利用SUMO表达系统高效表达猪O型口蹄疫病毒VP0、VP1、VP3基因

根据GenBank中公布的猪O型口蹄疫病毒(foot and mouth disease virus,FMDV)全基因合成了FMDV结构蛋白前体蛋白P1基因,同时设计了扩增FMDV结构蛋白VP0、VP1和VP3基因的引物。以P1基因为模板,分别经PCR扩增获得FMDV VP0、VP1和VP3基因。扩增产物克隆于Blunt载体中,酶切后将目的片段连接到原核表达载体SUMO中,构建重组表达质粒SUMO-VP0、SUMO-VP1和SUMO-VP3,将重组质粒转化大肠杆菌BL21(DE3)plysS进行诱导表达。经SDS-PAGE电泳可见融合蛋白均获得高效表达,融合蛋白表观分子质量分别约为55、48和40 ku。在IPTG浓度为1.0 mmol/L,温度为37℃,诱导5 h时融合蛋白表达量最大。Western blotting结果表明,融合蛋白均可被FMDV阳性血清识别,反应原性良好。

禽脑脊髓炎病毒VP1、VP3、VP0基因表达载体的构建

根据AEV-NH937基因组全序列设计3对引物,应用RT-PCR方法,扩增出AEV的外壳蛋白VP1、VP3、VP0基因,将它们克隆于pUCm-T载体上进行序列分析。结果显示,AEV-NH937病毒株VP1基因全长810 bp、编码270个氨基酸,VP3基因全长735 bp、编码245个氨基酸,VP0基因全长726 bp、编码242个氨基酸;与Calnek疫苗株相比,AEV-NH937病毒株VP1、VP3、VP0基因的核苷酸同源性分别为90.62%、93.88%、95.45%,氨基酸同源性分别为88.89%、97.96%、98.35%。将VP1、VP3、VP0基因分别插入载体pcDNA4/His Max构建表达重组质粒并转入宿主菌DH5α中,使基因得以表达。

O型口蹄疫病毒VP0和VP1蛋白的可溶性表达与反应原性分析

为了高效可溶性表达O型口蹄疫病毒(FMDV)VP0、VP1结构蛋白,根据大肠杆菌密码子的偏爱性优化合成VP0和VP1基因片段,并将其克隆到p E-SUMO载体中,构建重组质粒SUMOVP0和SUMO-VP1,将重组质粒转化到大肠杆菌BL21(DE3)感受态细胞中进行诱导表达,并优化诱导温度、时间和IPTG浓度等表达条件。结果显示,SUMO-VP0可溶性蛋白表达的最佳条件为:20℃条件下,0.1 mmol/L IPTG诱导表达8 h;SUMO-VP1可溶性蛋白表达的最佳条件为:37℃条件下,0.1 mmol/L IPTG诱导表达12 h。SDS-PAGE电泳和Western blot结果表明,表达的SUMOVP0、SUMO-VP1可溶性蛋白能够被抗FMDV的阳性血清识别,具有很好的反应原性。

猪嵴病毒结构蛋白VP0与VP1原核表达及间接ELISA方法的建立

分别建立基于猪嵴病毒(PKV)结构蛋白VP0与VP1的间接ELISA检测方法。对PKV结构蛋白VP0与VP1的基因进行合成并连接至原核表达载体pET-32a后,转入感受态细胞BL21中,用IPTG诱导表达的重组蛋白经Ni柱纯化后,分别以重组蛋白pET-32a-VP0与pET-32a-VP1作为包被抗原,采用棋盘滴定法建立两种间接ELISA检测方法,并进行重复性、敏感性、特异性实验和临床检测。结果显示重组蛋白pET-32a-VP0与pET-32a-VP1抗原的最佳包被浓度分别为2 mg/mL和2.5 mg/mL,反应条件均为37℃、1 h;封闭液最佳条件为5%脱脂乳,37℃、2 h;血清最佳孵育条件为1∶200,37℃、1 h;二抗最佳孵育条件分别为1∶20000和1∶15000,37℃、1 h;最佳显色时间为10 min,两种间接ELISA检测方法临界值分别为0.306和0.277。试验结果表明本研究成功建立了能够有效检测PKV血清特异性抗体的间接ELISA方法,且方法具有重复性好、敏感性高、特异性强、稳定性好等特点,对今后PKV的临床诊断及抗体检测试剂盒的开发奠定了重要基础。

