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。     

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。     

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.     

霍乱肠毒素B亚单位在转基因番茄中表达的研究

将霍乱肠毒素B亚单位（CT－B）基因及内质网引导序列（SEKDEL）克隆到质粒pRTL2和pBI121中,分别构建植物双元表达载体pBI-CTB和pBI-CTBK,CT-B基因由Ca35S启动子控制表达。采用叶盘法经根癌农杆菌介导转化番茄（金丰1号,Jinfeng1)各表达载体得到一批转基因植株。经PCR和Southern blot分析表明CT－B基因整合到了番茄基因组中;ELISA和Western blot分析表明pBI-CTB和pBI-CTBK的转基因植株能够有效表达CT－B多肽,分别占番茄叶片可溶性蛋白的0.055%和0.084%。     

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。     

利用柱花草为受体表达口蹄疫病毒外壳蛋白VP1的研究

将口蹄疫病毒外壳蛋白VP1基因克隆到植物表达载体pBI121,并转化到根癌农杆菌(Agrobacteriumtumefaciens)菌株LBA4404中,采用叶盘转化法转化柱花草(Stylosanthesspp.)栽培品种热研二号柱花草(S.guianensiscv.ReyanⅡ),获得了转基因植株,经PCR、PCR-Southern blot和Southern blot分析表明VP1基因已整合到转基因柱花草植株的核基因组中。经RT-PCR、Northern blot分析表明VP1基因已在转基因柱花草中获得转录。     

TaNHX2基因植物表达载体的构建及在拟南芥中的功能分析

将TaNHX2基因重组于质粒pBIN438的CaMV 35S启动子下游,构建含TaNHX2基因的植物双元表达载体pBIN438-TaNHX2。采用根癌农杆菌介导的真空渗透法转化拟南芥,得到T0代转基因拟南芥种子。经含Kan的平板筛选及PCR鉴定,获得54株阳性植株,选取生长一致的转基因阳性植株进行耐盐、耐旱分析,结果表明TaNHX2能够提高转基因植株的耐盐性和耐旱性。     

轮状病毒外壳蛋白VP7在转基因番茄果实中的特异表达

将轮状病毒外壳蛋白VP7基因克隆到含有番茄果实特异性启动子TFP的植物表达载体pTF ,并转化到根癌农杆菌 (Agrobacteriumtumefaciens)菌株EHA10 5中 ,采用叶盘转化法转化番茄 (LycopersiconesculentumMill.)栽培品种TX0 0 14 ,获得了转基因植株。经PCR、PCR Southernblot和Southernblot分析表明VP7基因已整合到转基因番茄植株的核基因组中 ,RT PCR、Westernblot结果表明VP7蛋白在果实中获得了特异表达     

O型口蹄疫病毒VP1 T细胞、B细胞表位双拷贝基因与大肠杆菌LTB、STⅠ肠毒素基因融合表达产物的免疫应答

合成O型口蹄疫病毒VP1蛋白中与细胞免疫(21～40表位肽)及体液免疫(141～160表位肽)相关的基因序列2020VP1,运用基因工程技术构建了含有肠毒素大肠杆菌LTB、STⅠ基因及双拷贝2020VP1的融合表达载体r2020-B-2020-STⅠ,转化宿主菌 BL21(DE3) RIL后的表达产物经SDS-PAGE分析,结果显示重组融合蛋白的分子量约为45 kDa,表达量较高.ELISA实验结果显示,融合蛋白能与霍乱毒素(cholera toxin)CTB抗体特异结合.动物实验表明,融合蛋白能够诱发兔体产生较强的FMDV中和抗体,免疫豚鼠在低浓度FMDV刺激下能够产生特异性T淋巴细胞增殖反应,说明融合蛋白能诱导机体产生FMDV特异性细胞及体液免疫反应;同时,融合蛋白免疫雌鼠能够抵抗大肠杆菌强毒株攻击,免疫兔体能够产生STⅠ中和抗体,且融合蛋白不具STⅠ毒性,证明融合蛋白具有良好的LTB、STⅠ免疫原性.实验结果表明,此融合蛋白具有开发成为口蹄疫及肠毒素腹泻联合疫苗的应用价值.     

