【作者】 李丽敏；

【导师】 刘福英；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 目的:犬传染性肝炎(Infectious canine hepatitis,ICH)是由犬传染性肝炎病毒(Infectious canine hepatitis virus,ICHV)引起的一种常见的急性败血性传染病。本病多发生于1岁以内的幼犬,死亡率高达40%,对实验动物犬和其它养犬业均有较大的影响。该病的紧急预防或治疗,可使用同型或异型免疫血清或丙种球蛋白,但对犬的保护效果有限。目前世界上用于紧急预防和治疗犬病毒性传染病,如犬瘟热、犬细小病毒感染等,最好的方法是使用具有中和病毒作用的单克隆抗体(monoclonal antibody,McAb)。目前尚未见成功制备ICHV六邻体蛋白单克隆抗体的报道。ICHV,又称犬腺病毒(Canine adenovirus, CAV),属于腺病毒科哺乳动物腺病毒属成员,是在已发现的哺乳动物腺病毒属中致病性最强、形态结构也研究得比较清楚的一种动物病毒。ICHV是无包膜的球形结构,衣壳由六邻体蛋白、五邻体蛋白和纤维蛋白组成。对六邻体蛋白的结构和功能研究表明六邻体蛋白可以引发极强的中和反应,中和抗原决定簇主要存在六邻体蛋白上,当六邻体蛋白被替代或突变将导致中和免疫的缺失。六邻体分子有一个五面体的基底和三角形的塔尖,基底包含两个区域P1、P2区,塔区由4个环构成即Loop1、Loop2、Loop3、Loop4。Loop1是最长、最复杂的环,自身折叠许多倍,与周围环境相互作用。对15个不同型的腺病毒的六邻体蛋白的序列研究表明,基底区P1、P2是保守的,变化区主要集中在Loop1、Loop2上。Loop1、Loop2存在有7个独特的超变区(Hypervariable Region,HVR),在这7个HVRs中,HVR1- HVR6出现在Loop1中,HVR7出现在Loop2的塔尖。其中HVR1、HVR2、HVR4、HVR5、HVR7中都含有中和抗原决定簇,表明六邻体中主要抗原决定簇就集中在Loop1区和Loop2区。本室参考已发表的Ⅰ型犬腺病毒基因序列设计了引物,克隆了ICHV分离株六邻体蛋白Loop1、Loop2基因,连接了Loop1、Loop2基因并将其定向克隆到原核表达载体pET28a中,命名为pET28aLoop。本研究将pET28aLoop转化到工程菌BL21(DE3)中,诱导表达目的蛋白,利用金属镍螯合的Ni-NTA亲和层析柱对重组的Loop蛋白进行纯化,以纯化的重组蛋白免疫BALB/c小鼠,取脾细胞与SP2/0细胞融合,筛选分泌犬肝炎病毒特异性单克隆抗体的杂交瘤细胞,接种至小鼠腹腔得到含有单克隆抗体的腹水,筛选具有中和ICHV作用的单克隆抗体。方法:1将原核表达质粒pET28a(+)Loop转化大肠杆菌BL21(DE3),涂琼脂板,挑单克隆菌,摇菌并用异丙基-β-D-硫代半乳糖苷酶(IPTG)诱导重组Loop蛋白的表达,SDS-PAGE电泳观察结果,Western blotting鉴定。菌体超声破碎,取上清和沉淀用于SDS-PAGE电泳,进行重组蛋白的可溶性分析。用尿素溶解沉淀,采用镍离子亲和层析柱纯化重组蛋白,透析后浓缩目的蛋白。2重组蛋白联合弗氏佐剂免疫雌性BALB/c小鼠三次,每次免疫间隔为两周,融合前3天用抗原蛋白加强。以病毒为包被抗原,采用ELISA方法检测小鼠血清抗体效价。取免疫鼠脾细胞与骨髓瘤细胞SP2/0在50% PEG4000下进行融合,经HAT培养基选择培养,用间接ELISA法筛选阳性克隆,有限稀释法进行克隆化。获得稳定分泌抗ICHV单克隆抗体的杂交瘤细胞株,接种小鼠腹腔制备腹水。3单克隆抗体鉴定:以ICHV为抗原包被,采用间接ELISA法测定腹水单克隆抗体效价;杂交瘤细胞染色体分析;检测杂交瘤细胞腹水的血凝抑制抗体效价及中和抗体效价;免疫印迹分析单克隆抗体特异性;采用间接ELISA相加实验方法分析单克隆抗体的抗原表位;采用辛酸-硫酸铵法纯化腹水单克隆抗体,SDS-PAGE鉴定单克隆抗体纯度。结果:1重组质粒pET28aLoop转化表达菌BL21(DE3)诱导表达后,经SDS-PAGE电泳,诱导表达菌在36kDa处出现一条蛋白表达条带,对照组无此条带。2 Western blotting结果显示诱导表达蛋白与抗六聚组氨酸抗体特异性结合。3蛋白可溶性分析:超声破菌离心后,沉淀中含有大量分子量为36kDa的蛋白,上清中蛋白量较少。4诱导表达的Loop蛋白经镍离子亲和层析纯化,纯度可达95%以上。5以重组蛋白作为抗原免疫小鼠,ELISA方法检测血清抗体效价高于1:320。6经2次细胞融合,960孔中有201孔长出杂交瘤细胞,融合率为21%。间接ELISA检测其中2孔为阳性。经克隆化培养筛选得到35株杂交瘤细胞株,液氮冻存。7杂交瘤细胞培养上清抗体最高效价达1:1.28×103,腹水抗体最高效价达1:104。8杂交瘤细胞染色体计数结果为92～100条之间。9血凝抑制实验表明细胞株2-7F9和2-1B1腹水血凝抑制抗体效价分别为1:640和1:320;细胞株1-7G3和1-8E6腹水未发现有血凝抑制作用。10 2-7F9 McAb的中和效价为1:64,2-1B1中和效价为1:32,1-7G3和1-8E6无中和效价。11 McAb表位分析结果:2-7F9和2-1B1两株McAb相加指数AI为47%。12 McAb的特异性:Western blotting结果为2-7F9和2-1B1 McAb与诱导菌在36kDa处有特异性条带;与ICHV在约101kDa处有特异性条带;对照组均未出现相应条带。13腹水纯化:取1ml腹水纯化后行SDS-PAGE电泳,结果可见在25kDa和50kDa有抗体轻链、重链的特异条带,并且在97.4kDa与66.2kDa之间、大于97.4kDa各有一条电泳条带;而腹水电泳条带较多。结论1本课题成功诱导、表达、纯化并鉴定了ICHV重组Loop蛋白,为深入研究ICHV Loop1、Loop2基因及其编码蛋白的功能提供了材料,同时为开发新的ICH基因工程产品奠定了良好的基础。2重组Loop蛋白免疫小鼠后,血清中产生高效价的抗ICHV抗体,表明重组Loop蛋白具有良好的免疫原性。3通过细胞融合,成功筛选出2株杂交瘤细胞株,能稳定地分泌具有中和ICHV作用的单克隆抗体,为进一步将其应用于ICH疾病的治疗及利用该单克隆抗体建立ICH诊断方法、制备ICH蛋白疫苗奠定了基础。更多还原

【Abstract】 Objective: Infectious canine hepatitis virus (ICHV) is the causative agent of infectious canine hepatitis (ICH) that is a common acute infectious disease. The mainly host for this virus is the puppy of not more than one years old. Mortality has been reaching to 40%. ICH has spread throughout the world and has become one of the most economically important diseases of the canine cultivation. Immunized serum (isotype or allotype) or gamma globulin is used to sick dogs for emergent prevention and treatment. But its efficiency is limited. At