Genetic Analysis of the VP1 Region of Human Enterovirus 71 Strains Isolated in Fuyang,China,During 2008

Enterovirus 71 （EV71） is a common cause of Hand, foot, and mouth disease （HFMD） and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences （891 nueleotides） from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous tO members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine（H） at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.

RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A–D from Clinical Specimens

Objective To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses(HEVs)from clinical samples and to contribute to etiological surveillance of HEV-related diseases.Methods A panel of RT-nPCR assays,consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D,was established in this study.The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 perμL and copies perμL,and the newly established methods were tested in clinical specimens collected in recent years.Results The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 perμL and 10 virus copies perμL,and for the complete VP1 gene was 1 CCID50 perμL and 100 virus copies perμL,using serially-diluted virus stocks of five serotypes.As a proof-of-concept,25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons.Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D,providing rapid,sensitive,and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Comparison of Immune Responses against FMD by a DNA Vaccine Encoding the FMDV/O/IRN/2007 VP1 Gene and the Conventional Inactivated Vaccine in an Animal Model

Foot-and-mouth disease virus （FMDV） is highly contagious and responsible for huge outbreaks among cloven hoofed animals. The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/OflRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model. The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the peDNA3.1＋ and pEGFP-N1 vectors to construct the VPI gene cassettes. The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector, which solely expressed the GFP protein. Six mice groups were respectively immunized by the sub-cloned pcDNA3.1＋-VP1 gene cassette as the DNA vaccine, DNA vaccine and PCMV-SPORT-GMCSF vector （as molecular adjuvant） together, conventional vaccine, PBS （as negative control）, pcDNA3.1＋ vector （as control group） and PCMV-SPORT vector that contained the GMCSF gene （as control group）. Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together, the DNA vaccine alone and the conventional inactivated vaccine （P〈0.05）. Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone, but this response was the most for the conventional vaccine group. However, induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine, but T-cell proliferation and IFN-？ concentration were the most in DNA vaccine with the GMCSF gene. Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene, showed protective neutralizing antibody response and the most Thl cellular immunity.

Cloning of Chicken Anemia Virus vp3 Gene and Apoptosis Inductive Effect of vp3 Gene In Vitro

Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE TM mediated transfection in vitro with pcDNA vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L 02, and RT PCR was performed afterward. The results of RT PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.

Analysis of Genetic Variation of VP2 Gene in 6 FPV Strainsin China

In order to understand the variation of FPV strains in the Jinan area, Shandong Province, China, the VP2 gene of 6 FPV strains was sequenced, and the analysis of the genetic relationship, evolution and main functional site variation was carried out. It was found that FPV-XY2, FPV-XY3 and FPV-XY6 were the same strain with 100% homology, and also close to FPV-XY1, and the homology between FPV-XY4 and FPV-XY5 was close. The homology between the reference strain and the test strain was over 99.3%. According to the evolutionary analysis, the genetic relationship among FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 was close, and the genetic relationship between FPV-XY4 and FPV-XY5 was close, and the result was similar to the homologous result. Compared with the VP2 amino acid sequence of the standard strain FPV-CU4, the VP2 protein of all the test strains changed from I to t on the 101st Amino acid, this may be the cause of immune failure in these 6 cases;the change of a to s in the 91st amino acid position of FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 may be the cause of enhanced virulence of FPV. This study provides a reference for exploring the epidemic law of FP in the Jinan area, the standard of FP treatment plan and the research and development of FPV subunit vaccine.

Cloning and Expression of the vp39 Gene of Bombyx mori Nuclear Polyhedrosis Virus in E.coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction.

Two novel mutations in the VPS33B gene in a Chinese patient with arthrogryposis,renal dysfunction and cholestasis syndrome 1:A case report

BACKGROUND The VPS33B(OMIM:608552)gene is located on chromosome 15q26.1.We found a female infant with autosomal recessive arthrogryposis,renal dysfunction and cholestasis syndrome 1(ARCS1)caused by mutation in VPS33B.The child was diagnosed with ARCS1(OMIM:208085)after the whole exome sequencing revealed two heterozygous mutations(c.96+1G>C,c.242delT)in the VPS33B gene.CASE SUMMARY We report a Chinese female infant with neonatal cholestasis disorder,who was eventually diagnosed with ARCS1 by genetic analysis.Genetic testing revealed two new mutations(c.96+1G>C and c.242delT)in VPS33B,which is the causal gene.The patient was compound heterozygous,and her parents were both heterozygous.CONCLUSION This study extends the mutational spectrum of the VPS33B gene to provide a molecular basis for the etiological diagnosis of ARCS1 and for genetic counseling of the family.

