摘要
E.coli O157:H7在临床感染中常常引起血样腹泻,贫血和肾脏的衰竭,严重者常常危及患者生命。由于采用抗生素疗法能够导致E.coli O157:H7的细胞壁溶解促进细胞毒素的释放而加重病程,在临床中针对E.coli O157:H7的感染以预防和保守疗法为主。为了有效预防E.coli O157:H7在临床中的感染,本文针对大肠杆菌O157:H7主要免疫原EspA进行了口服植物疫苗研究。
由于E.coli O157:H7的主要侵入途径是消化道,因此在疫苗的研究中如何能够激起机体有效的粘膜免疫是研究的重点。注射免疫途径的疫苗往往不能有效诱导肠道内的特异免疫反应。如果口服疫苗抗原没有经过特殊的处理而直接进行口服免疫时,能够有效到达肠道并激起肠道内淋巴系统特异反应的抗原量太低,也不能有效诱导肠道内的免疫反应。近十年来的研究表明,植物疫苗能够很好的解决这些免疫过程中遇到的困难。在植物表达重组抗原的过程中,由于植物的细胞壁能够对在植物细胞内表达的重组目的蛋白进行“胶囊”样包裹,因此在植物细胞壁的保护下,表达的抗原能够顺利进入肠道,并在消化系统对植物细胞进行肠道内消化时,释放出目的抗原,从而激发有效的粘膜免疫。生菜作为种植广泛,生长期短,成本低廉的常见蔬菜,具有可以生食的特点,因此在采用转基因生菜免疫时,避免了采用烟草、土豆、水稻等植物表达的重组蛋白在口服免疫时需要对植物疫苗进行预处理的缺点,生菜转基因疫苗能够将在植物细胞内表达的蛋白顺利地运送到肠道,刺激肠道淋巴系统的反应,因此生菜口服植物疫苗的研制在临床使用中具有可行性好,免疫成本低,免疫途径简单,免疫效果可靠的特点,便于疫苗的推广。
本研究通过对生菜中表达的来源于E.coli O157:H7的EspA生物学特性的研究,证实了大肠杆菌的EspA可以作为植物疫苗研究中的免疫原。本研究首次在植物细胞内进行了E.coli O157:H7表面蛋白EspA的瞬时表达及稳定表达研究,并分别对以两种体系中表达的重组蛋白进行了动物实验。实验通过试验小鼠的多克隆EspA抗体的体内及体外保护性试验,证实了免疫试验小鼠产生的多克隆EspA抗体的对HeLa细胞的保护性作用,并通过E.coli O157:H7⊿stx-B突变株的小鼠攻毒后的肠道病理研究发现EspA免疫后的小鼠具有对E.coli O157:H7⊿stx-B的抵抗力。
第一部分:
致病性大肠杆菌基因组中移动的基因元件决定了致病性的细菌的毒力,他们在病原菌侵入动物或植物后发挥毒性的过程中起着关键的作用。其中毒力岛PAI基因簇上20多个基因编码蛋白的功能集合体—Ⅲ型分泌系统(T3SS),在细菌的致病过程中发挥重要的作用。在T3SS系统中,很多与毒力相关的亚单位的装配机制与细菌鞭毛的装配机制类似,其致病蛋白的装配过程均发生在细胞的外壳上。大肠杆菌黏附过程中分泌的蛋白如EspA,EspB,EspD和EspF等蛋白都是T3SS的功能蛋白。EspB及EspD蛋白位于EspA微丝的末端,这些蛋白与宿主的细胞膜相互作用,在宿主的细胞膜上形成小孔后,效应因子如Tir,EspB,EspG,EspF和Map通过形成的孔道进入宿主的细胞。本试验首次在生菜中采用瞬时表达体系表达了E.coli O157:H7的EspA蛋白。
将构建好的pBI121-espA,pBI121-ΩespA载体通过液氮冻融法分别导入农杆菌AGL1,EHA105,LBA4404,GV3101和C58C1后,将带有重组质粒的农杆菌在卡那抗性和利福平抗性的YEB培养基中扩大培养,采用真空法转染培养14天的无菌生菜小苗,转染生菜在22℃培养3天后,检测各菌株转染生菜后EspA的表达水平,试验发现农杆菌AGL1转染的生菜中EspA的表达量最高,农杆菌LBA4404转染的生菜的EspA的量最低,通过检测pBI121-espA和pBI121-ΩespA转化农杆菌GV3101后通过真空法转染生菜,发现pBI121-ΩespA组具有比较高的EspA表达量,通过对瞬时体系中重组蛋白的表达研究,确定了不同农杆菌菌株在生菜瞬时表达系统中的不同的转染能力,这是首次在生菜瞬时表达的系统中进行此类综合研究。通过免疫小鼠的实验证实生菜细胞内瞬时表达的EspA能够刺激小鼠产生高水平的IgG和IgA抗体,这是首次关于植物表达E.coli O157:H7的主要免疫原EspA的疫苗报道。
第二部分:
在对致病性大肠杆菌的研究中,通常根据大肠杆菌对HeLa的黏附特性,来区分大肠杆菌EPEC和EHEC。Fluorescence actin assay试验(FAS)是鉴定E.coliO157的主要方法,同时HeLa细胞也成为E.coli O157进行细胞毒性试验的常用模式细胞。
在本部分中,乳酸菌免疫小鼠产生的多克隆抗体及生菜瞬时表达系统表达的重组蛋白免疫小鼠产生的抗体分别用来进行HeLa细胞的黏附试验、体外抗体与E.coli O157:H7⊿stx-B结合试验和抗体抑制菌落的生长试验。通过这3个实验证实EspA特异的抗体能够阻断E.coli O157:H7⊿stx-B向细胞内毒性蛋白的转移,并能够抑制E.coli O157:H7⊿stx-B感染细胞产生的特异性的细胞骨架改变,但在体外的实验中,抗体与细胞相互作用后,再用E.coli O157:H7⊿stx-B感染细胞,并不能阻止细菌与细胞的黏附。试验的多克隆抗体能够结合E.coliO157:H7⊿stx-B表面的EspA微丝,采用特异性的荧光标记的羊抗鼠二抗能够在荧光镜下观测到特异性的荧光。在以前的报道中,原核表达的EspA免疫小鼠产生的多克隆抗体能够通过上述3个实验得出EspA抗体具有对细胞的保护性作用。本试验首次对乳酸菌和生菜异源表达的EspA免疫小鼠产生多克隆抗体进行类似的实验,证实了乳酸菌和生菜表达的EspA具有与E.coli O157:H7的EspA微丝相同的抗原决定簇,并能够应用于E.coli O157:H7的口服疫苗研究。
第三部分:
细胞培养又严格地分为细胞培养、组织培养和器官培养,三者是密不可分相互联系的。组织细胞的研究已广泛应用于生物学、医学的各个研究领域。运用组织培养细胞具有条件可控,误差小,能进行单因素分析的优点。由于培养条件可以人为控制,所以便于进行各种物理、化学和生物的外界因素来研究细胞生命活动规律。