present monoclonal antibody with neutralization activity is the most effective way for prophylaxis and treatment of infectious canine disease, like canine distemper disease and canine parvovirus disease. Surprisingly little was known on infectious canine hepatitis monoclonal antibody.Infectious canine hepatitis virus is also called canine adenovirus typeⅠ(CAV-Ⅰ), belongs to Adenoviridae, Mammalian Adenoviruses, is a kind of virus which has the most pathogenicity in discoverable Adenoviruses. Canine adenovirus is a nonenveloped icosahedral capsid structure. The capsid has three principal protein components: the hexon, the penton, and the fiber. Hexon protein has been extensively studied for structure and function, which can cause stronger neutralization responses. Neutralization antigen determinant exist mainly in hexon protein. The neutralization immunization would be absence if the hexon was replaced or mutation. The hexon has a hexagonal pedestal base from which a tower region projects outward into the solvent. The pedestal base consists of the P1 and P2 region. The tower regions are formed by four loops, L1, L2, L3 and L4. The Loop1 is the longest and most complex, folding back on itself several times and projecting furthest into the solvent, providing maximal interaction with the environment. The sequence analysis of fifteen adenovirus hexon proteins showed that the p1 and p2 regions are conserved, and variable regions exist in Loop1 and Loop2. Seven discrete HVR exist in Loop1 and Loop2. HVR 1 to 6 are found in Loop1, and HVR7 is found at the tip of Loop2. Epitope generating neutralizing antibody are within HVR1, HVR2, HVR4, HVR5 and HVR7.In previous reaseach, two pairs primers were designed and synthesized according to the reported complete nucleotide sequence of canine adenovirus typeⅠin GenBank. The gene of Loop1 and Loop2 were amplified by PCR from ICHV genome obtained from infected dog feces. The two fragments were ligated each other and inserted into vector pET28a (named as pET28aLoop). In this study the plasmid pET28aLoop was transformed into E.coli BL21(DE3), and the recombinant Loop protein was induced by IPTG and purified by the Ni-NTA affinity chromatography column, then using the purified recombinant protein as immunogens to immunize BALB/c mice. Splenocytes were used to fusion with SP2/0 cells. After ELISA screening and subcloning, stable hybridoma cells lines secreting monoclonal antibodies against ICHV were obtained. Positive hybridoma cells were injected into the abdominal cavity of BALB/c mice, and the ascitic fluid was prepared. Screened the anti-ICHV neutralization monoclonal antibodies.Methods:1. The prokaryotic expression plasmid pET28aLoop was transformed into E.coli BL21(DE3) and selected monoclone. The E.coli. BL21(DE3) transformed with the recombinant vectors were cultured and induced with IPTG to express Loop protein. The expressional product was analyzed by SDS-PAGE electrophoresis and its antigenicity was confirmed by Western blotting with the His-specific McAb. The E.coli. BL21(DE3) harboring the recombinant vector of pET28aLoop was lysated with supersonic wave. The supernatant and sediment sample were checked by SDS-PAGE to analysis the solubility of the expressed product. The result was that the protein was expressed in the form of inclusion body. The inclusion bodies were solubilized in 8M urea-NTA0, the supernatant were collected after centrifugate. The fusion protein was purified by Ni-NTA affinity chromatography column. After that, the purified protein was gently renatured by dialyzation in PBS with 6M, 4M, 2M, 0M urea, and concentrated by PEG6000. 