O型口蹄疫病毒VP1 T细胞、B细胞表位双拷贝基因与大肠杆菌LTB、STI肠毒素基因融合表达产物的免疫应答

合成O型口蹄疫病毒VP1蛋白中与细胞免疫（21～40表位肽）及体液免疫（141～160表位肽）相关的基因序列2020VP1,运用基因工程技术构建了含有肠毒素大肠杆菌LTB、STI基因及双拷贝2020VP1的融合表达载体r2020-B-2020-STI,转化宿主菌BL21(DE3)RIL后的表达产物经SDS-PAGE分析,结果显示重组融合蛋白的分子量约为45kDa,表达量较高。ELISA实验结果显示,融合蛋白能与霍乱毒素（choleratoxin）CTB抗体特异结合。动物实验表明,融合蛋白能够诱发兔体产生较强的FMDV中和抗体,免疫豚鼠在低浓度FMDV刺激下能够产生特异性T淋巴细胞增殖反应,说明融合蛋白能诱导机体产生FMDV特异性细胞及体液免疫反应;同时,融合蛋白免疫雌鼠能够抵抗大肠杆菌强毒株攻击,免疫兔体能够产生STI中和抗体,且融合蛋白不具STI毒性,证明融合蛋白具有良好的LTB、STI免疫原性。实验结果表明,此融合蛋白具有开发成为口蹄疫及肠毒素腹泻联合疫苗的应用价值。     

Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells

Background

Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.

Principal Finding

Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID₅₀ of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h.

Conclusion

RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.     

Protective Immune Response to Foot-and-Mouth Disease Virus with VP1 Expressed in Transgenic Plants

It has been reported recently that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. The structural protein VP1 of foot-and-mouth disease virus (FMDV), which has frequently been shown to contain critical epitopes, has been expressed in different vectors and shown to induce virus-neutralizing antibodies and protection in experimental and natural hosts. Here we report the production of transformed plants (Arabidopsis thaliana) expressing VP1. Mice immunized with leaf plant extracts elicited specific antibody responses to synthetic peptides representing amino acid residues 135 to 160 of VP1, to VP1 itself, and to intact FMDV particles. Additionally, all of the immunized mice were protected against challenge with virulent FMDV. To our knowledge, this is the first study showing protection against a viral disease by immunization with an antigen expressed in a transgenic plant.     

猪口蹄疫病毒VP1结构蛋白抗体间接ELISA方法的建立

将口蹄疫病毒(FMDV)的VP1基因,通过pPROex-HT表达载体在大肠杆菌BL21(DE3)中成功表达,获得大小为31ku的融合蛋白,Westernblot检测证实表达的该蛋白具有良好的生物学活性。以纯化的融合蛋白为抗原建立了猪FMDVVP1蛋白间接ELISA检测方法。通过对80份田间血清样品的检测表明,该方法与FMDV液相阻断ELISA(国标试剂盒)的总符合率为96.25%,表明建立的VP1蛋白间接ELISA检测方法具有很好的特异性和敏感性。     

FMDV vp1基因在豆科牧草百脉根中的转化与表达

通过根癌农杆菌介导法,将FMDV阿克苏(Akesu/O/58)株结构基因vp1转化豆科牧草百脉根子叶和子叶柄,其愈伤、芽和生根等过程经50 mg/L Kan筛选后,获得Kan抗性百脉根植株。对抗性植株进行vp1基因的PCR、RT-PCR检测和VP1蛋白的Western-blotting杂交。结果表明:vp1基因转入百脉根中,检测有转录活性;目的蛋白获得了正确表达;扩繁和移栽后获得了批量转基因百脉根,为下一阶段的动物试验提供了实验材料。     