在本部分中,对生菜的组织培养进行了探讨,建立了生菜的稳定表达培养体系,在生菜的愈伤组织的培养过程中,发现0.2 mg-0.8 mg的6-BA均能诱导生菜生成愈伤组织,而在6-BA 0.8 mg/l,NAA 0.12 mg/l;6-BA 0.3 mg/l,NAA 0.12 mg/l两种激素水平存在的条件下,愈伤组织的分化出现部分的褐化。在同样培养条件下,0.2,0.4 mg/l 6-BA的激素水平培养下生菜的愈伤组织生长良好,说明0.3 mg/l6-BA的培养条件下,生菜细胞出现的褐化现象不是由6-BA的浓度引起的,而在6-BA 0.8 mg/l,NAA 0.12 mg/l;6-BA 0.3 mg/l,NAA 0.12 mg/l中NAA的浓度均大于其余的两组。最终通过试验研究发现,0.5 mg/l 6-BA和0.08 mg/l NAA的MS培养基是生菜颗粒状愈伤组织和不定芽分化培养的理想培养基。25 d时观察统计在含有浓度为0.3 mg/l的NAA生根培养基上,不仅生根率为100%,平均每株生根数为3-4条,而且生根试管苗株高为5-6 cm,生长旺盛。经抗生素培养基筛选后生成的愈伤组织的百分比发现各农杆菌转染的水平分别为45.6%(AGL1),26.6%(C58C1),22.7%(EHA105),25%(GV3101),17.7%(LBA4404)。转染成功率高的农杆菌菌株依次为AGL1＞C58C1＞GV3101＞EHA105＞LBA4404。
通过同样的方法建立了EspA在生菜愈伤组织中的稳定表达。通过连续传代3次后,用PCR、ELISA和Western-blot对生菜愈伤组织中的基因和蛋白分别进行检测,在生菜愈伤组织提取的基因组中检测到espA基因,并通过ELISA检测后发现生菜愈伤组织中表达的EspA蛋白的量为1.6μg/ml,将表达EspA蛋白的生菜愈伤组织饲喂小鼠免疫后产生了高水平的抗体水平,用E.coli O157:H7⊿stx-B突变株进行动物攻毒试验发现生菜表达的EspA愈伤组织免疫的小鼠的死亡率为50%,而对照组的死亡率为75%。通过对试验小鼠肠道组织的病理切片检测发现,生菜免疫组的小鼠肠道微绒毛完整,肠道基底细胞排列紧密,而空白对照组,肠道内容物增多,肠道微绒毛脱落,肠道基底细胞排列疏松,错乱不齐。该实验首次从病理组织学的研究方向论证了EspA植物疫苗对试验动物模型的保护作用。
第四部分:
EHEC O157主要产生两种毒素,分别称为志贺毒素样Ⅰ(Stx1)和志贺毒素样Ⅱ(Stx2)。在细菌体内,Stx2为分泌型表达,Stx1为胞内表达。两种毒素均由1个A亚单位和5个B亚单位组成,A亚单位具有细胞内毒性,能与28S rRNA作用导致蛋白质合成停止,是大肠杆菌O157H7引起临床表现的病理基础;B亚单位具有细胞结合特性,能与具有特定受体(Gb3)的细胞结合,从而引导A亚单位发挥作用。
针对B亚单位在EHEC疾病发展过程中的重要作用,在本部分中采用分子生物学方法对stx1B进行了基因克隆、原核表达和初步纯化,同时在本试验中将B亚单位与espA基因进行融合表达,期望两种蛋白能够在生菜细胞中同时表达,建立多抗原疫苗。但在随后的动物试验中虽然检测到特异的EspA特异的IgA抗体,但并没有检测到特异的IgG抗体,分析其原因可能是B亚单位与espA基因之间没有加入连接片段,而直接采用酶切位点直接将两基因连接,因此限制了重组蛋白的表达,造成了重组蛋白的错误折叠。
E.coli O157:H7 always brought blood diarrhea,anemia and hemolysis urine syndrome(HUS) in clinic infection.E.coli O157:H7 could release the cytotoxin during invading procession and the antibiotic could speed this cytotoxin release by the dissolution of cell wall,therefore the therapy of E.coli O157:H7 was conservative therapeutics.In order to prevent its occurrence,this study is carried in inspect of plant oral vaccine.
The main invade path of E.coli O157:H7 is enteron,and the arising of effect mucous membrane immunization is the most emergent question in vaccine study.In the previous studies,the inject immunization was incapable to induce the efficient mucosal immune,and antigen without protection could not arrive the intestines effectively.Another question in oral vaccine study is the quantity of antigen arriving at the intestines is too low to raise the intestinal mucosal immunization.Aimed at those matters,plant vaccine could solve these questions of the immunization.During the protein expression in plant cell,plant cell wall acts as a capsule and provides protection to the recombinant protein,so recombinant protein in the protection of plant cell could pass the upper gastrointestinal,and release the antigen during the digestive in the intestine.Lettuce as a vegetable was planted widely,and it could be harvest in 50 days,so its cost is low as a plant candidate.Because lettuce could be eat in raw,and avoid antigen denaturing in the immunization,it has the unique advantages compared to tobacco,potato and rice.
