Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

A novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini‐intein variant engineered for hyper‐N‐terminal autocleavage is covalently linked to the foot‐and‐mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an ‘IntF2A’ self‐excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POIs) from a ‘polyprotein’ precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function and accumulated at similar levels inside the cells. Importantly, no C‐terminal F2A extension remains on the released POIs. We demonstrated co‐expression of as many as three proteins in plants without compromising expression levels when compared with those using single‐protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti‐His Tag antibody in N. benthamiana leaves. The IntF2A‐based polyprotein transgene system overcomes key impediments of existing strategies for multiprotein co‐expression in plants, which is particularly important for gene/trait stacking.     

口蹄疫病毒基因组内部核糖体进入位点一级和二级结构分析

以口蹄疫病毒(foot-and-mouth disease virus,FMDV)强毒China/99株牛舌水泡皮为材料,用RT-PCR法提取RNA及扩增目的cDNA,然后与pGEM-T Easy载体连接并转化JM109菌株,再经重组质粒电泳、PCR和EcoRI酶切鉴定.用DNAstar软件比较了内部核糖体进入位点(IRES)的序列差异,并用RNAdraw软件绘制和分析了该区段的二级结构.8株FMDV IRES核苷酸序列比较表明该区段较为保守,并对非保守区域进行了分析.二级结构分析表明,FMDV IRES至少有3种二级结构图形:第一型有5个结构域,与Pilipenko等报道的一致;第二和三型分别有6和11个结构域,与Pilipenko等报道的结果不同.无论FMDV IRES二级结构如何不同,但单链区大部分核苷酸序列或基序相同,如AACUCC、GAAA、CUUU、AGG、AACC、GUAA等.茎环柄部核苷酸对维持二级结构的空间构像具有十分重要的作用,环中或单链区序列(基序)在维持其功能方面具有很重要的作用,如GAAA和CUUU基序分别是三级结构的组件和嘧啶区结合蛋白的结合位点.     

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。     

The Solution Structure of the Immunodominant and Cell Receptor Binding Regions of Foot-and-mouth Disease Virus Serotype A,Variant A

The solution structure of a 20 amino acid long peptide corresponding to the region 141–160 of the envelope protein Vp1 from foot-and-mouth disease virus (FMDV) serotype A, variant A, has been determined by a combination of NMR experiments and computer calculations. The peptide contains both the immunodominant epitope as well as the sequence (RGD) used by the virus to bind the cell receptor in the initial stages of infection. These two sites have been shown to partially overlap. One hundred and thirty-five NMR distance constraints were used to obtain a set of 11 structures by distance geometry, minimization and molecular dynamics simulations. These structures were divided into two homogeneous families based upon backbone superimposition. The first and most populated family was characterized by a backbone RMS of 1.5±0.4 Å, the second by a backbone RMS of 0.8±0.2 Å. The two families had similar structural features and differed mainly in the backbone angles of G149. In the larger of the two families these angles favoured the formation of a loop comprising residues 147 to 152 and stabilized by a H-bond between the NH of D147 and the CO of A152. In the second family, where this bond was absent, the peptide adopted in this region the shape of an irregular helix. The C-terminal half of the peptide (152–159) was similar in both families and largely helical. Similar structural features were also found within the VRGDS sequence (144–148) which was assigned to a β-turn type IV. The features of the two families of structures were found to be different from those of the recently published X-ray structure of the antigenic loop of a chemically modified form of FMDV. Proposals accounting for these differences are provided which take into account the dual activity of the 141–160 sequence (i.e. antibody binding and cell invasion through receptor binding).     

含RGD受体结合位点口蹄疫病毒Asial/JS/China/2005株的拯救及初步鉴定

[目的]构建含有精氨酸.甘氨酸.天冬氨酸(RGD)受体结合位点口蹄疫病毒(FmDV)Asial/JS/Chind2005株的全长感染性cDNA克隆.[方法]采用定点突变方法,构建Asial型FMDV含有预期突变的全长cDNA克隆pFMDV-RGD.pFMDV-RGD重组质粒经NotI线化后,与表达T7 RNA聚合酶的真核质粒peDNATIP共转染BHK-21细胞,进行FMDV-RGD病毒拯救.[结果]序列测定结果表明成功构建了FMDV含有RGD受体位点的Asial/JS/China/2005全长cDNA克隆.共转染试验获得拯救病毒,对拯救的病毒分别进行序列测定、间接免疫荧光、电子显微镜观察和乳鼠致病性分析,表明成功拯救了含有RGD受体结合位点的Asial/JS/China/2005株FMDV.[结论]该试验为进一步研究含有RGD和RDD受体结合位点2个拯救病毒生物学特性的差异奠定了基础.     