鹅细小病毒VP1与VP3非重叠序列的克隆与原核表达

根据鹅细小病毒 GPV H1株基因组核苷酸序列设计了一对引物 ,对其结构蛋白 VP1与 VP3非重叠核苷酸序列进行 PCR扩增 ,并克隆到表达性载体 p GEX- 6 p- 1,经酶切鉴定筛选出阳性克隆 ,并转化到大肠杆菌 BL 2 1(ED3) plys S进行原核表达 ,并用 SDS- PAGE鉴定 ,结果表明融合蛋白在表达性细菌 BL2 1中重获得了高效表达。

鹅细小病毒VP1-VP3非重叠区基因的克隆与序列分析

通过克隆鹅细小病毒(Goose parvovirus,GPV)分离株VP1-VP3非重叠区基因,并对其进行序列分析,为鹅细小病毒感染与疫苗免疫的鉴别诊断奠定理论基础。进行鹅胚病毒增殖,收集尿囊液,提取基因组,参考发表的B株序列,设计合成一对引物,经PCR扩增,克隆VP1-VP3基因,筛选阳性克隆,对其进行序列测定及同源性分析。结果表明,克隆的基因片段为901 bp,VP1-VP3基因共594 bp,编码198个氨基酸;弱毒株之间亲缘关系很近,核苷酸同源性99.5%～100%, 氨基酸同源性98.5%～100%; 强毒株与B株亲缘关系较近, 核苷酸同源性96.6%,氨基酸同源性97.5%;弱毒株与强毒株亲缘关系较远,核苷酸同源性92%～93%,氨基酸同源性96%～97.5%;弱毒株与B株亲缘关系最远,核苷酸同源性92.5%～93%,氨基酸同源性93.9%～95.5%。说明强毒株与弱毒株之间核苷酸序列存在差异。

鹅细小病毒VP1-VP3非重叠序列地高辛探针的制备和应用

根据 GPV H1株核苷酸序列 ,设计了扩增 VP1- VP3基因非重叠序列的 1对引物 ,对其结构蛋白 VP1与 VP3非重叠核苷酸序列进行 PCR扩增 ,将 PCR产物纯化、回收后制备出 GPV VP1- VP3基因 DIG标记核酸探针 ,其标记效率达到0 .1pg/μl。特异性检测结果表明 ,该探针能与 GPV不同毒株核酸发生特异性杂交 ,而与对照的 DPV、GPMV等病毒的核酸杂交反应均为阴性 ;敏感性检测结果表明该探针对 GPV的最低检出量为 0 .0 32 ng。上述试验结果表明该探针可以用于

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.

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.

1株3型鸭甲肝病毒的VP1和VP3基因的密码子使用偏性分析

为了解临床分离的1株3型鸭甲肝病毒的结构蛋白VP1和VP3基因的密码子使用特性,本文对该毒株的VP1和VP3基因与从NCBI下载的同源性较高的3型鸭甲肝病毒进行了密码子偏性分析。结果表明,该毒株的VP1和VP3与所比对的5株3型鸭甲肝病毒VP1和VP3基因密码子使用模式基本一致,分别确定了12种和13种高频密码子,且偏爱以A或U结尾。通过6株3型鸭甲肝病毒的VP1和VP3基因的密码子偏性比较和聚类分析发现,其中4株分类地位相近。

