨在体外原核表达猫传染性腹膜炎病毒(feline infectious peritonitis virus,FIPV)SH2021株核衣壳蛋白(nucleocapsid,N),制备兔源多克隆抗体。根据FIPV SH2021株N基因序列设计引物,构建pCold-I-N重组表达质粒。随后,将重组质粒转化至表达感受态细胞BL21(DE3),在终浓度为1.0 mmol·L^(-1)的IPTG和16℃的诱导条件下进行表达。最后,利用His层析柱进行纯化,纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体。结果表明,纯化获得重组N蛋白大小约为45 ku,制备的N蛋白兔多克隆抗体效价可达1∶512000,该抗体对N蛋白具有良好的反应原性。本研究成功所制备了FIPV N蛋白兔多克隆抗体,该抗体具有良好的反应原性和特异性,为FIPV抗原抗体检测以及研究N蛋白生物学功能研究提供重要工具。

CYP2C19基因多态性、血清NT-proBNP水平与急性脑梗死静脉溶栓预后不良的关系

目的探讨CYP2C19基因多态性、血清N末端B型脑利钠肽前体(NT-proBNP)水平与急性脑梗死(ACI)患者静脉溶栓预后不良的关系。方法选择210例ACI患者,均行阿替普酶静脉溶栓治疗,治疗90 d后根据改良Rankin量表(mRS)评分将患者分为预后不良组(mRS评分≥3分)52例、预后良好组(mRS评分<3分)158例。治疗前采用实时荧光定量PCR法检测CYP2C19基因多态性,时间分辨荧光免疫层析法检测血清NT-proBNP。用Pearson或Spearman分析CYP2C19基因多态性、血清NT-proBNP水平与mRS评分的相关性;用多因素Logistic回归分析ACI患者静脉溶栓预后的影响因素;用受试者工作特征曲线分析CYP2C19基因多态性、血清NT-proBNP水平对ACI患者静脉溶栓预后不良的预测价值。结果预后不良组年龄、高血压比例、糖尿病比例、入院美国国立卫生研究所脑卒中(NIHSS)评分、空腹血糖水平高于预后良好组(P均<0.05)。预后不良组和预后良好组CYP2C19基因多态性比较差异有统计学意义(P<0.05),预后不良组血清NT-proBNP水平高于预后良好组(P<0.05)。ACI患者CYP2C19基因多态性(r_(s)=0.362)、血清NT-proBNP水平(r=0.426)与mRS评分均呈正相关(P均<0.05)。高NIHSS评分、CYP2C19基因慢代谢型、高血清NT-proBNP水平是ACI患者预后不良的危险因素(P均<0.05)。CYP2C19基因多态性、血清NT-proBNP水平单独及联合预测ACI患者静脉溶栓预后的曲线下面积分别为0.752、0.786、0.861,二者联合预测的曲线下面积高于单独预测(P均<0.05)。结论CYP2C19基因多态性和高水平NT-proBNP是ACI患者静脉溶栓预后不良的危险因素,二者联合对不良预后有较高的预测价值。

Cloning and Sequencing of a Gene Encoding Aminopeptidase N in the Midgut of Helicoverpa armigera(Hubner)

A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACE technique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp in length, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108. 3 kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features with aminopeptidase N from several moth species, including the consensus zinc-binding motif HEXXHX18E and the GAMEN motif common to gluzincin aminopeptidases. The first 20 amino acid residues at N-termini is hydrophobic transmembrane helix. The sequence of APN-Harm was deposited in GenBank and the accession number is AY181026.