口蹄疫病毒VP1基因在番茄中的表达及转基因番茄对豚鼠诱导免疫保护

构建克隆有O型口蹄疫病毒China99株VP1基因的植物双元表达载体pBin438/VP1。通过农杆菌介导法转化番茄子叶,经卡那霉素抗性筛选,获得60株抗性植株。对抗性植株分别做PCR、RT-PCR检测目的基因的整合与转录,ELISA筛选约40%的卡那抗性植株阳性,分别提取两株ELISA和Western blot检测阳性的转基因番茄叶片蛋白与弗氏佐剂乳化,于0、15、30d经肌肉途径免疫豚鼠,第三次免疫后28d用100ID50的同源强毒攻击,根据豚鼠抗体水平的消长动态和免疫豚鼠抗强毒攻击的保护率进行转基因植物疫苗免疫原性的评估。结果表明,双元表达载体pBin438/VP1构建正确,PCR、RT-PCR结果证实口蹄疫病毒VP1基因已整合到番茄基因组并在转录水平表达,ELISA和Western blot检测重组蛋白能够与FMDV阳性血清反应。转基因番茄第三次免疫豚鼠后21d血清效价最高可达1∶64,攻毒后两组免疫豚鼠保护率分别达80%和40%,证明转基因番茄表达的VP1蛋白具有良好的免疫原性。     

FMDV vp1基因在烟草中表达及转基因烟草的免疫效果

通过三亲杂交法,构建克隆有阿克苏(Akesu/58)O型口蹄疫病毒vp1基因的双元表达载体pBin FMDV VP1.采用农杆菌介导法转化NC89烟草叶盘,经卡那霉素筛选,共获得49株抗性植株.对抗性植株总DNA进行目的基因的PCR检测,有40株阳性植株.对阳性植株总RNA进行目的基因的RT-PCR检测,有21株阳性植株.将7株ELISA和Western-blot检测阳性植株叶片提取物分别与弗氏佐剂乳化,在0、15、30和45d腹膜腔接种Balb/C小白鼠,于第4次免疫后第9d进行血清抗体检测;第12d用10 4SM\-\{50\}LD的同源强毒进行攻击;攻毒后24h采血,通过乳鼠病毒血症试验判定攻击Balb/C小白鼠的发病和保护情况.结果表明双元表达载体pBin FMDV VP1构建正确;vp1基因转入NC89烟草并获得表达;7组中有2组Balb/C小白鼠血清抗体呈阳性,攻毒保护率分别为100%和63%.证明2株转基因烟草表达的VP1蛋白具有较好的免疫原性,所免疫的2组Balb/C小白鼠对同源强毒攻击有一定的抵抗能力.     

FMDV vp1基因在烟草中表达及转基因烟草的免疫效果

通过三亲杂交法,构建克隆有阿克苏(Akesu/58) O 型口蹄疫病毒vp1 基因的双元表达载体pBin FMDVVP1。采用农杆菌介导法转化NC89烟草叶盘,经卡那霉素筛选,共获得49株抗性植株。对抗性植株总DNA进行目的基因的PCR检测,有40株阳性植株。对阳性植株总RNA进行目的基因的RT PCR检测,有21株阳性植株。将7株ELISA和Western blot检测阳性植株叶片提取物分别与弗氏佐剂乳化,在0、15、30 和45d 腹膜腔接种Balb/C小白鼠,于第4次免疫后第9d进行血清抗体检测;第12d用104SM50 LD的同源强毒进行攻击;攻毒后24h采血,通过乳鼠病毒血症试验判定攻击Balb/C小白鼠的发病和保护情况。结果表明:双元表达载体pBin FMDVVP1构建正确;vp1基因转入NC89烟草并获得表达;7组中有2组Balb/C小白鼠血清抗体呈阳性,攻毒保护率分别为100%和63%。证明2株转基因烟草表达的VP1蛋白具有较好的免疫原性,所免疫的2 组Balb/C小白鼠对同源强毒攻击有一定的抵抗能力。     