In this study,lettuce was used to express EspA and certified it might be the candidate immunogen in E.coli O157:H7 vaccine study.EspA was expressed in plant cells by the transient expression and stable expression for the first time.After EspA expression,BALB/C mice were immuned and specific antibodies were valued.The protections of the polyclonal antibodies of immuned mice were carried on HeLa cell monplays,and mice intestinal histopathologic slides were made after E.coli O157:H7 challenge.It was found the polyclonal antibody of EspA-immuned mice could protect the mice from E.coli O157:H7 infection.
The first part:
Mobile genetic elements decided the virulence of the pathogenic bacteria,and play an important role in the animal and plant incursion.TypeⅢsecretion systems (T3SS) are complex assemblies that require the function of more than 20 genes for their activity.The complex is composed of Esc/Sep proteins on which a filamentous structure of polymerized EspA is assembled.EspB and EspD are located at the distal end of the EspA filament,and it has been suggested that these proteins are involved in pore formation in the host cell membrane.Effector proteins such as Tir,EspB,EspG, EspF,and Map are translocated through this structure.Translocation of these molecules into host cells results in changes of cytoskeleton of the underlying epithelial cells.
EspA was transient expressed in lettuce for the first time,and PBI121-espA, PBI121-ΩespA was introduced into Agrobacterium AGL1,EHA105,LBA4404, GV3101 and C58C1.Lettuces were cultured for 14 days and transfected by the Agrobacterium.After cultured for 3 days in 22℃,the concentration of EspA in lettuce extract was valued.The AGL1 group had the highest EspA concentration,and the lowest was LBA4404 group.By the detection of the PBI121-espA,PBI121-ΩespA group,the PBI121-ΩespA group had the higher EspA concentration.In this study,the transfection ability of Agrobacterium AGL1,EHA105,LBA4404,GV3101,C58C1 were valued and this is the first report of the transfection ability study in the lettuce. By the mice immune,BALB/C mice which were immunized with lettuce-derived EspA produced specific antibody titers with the highest IgA titer of 3,200 and IgG titer of 12,000.