辽宁沈阳株猫泛白细胞减少症病毒VP2基因扩增及生物信息学分析

【目的】通过生物信息学方法分析1株2022年辽宁沈阳地区猫泛白细胞减少症病毒(Feline panleukopenia virus, FPV)LN3株VP2蛋白的分子特征。【方法】利用FPV胶体金试纸条对临床上表现呕吐、腹泻等症状的患病猫粪便进行检测,提取该病猫粪便的病毒DNA进行FPV VP2基因PCR扩增及测序,使用Seqman软件对序列进行拼接。将所获序列与NCBI数据库中FPV和犬细小病毒(Canine parvovirus, CPV)的VP2基因进行相似性比对及遗传进化分析。使用生物信息学软件对该毒株VP2蛋白进行预测,包括理化性质、亲/疏水性、跨膜区、糖基化位点、亚细胞定位、二级结构、三级结构等。【结果】FPV LN3株VP2基因全长1 755 bp,编码584个氨基酸;与GenBank上登录的15株FPV同属一个大分支,相似性为98.9%~99.7%;与CPV处于不同分支上。生物信息学软件预测显示,FPV LN3株VP2蛋白为亲水性蛋白,无跨膜结构;含有7个潜在N-糖基化位点、86个O-糖基化位点和50个磷酸化位点;VP2蛋白在细胞质、细胞核、线粒体中的可能性分别为43.5%、34.8%和21.7%;VP2蛋白二级结构中无规则卷曲、α-螺旋、延伸链、β-转角分别占61.82%、8.90%、24.32%及4.97%,三级结构预测结果与其一致;VP2蛋白共有20个抗原表位。【结论】VP2是FPV遗传变异的关键基因,FPV LN3株VP2蛋白属于亲水性、非跨膜稳定蛋白,共存在20个抗原表位。试验结果为进一步研究FPV VP2蛋白功能及研发新型疫苗提供理论依据。

Molecular Detection and Disease Resistance Identification of Transgenic Wheat with pti5-vp16 Gene

The immature embryos of wheat cultivars Liaochun10, Tiechun1 and Fengqiang3 were bombarded with gold particles coated with pti5 vp16 by gene gun and disease resistant regenerated plants were attained. In order to confirm that the plants are genuine transformed ones, a series of molecular tests were conducted as follows. Firstly, transient GUS expression test on embryos two days after bombardment was done. There were many obvious blue spots produced on the surface of bombarded embryos after GUS staining, in which the maximum reached to 85 blue spots per embryo. Secondly, PCR test was performed with DNA from the regenerated plants obtained after double selection with ppt. 6 plants were found PCR test positive. At last, further verification analysis using dot hybridization and southern blotting was carried out on those PCR positive plants and the strong hybridization signals appeared as expected. All the above tests were uniformly indicated that the disease resistant regenerated plants were true transgenic plants. When inoculated with Blumeria graminis, transgenic wheat plants of PCR positive results were mostly resistant(R) after 7 days, and resis tant, moderate resistant(MR), moderate susceptible(MS) at 14 days respectively. The disease severity of them was distinctively lighter than that of control.

Isolation of a feline-derived feline panleukopenia virus with an A300P substitution in the VP2 protein and confrmation of its pathogenicity in dogs