共表达口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因牛肾细胞株的筛选及稳定性

[目的]筛选稳定表达口蹄疫病毒衣壳蛋白的牛肾细胞(Madin-Darby bovinekidney,MDBK)株.[方法]采用聚合酶链式反应(Polymerase chain reaction,PCR)方法从重组质粒pMD-P1-2A和pMD-3C中分别扩增口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因,将两基因依次插入逆转录病毒载体pBABE-puro.重组逆转录病毒载体pBABE-puro/P1-2A-3C和pVSV-G质粒载体用脂质体介导共转染GP2-293包装细胞.产生的重组逆转录病毒感染MDBK细胞后使用嘌呤霉素筛选抗性细胞.利用克隆环套取法得到单克隆细胞.经间接免疫荧光和酶联免疫吸附测定(Enzyme-linkedimmunosorbent assay,ELISA)方法检测MDBK细胞中衣壳蛋白的表达,并在电镜下观察口蹄疫病毒空衣壳.[结果]成功筛选到稳定表达口蹄疫病毒衣壳蛋白的MDBK细胞株,衣壳前体蛋白P1-2A在蛋白酶3C裂解作用下正确组装成空衣壳.[结论]该研究为口蹄疫亚单位疫苗的研制提供了实验材料.     

口蹄疫病毒多基因重组质粒的体外表达及衣壳组装

PCR扩增获得包含口蹄疫病毒P1、2A、3C、3D及部分2B编码区的目的基因片段P12X3C3D,将P12X3C3D经AflⅡ和XbaⅠ双酶切后,定向克隆于真核表达质粒载体pcDNA3.1( );另将PCR扩增获得的P12X3C3D直接与真核表达质粒载体pTARGET^TM连接,进行筛选、鉴定及DNA序列分析,分别获得重组质粒pcDNA3．1／P12X3C3D、pTARGET／P12X3C3D。将重组质粒分别转染BHK-21细胞,通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中表达的口蹄疫病毒抗原,用磷钨酸负染,以电子显微镜观察转染重组质粒的细胞中组装的口蹄疫病毒空衣壳。结果表明,口蹄疫病毒基因组片段正确克隆到真核表达质粒载体上,重组质粒pcDNA3．1／P12X3C3D、pTARGET／P12X3C3D均可在BHK-21细胞中表达FMDV目的蛋白。其中重组质粒pTARGET／P12X3C3D表达的口蹄疫病毒抗原蛋白,能够在细胞内正确组装成病毒空衣壳。     

番茄花叶病毒移动蛋白和番茄抗病基因Tm-2^2的互作诱导烟草转化体程序性细胞死亡

番茄的抗病基因Tm -2 2 与番茄花叶病毒 (ToMV)的移动蛋白MP基因是一对互作的基因 ,Tm- 2 2 基因和ToMV MP基因同时在烟草中表达 ,并分别获得单一基因整合的纯合转化体植株。病毒接种试验表明 ,Tm -2 2 基因转化体与Tm- 2 2 番茄对Tobamavirus病毒的特异抗性结果一致 ;Tm -2 2 转基因植株和ToMV MP转基因植株杂交试验及其农杆菌注射试验均证明 :(1)Tm -2 2 基因与ToMV- MP在转基因烟草上保持“基因对基因”的互作关系 ;(2 )在外源乙烯的参与下 ,ToMV的移动蛋白与Tm -2 2 基因编码蛋白的互作能够诱导转化体程序性细胞死亡。这一结果为今后研究Tm -2 2 与MP互作的分子机制奠定了基础。     

表达黄瓜花叶病毒外壳蛋白的转基因番茄抗黄瓜花叶病毒侵染

通过土壤农杆菌(Agrobacterium tumefaciens)介导将黄瓜花叶病毒外壳蛋白(CMV CP)的cDNA成功地引入番茄(Lycopersicon esculentum)植株中,并得到转基因植株。用强致病力CMV株系接种后,表达CMV外壳蛋白的转基因植株表现出对CMV侵染的抗性。转基因植株RI代的抗性基因以接近3:1比例分离。对R_1代接种CMV后,表达CMV CP的植株病症减轻,发病率、病情指数及病毒积累量明显低于对照。病症出现推迟1个多月。     