口蹄疫病毒结构蛋白P1-2A及蛋白酶3C基因的转基因番茄对豚鼠免疫反应的初步研究

构建了O型口蹄疫病毒China99株结构蛋白P1-2A、非结构蛋白3C以及部分2B基因(P1-2X3C)的植物双元表达载体pBin438/P1-2X3C,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40余株抗性植株,对得到的抗性植株进行分子生物学检测,65%的再生植株PCR检测阳性;RT-PCR结果证实P1-2X3C基因在转基因番茄中能够有效转录;ELISA和Western blot检测表明转基因植株中表达的目的蛋白具有免疫反应性。转基因番茄叶片蛋白粗提液经肌肉途径免疫豚鼠,于第3次免疫后28d用100ID50/0.2mL的同源强毒攻击,结果表明口蹄疫病毒P1-2X3C基因的转基因番茄表达产物具有良好的免疫原性,豚鼠3免后血清效价可达1:64~1:128,攻毒后两组免疫豚鼠保护率分别达3/5和5/5。     

口蹄疫病毒3D基因的克隆表达及功能分析

利用RT-PCR技术扩增了口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因,并将其克隆到原核表达质粒载体pET-28a( )中。3D基因经测序确认后在大肠杆菌BL-21中表达,表达产物纯化的目的蛋白进行Western-blotting检测,获得分子量约55KDa的单一3D基因表达产物。利用RNA体外复制体系和荧光定量PCR技术,证明纯化的3D基因表达产物RNA依赖的RNA聚合酶具有较高的酶活性,可以在体外从头合成FMDVRNA,且主要以引物依赖的方式合成病毒基因组。     

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。     

核糖体展示口蹄疫单链抗体库的构建

目的:构建库容量大、多样性好的核糖体展示口蹄疫单链抗体(scFv)库。方法: 分离口蹄疫病毒免疫的兔脾细胞,提取总RNA,用RT-PCR扩增兔抗体的重链可变区(VH)基因和轻链可变区(VL)基因,同时扩增作为间隔区的兔抗体Ck基因;采用重叠延伸PCR (简称SOE-PCR)技术连接VH-VL基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接pMD18-T载体转化E.coli DH5α大肠杆菌,挑取阳性克隆测序以鉴定scFv组装。结果:成功构建了库容量达8.21×10¹³的兔源口蹄疫核糖体展示scFv库。结论: 构建的大容量兔源性口蹄疫核糖体展示抗体库可以成为进一步筛选特异性口蹄疫单链抗体的实验平台,为开发诊断性口蹄疫单链抗体奠定了很好的实验基础。     

FMDV OH99株基因组全序列的测定及其基因特征研究

采用RT-PCR方法对FMDV OH99株基因组全序列进行了分子克隆与测序。结果表明OH99株基因组全基因组序列长8040nt,其中5’NCR长1026nt,前导蛋白(L)编码区长603nt。该毒株结构蛋白与非结构蛋白编码区的核苷酸序列为6318nt,3’NCR长93nt,其后是poly(A)尾巴,测序结果表明该结构至少含有56个A。应用分子生物学软件,将OH99株与其它参考毒株进行了序列比较,并对其基因特征、推导的氨基酸序列进行了研究分析。结果显示,在分类地位上OH99株归属于O型FMDV,与OTY TW／97具有较高的同源性,而与其他参考毒株的差异性比较大,而且在基因组功能未知区域和3A编码区域具有两处明显的基因片段缺失现象,其中3A编码区缺失30nt,与OTY TW/97株相同,但功能未知区域的缺失状况与OTY TW／97稍有差异。根据VP1基因序列,对OH99株与参考毒株进行了系统发生树分析,分析结果表明OH99株与0TY TW／97株在同一基因型内,其遗传关系最近,而与其毒株遗传关系较远。