Feline panleukopenia virus(FPV)is a single-stranded DNA virus that can infect cats and cause feline panleukopenia,which is a highly contagious and fatal disease in felines.The sequence of FPV is highly variable,and mutations in the amino acids of its capsid protein play crucial roles in altering viral virulence,immunogenicity,host selection,and other abilities.In this study,the epidemiology of FPV was studied using 746 gastrointestinal swab samples derived from cats that presented gastrointestinal symptoms specifcally,diarrhea or vomiting during the period spanning from 2018 to 2022.The overall prevalence of FPV-positive patients among these samples was determined to be 45.4%.Capsid(virion)protein 2(VP2)gene of each FPV-positive sample was sequenced and amplifed,yielding 65 VP2 sequences.Among them,six VP2 gene sequences were detected in the majority of the samples test positive for FPV,and these positive samples originated from a diverse range of geographical locations.These isolates were named FPV-6,FPV-10,FPV-15,FPV-251,FPV-271 and FPV-S2.Additionally,the substitution of Ala300Pro(A300P)in VP2 was detected for the frst time in feline-derived FPV(FPV-251).FPV-251 isolate,with this substitution in VP2 protein,exhibited stable proliferative capacity in Madin-Darby canine kidney(MDCK)cells and A72 cells.FPV-271 was selected as the FPV control isolate due to its single amino acid diference from VP2 protein of FPV-251 at position 300(FPV-271 has alanine,while FPV-251 has proline).After oral infection,both FPV-251 and FPV-271 isolates caused feline panleukopenia,which is characterized by clinical signs of enterocolitis.However,FPV-251 can infect dogs through the oral route and cause gastrointestinal(GI)symptoms with lesions in the intestine and mesenteric lymph nodes(MLNs)of infected dogs.This is the frst report on the presence of an A300P substitution in VP2 protein of feline-derived FPV.Additionally,FPV isolate with a substitution of A300P at VP2 protein demonstrated efcient replication capabilities in canine cell lines and the ability to infect dogs.

重组传染性法氏囊病病毒VP2/VP243基因表达及保护性和免疫原性

目的在重组鸡痘病毒中表达传染性法氏囊病毒 VP2/VP243基因,并研究表达产物的保护性和免 疫原性。方法以 FPV 282E_4株 TK基因为侧翼,分别将鸡法氏囊病病毒(Infectious Bursal Disease Virus,IBDV) VP2 和VP243目的基因克隆到牛痘病毒A型包涵体(ATI)和痘苗病毒P7.5复合型启动子下游,构建成2个重组表达质 粒,命名为pUTALacVP和pUTA LacVPO。用这2个质粒转染CEF细胞,用X-gal染色法筛选出2株重组病毒vUTA lacVP2和 vUTALacVP0。用这 2株病毒免疫 1周龄 SPF鸡,以常规疫苗为对照。结果 ELISA、SDS-PAGE和 Western blot表明表达产物可与IBDV特异性多克隆抗体反应,并诱导鸡保护性抗体。在使用vvIBDV攻击前,对照组抗体水 平显著高于实验组。但攻击5 d后,实验组抗体水平明显升高。结论vUTALacVP2和 VPO在 CEF细胞中实现了高 效表达,但重组疫苗的保护率低于常规疫苗。

番鸭细小病毒浙江分离株VP基因的克隆与序列分析

为明确番鸭细小病毒结构蛋白VP的特征,本研究运用PCR从已分离鉴定的番鸭细小病毒浙江分离株(MDPV-ZJ)中分段扩增出VP基因,并对其进行克隆测序和分析。结果表明,番鸭细小病毒浙江分离株VP基因全长为2 199bp,编码732个氨基酸。所编码的包膜蛋白大小为81.32ku、理论等电点为6.59、不稳定系数为37.49、亲水性平均系数为-0.667,属于亲水性稳定类蛋白,该编码的结构蛋白没有信号肽,为非分泌蛋白。根据VP基因特征从遗传进化上可将MDPV分为2个大的基因型:经典型和基因重组型,经典型MDPV可以进一步划分为台湾亚群、大陆亚群和欧洲群,MDPV各分离株在遗传进化上存在明显的地域性。本试验中MDPV浙江分离株株处于MDPV大陆亚群。