共表达O型口蹄疫病毒P1-2A基因和猪白细胞介素18的重组鸡痘病毒的构建

目的:为获得能有效预防O型口蹄疫病毒的重组鸡痘病毒活载体疫苗奠定基础。方法：在O型口蹄疫病毒P1-2A基因上游引入Kozak序列,下游通过Linker与细胞因子IL-18联结,获得P1-2A基因与猪IL-18基因融合表达基因盒P1-2A-IL-18,将该表达基因盒克隆至鸡痘病毒中间转移载体pUTAL-3C中,构建重组鸡痘病毒转移载体质粒pUTAL-3C- P1-2A-IL-18。通过脂质体转染法,将pUTAL-3C- P1-2A-IL-18与鸡痘病毒282E4株共转染鸡胚成纤维细胞（CEF）,通过BrdU三次加压筛选,挑选出单克隆重组病毒株。结果：经RT-PCR和间接免疫荧光法鉴定,证明所筛选的1株重组鸡痘病毒在CEF中能正确表达P1-2A-IL-18基因盒。结论：成功获得了一株共表达O型口蹄疫病毒P1-2A基因和猪白细胞介素18基因的重组鸡痘毒疫苗候选株rFPV-3C-P1-2A-IL-18。     

表达苏云金杆菌5－内毒素基因的转基因烟草的抗虫性

苏云金杆蓠(Bacillus thuringiensis)HD-1的δ-内毒索基因原始克隆THl2和TH48分别含有5．3kb和6．6kb型类同源异族基因。经定点突变改造其5’端,酶切对3’端进行缺失后,在大肠杆菌中仍能表达出有杀虫活性的被修饰的毒蛋白。将上述加工过的基因插入到双元载体pBin437(pBinl9的派生质粒)中,借助辅助Ti质粒(Pal,1404转人烟草,获得了抗卡那霉索的再生植株。经southern印迹和狭槽印迹(slot blot)杂交证明有1-5个拷贝的毒素基因整合至烟草染色体中。Northern印迹法分析结果表明这两类基医都在转基因植株中得到表达。育4株5．3kb类型,3株6．6kb类型的转基因植株对烟青虫有高抗性,与对照相比,这7株转基因烟草植株的杀虫率可达40一50％,并能显著抑制昆虫蜕皮和生长发育,表现明显的抗虫作用。结果表明这两类毒蛋白基因都可在植物中表达并赋予转基因植株以抗虫性。     

植物多基因干扰载体体系构建与效用分析

多数重要的功能基因属于多基因家族,这些家族成员间存在功能冗余,高效的多基因干扰体系对研究多基因家族成员的生物学功能及其分子调控机制具有重要意义。对pCAMBIA1301载体改造,构建了适用于植物的多基因干扰体系pCAMBIA1301m和pCAMBIA1301s。使用该多基因干扰体系构建了四基因的干扰载体pCAMBIA1301m：35S∷SlPP2C1-2-3-4,4个目标基因为来源于番茄PP2C家族A组的PP2C1、PP2C2、PP2C3和PP2C4,并通过遗传转化导入番茄,用GUS染色和PCR检测转基因阳性植株,再利用RT-qPCR技术检测T₁和T₂代转基因植株中目标基因的干扰效率,用T₂代种子分析转基因番茄对ABA敏感性。结果表明,应用该干扰体系成功获得了四基因干扰的转基因植株35S∷SlPP2C1-2-3-4。在转基因番茄中4个目标基因的表达量显著低于野生型,其干扰效率均高于70%,转基因番茄种子萌发具有强烈的ABA不敏感性。多基因干扰体系能高效地同时沉默多个目标基因。     

The Effect of Bovine IFN-α on the Immune Response in Guinea Pigs Vaccinatedwith DNA Vaccine of Foot-and-Mouth Disease Virus

Foot-and-mouth disease virus (FMDV) belongs to thegenus Aphthovirus of the family Picornavidae. The FMDVgenome is a copy of positive-sense, single-stranded RNA,which contains one large open reading frame (ORF). TheORF is translated into a polypeptide, which undergoesautoproteolytic cleavage to produce the structural and non-structural proteins and ultimately forms mature viral pro-teins [1,2]. FMD is caused by the FMDV, which is a highly conta-gious vesicular disease of cloven-hoofe…     