鸭3型甲肝病毒的分离鉴定与VP1基因序列分析

【目的】探明引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原及其遗传进化特征。【方法】对安徽省某鸭场的病死雏鸭中采集的出血肝脏开展鸭已知病原核酸检测、病原分离鉴定和动物回归试验,在明确其病原为鸭3型甲肝病毒(Duck hepatitis A virus type 3,DHAV-3)的基础上分析其VP1基因序列分子特征。【结果】细菌分离结果显示,未分离到细菌;经病毒核酸(RT-)PCR检测结果显示,鸭3型甲肝病毒(DHAV-3)核酸阳性,未检测出其他已知引起鸭肝出血的病毒核酸。将该阳性样品经鸭胚进行病毒分离与传代,发现接种后鸭胚发生死亡,胚体全身出血,对第5代尿囊液经RT-PCR检测为DHAV-3,将其命名为AH230225。经测定,该分离株的鸭胚半数致死量(Effective lethal dose 50,ELD_(50))为10^(−4.17)/0.1 mL。动物回归试验表明,该毒株对樱桃谷雏鸭的致死率为80%,且攻毒死亡鸭肝脏和肾脏的剖检病变与临床典型病变相近。对该分离毒的VP1基因核苷酸序列进行同源性分析,显示AH230225株的VP1基因核苷酸序列与AH07株DHAV-3(安徽分离株)的同源性最高,为98.8%,与GenBank登录的10株DHAV-3分离株VP1基因核苷酸序列同源性为90.4%~98.8%,而与DHAV-1和DHAV-2的VP1基因核苷酸序列同源性分别为62.1%~63.0%、64.6%~64.9%;基于VP1蛋白氨基酸序列的遗传进化显示,该分离株与AH07株DHAV-3处于同一小进化分支上,亲缘关系最近;而与SD01株、G株和韩国株(AP-04009、AP-03337)等亲缘关系较远,即远离DHAV-1和DHAV-2进化分支。【结论】引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原为鸭3型甲肝病毒DHAV-3,同时明确了该毒株VP1基因的分子特征及遗传进化规律,为深入研究DHAV-3的致病机制和制定防控措施提供科学依据。

2020—2023年山东地区13株3型鸭甲型肝炎病毒的分离鉴定及VP1基因序列分析

为研究2020—2023年山东地区3型鸭甲型肝炎病毒的流行变异情况,试验采集了山东地区13份疑似鸭肝炎病毒感染的病料,通过病毒分离、RT-PCR扩增鉴定,并进行VP1基因测序及遗传变异分析。结果显示:共分离到13株3型鸭甲型肝炎病毒,均属于GⅠa分支,且分属于GⅠa分支不同小分支,其中2020年分离株(20148、20149)与2021年商品肉鸭分离株(SP21042)核苷酸相似性在98.8%~100%;2023年商品鸭分离株(SP23012、SP23014、SP-2、SP-3、SP23009)核苷酸相似性在99.7%~100%;2021年种鸭分离株(21083)与2022年、2023年种鸭分离株(22443、22468、BL23028)、2023年商品鸭分离株(SP23010)核苷酸相似性在98.8%~99.4%,小分支内相似性均高于98.8%,不同小分支间相似性在96.9%~98.2%;2023年部分3型DHAV分离株VP1基因出现了个别氨基酸位点的突变。研究表明,山东地区分离的13株GⅠa分支鸭甲型肝炎病毒VP1基因出现了变异,需要加以关注。

Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1(DHAV-1) Isolates in Shandong Province of China

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD 50 s) and the median lethal doses(LD 50 s),respectively.The results showed that the ELD 50 s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10 6 /mL to 1.44 × 10 7 /mL,while the LD 50 s were 2.39 × 10 5 /mL to 6.15 × 10 6 /mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(aa 158-160,180-193 and 205-219) and other variable points in VP1 protein,but which didn't cause virulence of DHAV-1 change.

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.

Experimental Study on the Antitumor Effect of Chicken Anemia Virus vp3 Gene against Liver Carcinoma In Vivo

In order to testify the antitumor effect, especially its effect against liver carcinoma in vivo, of VP3 protein, one kind of protein coded by chicken anemia virus, recombinants pcDNA-vp3 containing chicken anemia virus vp3 gene, and control vector pcDNA3 were mixed with murine liver carcinoma cell lines H22 respectively. The mixture was injected subcutaneously into Balb/C mice. Some days later, the mice were killed and the solid tumor weighed. The antitumor efficiency was evaluated. The manners of VP3 protein in vivo inducing tumor cell death were identified by using TUNEL assay. All the results suggested that the injection of pcDNA-vp3 and H22 mixture resulted in a significant reduction of tumor growth in mice when compared with the results of control groups. TUNEL assay revealed that VP3 induced apoptosis in vivo. All these indicated that CAV vp3 might be a potential new gene in reducing the growth rate of tumor cells in liver carcinoma or in other kind of solid tumors in vivo.