猪O型口蹄疫病毒强弱毒株VP_1基因的克隆与序列分析

本研究根据口蹄疫病毒 (FMDV)VP1基因的序列 ,设计并合成了 1对用于扩增整个VP1基因的引物 (5P、P6 )。从细胞培养液或组织中提取总RNA ,通过PT_PCR扩增 ,从F2 9株、O3I3株和T5 0 9株中均获得了 1条约 740bp的DNA电泳带。将PCR产物双酶切后电泳回收 ,插入到相应双酶切的pUC18质粒中 ,获得了重组质粒。通过PCR鉴定 ,证明重组质粒pUCVP1/F2 9、pUCVP1/O313、pUCVP1/T5 0 9均插入了VP1基因。对上述 3个重组质粒进行测序后分析 ,F2 9强毒株与O3I3、T5 0 9弱毒株相比 ,其核苷酸序列同源性分别为 98.75 %和 99.0 6 % ;因F2 9株核苷酸发生 3个碱基缺失与 1个碱基替换 ,故推导的氨基酸序列同源性分别仅为 44 .13%和 41.32 % ;而T5 0 9株与O3I3株相比 ,其核苷酸、氨基酸序列同源性分别为 99.37%和 95 .31%。通过序列分析发现 ,本研究的 3个毒株与国内大多数毒株 (包括 1997年台湾暴发FMDV所分离的毒株 ,除O/A/ 5 8株外 )均属于同一基因型 ,核苷酸序列同源性为 85 %～ 94% ;而与国外毒株相比 ,属不同的基因型 ,核苷酸序列同源性仅为 81%～ 82 %。其推导的氨基酸序列 ,除F2 9株与O/HK/ 93株及 1997年台湾暴发FMDV分离的少数毒株的氨基酸序列同源性仅为 45 %～ 6 3%外 ,本研究的

犬细小病毒VP2蛋白的原核表达与纯化

为了研究犬细小病毒(canine parovirus,CPV)VP2蛋白的结构和功能,本试验对CPV VP2蛋白进行表达和纯化。采用大肠杆菌表达外源蛋白的方法,将CPVVP2基因插入原核表达载体pET-32a(+)中构建重组原核表达载体pET-VP2,转化至大肠杆菌BL21(DE3)感受态细胞中,在不同IPTG浓度、诱导温度和诱导时间条件下进行原核表达,确定最佳诱导条件。最佳条件下的诱导产物经超声破碎离心后,利用镍柱对重组蛋白进行纯化并用SDS-PAGE和Western blotting进行双重鉴定。结果显示,重组质粒pET-VP2经双酶切鉴定分别获得大小为5 900bp左右的载体条带和1 755bp左右的目的基因条带,成功构建了pET-VP2重组质粒;重组VP2蛋白的分子质量约为64ku,与预期大小一致,且在37℃、1.0mmol/L IPTG、诱导5h条件下表达量最高,条带最亮;蛋白超声破碎后经SDS-PAGE发现,只在沉淀中出现了目的条带,而上清中并未出现相应条带,说明重组蛋白均以包涵体的形式存在;纯化后获得的重组蛋白,经SDS-PAGE和Western blotting双重鉴定,均在64ku处出现条带,说明纯化后的蛋白为重组蛋白pET-VP2。本试验通过对CPV VP2蛋白的原核表达及纯化成功获得了大量纯度较高的CPV VP2蛋白,为今后制备CPV VP2蛋白多克隆抗体及进一步研究该蛋白在对治疗犬细小病毒病中的临床应用价值提供了基础理论依据。

番鸭细小病毒强、弱毒株VP2基因的序列测定比较

According to the complete nucleotide sequence of Muscovy duck parvovirus registered in the gene bank, two modified primers (LHMP7/LHMP8) were designed and, for each of them, a restriction endonuclease recognition site, SacⅡ or KpnⅠ, was included respectively. The DNA encoding VP2 structural protein of the wild strain MDPV Q and the attenuated strain MDPV 26 which had been derived by continued passage of virulent wild type (MDPV Q) in Muscovy duck embryo were amplified by PCR and recombined into T vector and sequenced respectively. It shows that both DNA encoding VP 2 structural protein of MDPV Q and MDPV which has been registered in the gene bank had the high homology (97.9%). MDPV 26 and MDPV Q displayed 99.7% identity, and shared 98.3% homology with that of reported MDPV.

传染性法氏囊病病毒超强毒株F9811VP_2基因的克隆及序列分析

根据 NCBIG Gene Bank记载的传染性法氏囊病病毒超强毒 ( vv IBDV) OKYM株的核苷酸序列 ,设计并合成 1对特异性扩增 IBDV主要宿主保护性抗原 VP2 基因的引物。从 IBDV F981 1感染发病鸡法氏囊组织中提取病毒 RNA,经 RT-PCR扩增出约 1 .5kb的基因片段 ,采用平端连接法将此基因片段克隆至p UC1 1 9质粒的 Sma 位点上。经核苷酸序列测定 ,并与国内外其他 vv IBDV VP2 基因序列进行比较分析 ,发现 F981 1与 OKYM在 VP2 基因上有 1 8个核苷酸不同 ,但在氨基酸序列上仅 2 1 2位存在差异 ,从而从分子生物学角度证明 F981 1为 vv IBDV。对 1 4 3～ 3 82氨基酸区域所作系统进化树分析表明 ,F981 1、G92 0 1与OKYM、UK661、HK4 6相近 ,而 F950 2、G93 0 3与 OKYM、U K661、HK4 6较远 ,说明我国存在许多不同的vv