重组鸡痘病毒和DNA疫苗对口蹄疫病毒的豚鼠免疫保护

将构建的携带FMDV衣壳蛋白P1-2A和蛋白酶3C编码基因的重组鸡痘病毒活载体疫苗vUTAL3CP1以及编码FMDVP1-2A基因和猪IL-18基因的重组DNA疫苗pVIRIL18P1,分别以单独和混合的方式给豚鼠进行2次免疫,然后测定FMDV特异性结合抗体、中和抗体和T淋巴细胞增殖反应,并用250ID50的FMDV进行攻击,观察其保护效果。结果表明这2种基因工程疫苗均能诱导豚鼠产生特异性的体液免疫及细胞免疫应答。其中以vUTAL3CP1两次免疫组的效果最好,其诱导的抗体水平已接近于常规灭活疫苗,而细胞免疫水平则比后者高得多。攻击保护结果表明该组完全保护率可达3/4,而另外两组也具有一定保护效果。上述研究结果为进一步进行大动物免疫攻毒试验,并最终筛选出最佳疫苗和免疫程序奠定了基础。     

牛α-IFN及FMDV P12A3C双表达载体的构建及其在BHK-21细胞中的表达

从口蹄疫病毒Asia I/Jiangsu毒株的细胞毒中提取总RNA,通过RT-PCR方法分别获得FMDV的P12A及3C基因;同时以pMD18-T-α-IFN质粒为模板,PCR扩增得到α-IFN基因.将α-IFN基因及FMDV P12A及3C基因连接至双启动子表达载体pBudCE4.1上,构建成双效表达质粒pBudCE4.1-α-IFN-P12A3C,经电泳、PCR、双酶切和DNA测序鉴定表明双效质粒载体构建成功.用此重组质粒转染BHK-21细胞后并对其表达情况进行检测,表明该双表达质粒在BHK-21细胞中能够成功表达.以此重组质粒免疫乳鼠后12 h,按100 TCID50/O.1 mL的量进行攻毒,结果发现该质粒能够抑制病毒的增殖,对乳鼠有一定的保护作用.结果表明成功构建了牛α-IFN及FMDV P12A3C组合基因双表达载体,为进一步研究口蹄疫基因疫苗提供前期基础.     

Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.     

口蹄疫病毒多基因重组质粒在BHK-21细胞中的表达

利用定点突变的原理,获得包含有口蹄疫病毒P1,2A,3C及部分2B编码区的目的基因片段,KpnⅠ和XbaⅠ双酶切后,定向克隆于真核表达质粒载体pcDNA3.1（+）,经筛选、鉴定及DNA序列分析后,将重组质粒pcDNA3.1/P12X3C转染BHK-21细胞,通过双抗体夹心ELISA方法和间接免疫荧光标记方法,检测细胞中表达的口蹄疫病毒抗原。结果表明,口蹄疫病毒基因片段正确克隆到真核表达质粒载体上,重组质粒pcDNA3.1/P12X3C可在BHK-21细胞中表达FMDV目的蛋白。     

Systemic wound signaling in tomato leaves is cooperatively regulated by systemin and hydroxyproline-rich glycopeptide signals

Hydroxyproline-rich glycopeptides (HypSys peptides) have been isolated recently from tobacco and tomato leaves that are powerful activators of protease inhibitor synthesis. The peptides are processed from polyprotein precursors, two from a single tobacco precursor and three from a single tomato precursor. The precursor genes are expressed in response to wounding and methyl jasmonate, similar to the expression of the systemin precursor prosystemin in tomato leaves. Here we investigate the relationships between systemin and the tomato HypSys peptides in regulating wound signaling in tomato plants. Analysis of transgenic tomato plants over-expressing sense and antisense constructs of the tomato HypSys precursor under the 35S CaMV promoter show that the transgenic plants regulate protease inhibitor gene expression in response to wounding in a manner similar to prosystemin. The evidence indicates that the expression of both the tomato HypSys precursor gene and the prosystemin gene in response to wounding are necessary for strong systemic signaling. The data supports a role for both genes in an amplification loop that up-regulates the octadecanoid pathway and the synthesis of jasmonates to effect strong systemic signaling of defense genes. This report provides the first demonstration of the involvement of two plant peptides derived from two unrelated genes in regulating long distance wound signaling in plants. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors () is Clarence A. Ryan.     

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS and NPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) using Agrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced the time required for production of transformed peanut plants. Transgenes were stably integrated into the peanut genome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanut were observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delay in symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. All transgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformed control plants and plants transformed with a gene cassette not containing the nucleocapsid gene became systemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate that protection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the sense RNA of the tomato spotted wilt virus nucleocapsid gene