The second part:
By E.coli adherence characterization,the EPEC and EHEC could be differentiated.HeLa cell was widely used in E.coli O157:H7 virulence study and the cytoskeleton aggregation was a characterization during E.coli O157:H7 invasion.In the study of polyclonal antibody protection,Fluorescence actin assay was frequently used.
In this study,BALB/C mice were immuned with EspA expressed in Lactococcus. Lactis NZ9000(L.Lactis NZ9000) and lettuce,respectively.The polyclonal mice antisera were valued with the bacterial adherence assay,Fluorescent actin staining (FAS) and labelling of EspA filaments.In the following study,immuned mice polyclonal antibody blocked E.coli O157:H7-induced host cell actin re-arrangement and could label E.coli O157:H7 EspA filaments in vitro.But the polyclonal antibody could not inhibit E.coli O157:H7 adherence to HeLa cell in vitro.The polyclonal antibody could recognize the natural EspA on the surface of E.coli O157:H7 and the specific fluorescence were observed with the help of the HRP-marked goat anti mouse serum.In the previous studies,the prokaryotic expressed EspA could have the same protection effect in FAS,but this is the first report of the immune effect of EspA expressed in Lactococcus.Lactis NZ9000(L.Lactis NZ9000) and lettuce,respectively. Then it was approved that both of EspA expressed in Lactococcus.Lactis NZ9000(L. Lactis NZ9000) and lettuce had the same antigenic determinant,and could be the candidate antigen in the vaccine study of E.coli O157:H7.
The third part:
Cell culture is divided into cell culture,tissue culture and organic culture,and each of them has close relation with each other.Now,the study of tissue culture is widely applied in biology and medicine.The tissue culture condition could be controlled easily and the error is low,so it is easy to study the physical,chemical and biological factor in tissue culture.
In this part,lettuce tissue culture was studied and the stable expression system was set.In the lettuce callus culture,0.2 mg-0.8 mg/l 6-BA could induce the lettuce callus,but callus could be browned in 6-BA 0.8 mg/l,NAA 0.12 mg/l;6-BA 0.3 mg/l, NAA 0.12 mg/l.At the same culture,callus grows well in 0.2,0.4 mg/l 6-BA,and it suggested the brown callus was not related with 6-BA.Adventive bud could differentiate into integrate plant in the 0.3 mg/l NAA MS culture medium.
The rate of lettuce callus were 45.6%(AGL1),26.6%(C58C1),22.7% (EHA105),25%(GV3101),17.7%(LBA4404) in Kan antibiotic medium after agrobacterium transfection.The transfect ability was AGL1＞C58C1＞GV3101＞ EHA105＞LBA 4404.With the same method,the stable expression of EspA in lettuce callus was set,and the callus was generated for three times,and then was detected with PCR,ELISA and Western-blot.The genome of the lettuce callus was extracted with genome extract kit,and espA gene was present by the PCR detection.EspA protein in the callus was also detected by ELISA and Western-blot.The EspA in lettuce callus was 1.6μg/ml and the mice produced high IgG specific antiserum.The mice were attacked with E.coli O157:H7⊿stx-B and 50%mice survived after 5 days. The intestinal microvilli were intact and the fundus cells arrange wass tightness,but microvilli of the control group were broken off and the fundus cells arrange was in disorder.This study demonstrated the protection of the EspA plant vaccine in the histopathology.
The fourth part:
EHEC O157 produced Stx1 and Stx2 while the Stx2 is secreted into the medium and the Stx1 is expressed in cytoplasm.Both of the two toxin are composed of A subunit and five B subunits.The A subunit has the cytotoxin by the interaction with 28Sr RNA,and this is the base of the pathogenic mechanism of E.coli O157;the B subunit could bind to Gb3 and initiate the A subunit.
According to the pathogenic mechanism of E.coli O157,the stx1-B gene was cloned from the pMD18-T-stx1 plasmid,and the expression vector pET15b-stx1B was constracted.The Stx1-B was purified by inclusion denaturing and renaturing. Meanwhile,espA gene and stx1-B fuse gene was cloned into pBI121,and the lettuce was transfected by Agrobacterium GV3101 harboring pBI121-espA-B.The mice were fed with transgenic plant,but the specific IgG was not detected in the mice serum. The possible reason might be the false folding of the fused protein and then caused the immune failure.
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