小麦Vp-1基因RNA干扰表达载体的构建及遗传转化

小麦成熟期穗发芽是世界性的自然灾害,严重影响小麦的产量和品质。Viviparous-1(Vp-1)是促进胚成熟和休眠的主要转录调节因子,与小麦穗发芽抗性有着密切的关系。本实验根据小麦Vp-1基因序列,以植物表达载体pAHC25为基础,成功构建了含有反向重复序列的RNA干扰表达载体pAHC-WVpRi。采用基因枪法轰击小麦品种新春9号幼胚材料1825个,共获得34株T0再生植株。利用Bar基因引物和干扰片段特异引物对再生植株进行PCR检测,获得Bar基因和干扰片段均为阳性的植株3株,转化率为0.16%。本研究为深入分析Vp-1基因功能,进而通过分子育种进行小麦穗发芽抗性的遗传改良提供了科学依据。

小麦VP基因的克隆、生物信息学分析及功能初探

为研究小麦液泡膜H^+转运无机焦磷酸酶基因VP在植物中的作用,根据大麦、水稻等植物VP基因序列设计简并引物,用RACE法扩增小麦VP基因全长,对VP蛋白进行生物信息学分析,构建VP基因植物表达载体,转化拟南芥,对阳性转基因纯合体株系进行筛选和耐盐性鉴定。结果表明,小麦VP基因编码区序列全长2 286 bp,共编码761个氨基酸,等电点为5.17。VP蛋白为无信号肽的疏水性蛋白,主要含α螺旋。转VP基因拟南芥在NaCl胁迫下的萌发率、绿苗率均高于野生型拟南芥,成苗生长状态优于野生型拟南芥,说明小麦VP基因提高了转基因拟南芥的耐盐性。

快速跳出局部最优的VPS-GEP算法

传统GEP(Gene Expression Programm ing)算法存在局部收敛方面的缺陷,为了解决这一问题,提出了可以使进化快速跳出局部最优的VPS-GEP(Various Popu lation Strategy GEP)算法,证明了在概率意义上GEP平均每代进化所耗时间与群体规模成正比,用两个标准测试函数和一个标准测试数据集测试了VPS-GEP算法的函数挖掘能力和效率。实验表明,VPS-GEP算法可以减少进化停滞代数55%以上。

携带pdcd5基因的增殖型腺病毒与VP-16杀伤K562细胞的协同作用

程序性细胞死亡因子5(programmed celldeath5,PDCD5)在多种肿瘤细胞中表达明显降低。携带pdcd5基因的靶向增殖型腺病毒SG611-pdcd5对白血病细胞具有杀伤作用,对正常细胞具有相对保护作用。本研究旨在观察联合应用SG611-pdcd5与低剂量化疗药物依托泊甙(etoposide,VP-16)对白血病细胞系K562的作用。以不同浓度梯度的药物或不同感染复数(multiplicity of infection,MOI)的病毒液作用于K562细胞,培养48小时后用MTT法检测细胞存活率。结果表明,与SG611-pdcd5(MOI=40)或VP-16(0.5μg/ml)单独作用组相比,SG611-pdcd5(MOI=40)+VP-16(0.5μg/ml)组细胞存活率明显降低(p均<0.05),分别为(59.45±4.12)%、(82.91±3.41)%及(42.00±5.75)%。SG611-pdcd5与VP-16的协同作用强于PDCD5蛋白或携带pdcd5基因的非增殖型腺病毒Ad-pdcd5与VP-16的协同作用(p均<0.05);VP-16浓度为4.0μg/ml时,VP-16+SG611-pdcd5(MOI=40)组、VP-16+proPDCD5(40μg/ml)组及VP-16+Ad-pdcd5(MOI=80)组细胞存活率分别为(37.09±1.89)%、(52.36±1.64)%及(73.64±4.33)%。结论:SG611-pdcd5具有促进低剂量VP-16杀伤K562细胞的作用,二者联合可增强对白血病细胞的杀伤作用。