猪口蹄疫病毒VP1结构蛋白抗体间接ELISA方法的建立

将口蹄疫病毒(FMDV)的VP1基因,通过pPROex-HT表达载体在大肠杆菌BL21(DE3)中成功表达,获得大小为31ku的融合蛋白,Western blot检测证实表达的该蛋白具有良好的生物学活性。以纯化的融合蛋白为抗原建立了猪FMDV VP1蛋白间接ELISA检测方法。通过对80份田间血清样品的检测表明,该方法与FMDV液相阻断ELISA（国标试剂盒）的总符合率为96.25%,表明建立的VP1蛋白间接ELISA检测方法具有很好的特异性和敏感性。     

Correlation between efficacy and structure of recombinant epitope vaccines against bovine type O foot and mouth disease virus

To develop recombinant epitope vaccines against foot-and-mouth disease virus (FMDV), genes coding for six recombinant proteins (rP1–rP6) consisting of different combinations of B cell and T cell epitope from VP1 capsid protein (VP1) of type O FMDV were constructed and the 3D structure of these proteins analyzed. This revealed a surface-exposed RGD sequence of B cell epitopes in all six recombinant proteins as that in VP1 of FMDV and rP1, rP2 and rP4 globally mimicked the backbone conformation of the VP1. rP1, rP2 and rP4 stimulated guinea pigs to produce higher level of neutralizing antibodies capable of protecting suckling mice against FMDV challenge. rP1 stimulated cattle to produce FMDV-neutralizing antibody. The data suggest that an efficient recombinant epitope vaccine against FMDV should share local similarities with the natural VP1 of FMDV.     

Foot and mouth disease virus polyepitope protein produced in bacteria and plants induces protective immunity in guinea pigs

The goal of this project was to develop an alternative foot and mouth disease (FMD) vaccine candidate based on a recombinant protein consisting of efficient viral epitopes. A recombinant gene was designed that encodes B-cell epitopes of proteins VP1 and VP4 and T-cell epitopes of proteins 2C and 3D. The polyepitope protein (H-PE) was produced in E. coli bacteria or in N. benthamiana plants using a phytovirus expression system. The methods of extraction and purification of H-PE proteins from bacteria and plants were developed. Immunization of guinea pigs with the purified H-PE proteins induced an efficient immune response against foot and mouth disease virus (FMDV) serotype O/Taiwan/99 and protection against the disease. The polyepitope protein H-PE can be used as a basis for developing a new recombinant vaccine against FMD.     

Asia Ⅰ型口蹄疫病毒中和表位与羊IgG重链恒定区的融合表达及免疫原性测定

VP1蛋白是口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)诱导机体产生抗病毒感染免疫的主要蛋白,含有病毒的若干中和表位.本研究设计和合成了由Asia Ⅰ型FMDV VP1蛋白136～160aa和198～211aa两个表位组成的重复串联表位的编码基因,并克隆了羊IgG重链恒定区编码基因.利用BamH I、EcoR I和Xho I位点将2个基因片段依次克隆到pPROExHTb载体,构建成重组质粒pPRO-FshIgG,将其转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达得到融合蛋白FshIgG.100μg FshIgG蛋白免疫豚鼠后刺激豚鼠产生了高效价的FMDV中和抗体,而且使这些免疫豚鼠在用200 ID_(50)剂量FMDV攻击时得到了完全保护.由此证明,羊IgG重链恒定区蛋白能够作为FMDV表位肽的载体,而融合蛋白FshIgG可成为一种口蹄疫表位疫苗候选物用于口蹄疫的预防.     

Immune potential of a novel multiple-epitope vaccine to FMDV type Asia 1 in guinea pigs and sheep

To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus(FMDV)type Asia 1 in sheep,a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized.The biologically functional molecule,the ovine IgG heavy constant region(oIgG)as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed.The recombinant proteins,RE and RE-oIgG,were expressed as a formation of inclusion bodies in E.coli.The immune potential of this vaccine regime in guinea pigs and sheep was evaluated.The results showed that IgG could significantly enhance the immune potential of antigenic epitopes.The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs,but confer complete protection in guinea pigs against virus challenge.Although the recombinant protein RE could not confer protection in the vaccinated animals,it could delay the appearance of the clinical signs and reduce the severity of disease.Inspiringly,the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination.Therefore,we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.