穗发芽率和发芽指数及STS标记Vp1B3在小麦抗穗发芽基因型鉴定中的应用

为了筛选抗穗发芽品种(系)并了解其抗性机制,应用整穗发芽法和发芽指数鉴定了33份小麦新品系的穗发芽抗性,以红粒品种京冬8号和京9428作为抗穗发芽对照,以白粒品种京411和中优9507作为感穗发芽对照,并结合共显性STS标记Vp1B3对其基因型进行检测。结果表明,穗发芽率(SGR)低于抗性对照京冬8号和京9428平均值(12.7%)的品系有7份,其中2份为白粒,5份为红粒。只有一份红粒品系CA0489的发芽指数(GI)低于抗性对照平均值(13.2%)。应用STS标记Vp1B3共扩增出849 bp、569 bp和652 bp三种片段,分别属于抗穗发芽基因型Vp1Bb和Vp1Bc及感穗发芽基因型Vp1Ba,其频率分别为3%、18%和79%。红粒抗穗发芽品系CA0489属于Vp1Bb基因型和红色种皮休眠型,CA0481属于Vp1Bc抗穗发芽基因型,另外三份红粒抗穗发芽品系的抗性机理还需要进一步研究;白粒抗穗发芽品系CA0509和CA0459的抗性为非Vp1B3类型,其抗性可能与穗部性状和颖壳抑制物有关。

柯萨奇病毒A16型VP1-VP4基因克隆及其表达产物的抗原相关性分析

目的克隆并表达柯萨奇病毒A16型VP1-VP4蛋白基因,初步分析其抗原相关性。方法抽提病毒RNA,经RT-PCR方法分别扩增出VP1-VP4蛋白基因片段,经克隆后,在QIA表达系统中表达,表达产物经8 mol/L尿素洗涤及镍柱亲和层析纯化后,用柯萨奇病毒A16型病毒免疫兔血清及肠道病毒71型病毒免疫兔血清对重组蛋白进行Western blotting及ELISA分析其抗原相关性及交叉反应性。结果成功构建的重组质粒pQE30a/VP1-VP4并经IPTG诱导,重组蛋白VP1-VP4高效表达并纯化成功,经Western blotting及ELISA证实重组蛋白VP1-VP4可以被CVA16免疫兔血清特异识别,部分与肠道病毒71型免疫血清存在交叉反应。结论在大肠杆菌M15中高效表达出柯萨奇病毒A16型VP1-VP4蛋白,经纯化产物具有较强的抗原反应性。

I型鸭肝炎病毒VP1、3D基因克隆及其在大肠杆菌中的表达

根据GenBank中的I型鸭肝炎病毒全基因序列设计了扩增I型鸭肝炎病毒VP1、3D基因的引物,用该特异性表达引物从I型鸭肝炎病毒cDNA模板中扩增得到目的基因VP1、3D,用相同的限制性内切酶酶切目的基因和表达载体pET32a后构建重组表达载体,转化宿主BL21(DE3),用不同浓度的IPTG诱导VP1、3D基因的表达,收集菌液进行SDS-PAGE电泳,Western-blotting分析蛋白免疫原性。结果表明,VP1、3D在大肠杆菌中表达量较高,表达产物的分子量约为48kD、68kD,并能被兔抗DHV-1血清所识别。I型鸭肝炎病毒VP1、3D蛋白在大肠杆菌中表达产物具有免疫原性。

柯萨奇B3病毒VP1基因在大肠杆菌中的表达

本文通过融合柯萨奇Ｂ３病毒（ＣＶＢ３））ＶＰ１基因与人凝血酌基因，仗ＣＶＢ３的ＶＰ１在大肠杆菌中得到稳定的表达。经蛋白扫描仪等测定，表达的融合蛋白占菌体可溶性蛋白的１１％左右。表达的ＶＰ１产物勺抗ＣＶＢ３ＶＰ１单克隆抗体和小鼠抗ＣＶＢ３血清产生较强的免疫反应，与天然的ＣＶＢ３蛋白抗原性相同。应用无关单抗和载体质粒对照均呈现阴性反应。应用表达的ＶＰ１作为抗原，我们对临床部分急性病毒性心肌炎患者血清进行了特异性ＩｇＭ免疫印迹法检测，其结果与组织培养的ＣＶＢ３制备的蛋白抗原对患者血清的特异性ＩｇＭ反应基本一致。采用基因上程方法制备ＣＶＢ３抗原产最高，易纯化，免疫原性没有改变。故可以试用表达的ＶＰ１抗原代替目的有实验室感染危险的病毒培养及抗原制备方法，快速、特异地诊断ＣＶＢ３感染。