2 The purified Loop accompanied with adjuvant were used as immunogen to immunize BALB/c mice for 3 times. The immune interval was two weeks. The titer of antiserum was tested by indirect ELISA used ICHV as the coated antigen. And the mouse was attacked by Loop protein three days before the hybridization. The spleen cells of the immunized mice were hybridized with the myeloma cell SP2/0 by 50% PEG4000. The hybridoma cells were selected by HAT culture medium. The positive hybridoma cells secreting the antibody against Loop were primary detected by the indirect ELISA. Thirty-five hybridomas producing monoclonal antibodies against ICHV were obtained by limiting dilution after 3 times subcloning. Positive hybridoma cell line cells were injected into the abdominal cavity of BALB/c mice. Every mouse was injected with 2×106 cells and the monoclonal antibody ascite was prepared.3 Characterization of monoclonal antibody: The titers of monoclonal antibody ascites and supernament were measured by indirect ELISA used ICHV as coated antigen. Karyotype analysis of hybridoma cell. Hemagglutination inhibition test and neutralization antibody test assays the titer of hemagglutination inhibition and neutralization of monoclonal antibody. The specificity of monoclonal antibody was analyzed by Western blotting. ELISA additivity test was employed to detect whether the corresponding monoclonal antibody recognize different epitype on ICHV. The ascites was purified by the method of caprylic acid-ammonium sulfate precipitation. The characterization of monoclonal antibodies was performed by SDS-PAGE.Results:1 The recombinant plasmid pET28aLoop was transformed into E.coli BL21(DE3) and induced by IPTG. SDS-PAGE analysis showed an induced product band about 36kDa;2 Western blot analysis indicated that the recombinant protein could be recognized by His-specific McAb;3 SDS-PAGE showed that the 36kDa recombinant protein most existed in the precipitation of the lysed bacteria broken by ultrasonic wave, only a little of them was soluble protein;4 The purity of Loop protein was about 95% after purification with Ni-NTA affinity chromatography column;5 The mouse was immunized successfully using recombinant Loop protein as antigen, the serum titer tested by indirect ELISA was up to 1:1.28×103;6 After 2 times of cell hybridation, 201 wells in 960 grow hybridoma cell, the fusion rate was 21%. 2 wells of antibody-secreting hybridoma cells against ICHV were obtained by HAT selection and antibody detection. 35 hybridoma cell lines can secret the McAb against ICHV continuously after coloning, the positive cell strain were subcultured and frozen;7 The titer of the specific antibody in supernatant and ascites were found by ELISA to be 1:1.28×103 and 1:104 respectively. 8 The chromosomal numbers of hybridoma cells were 92～100;9 Four hybridoma cell lines were named 2-7F9, 2-1B1, 7G3 and 8E6, of which monoclonal antibody 2-7F9 and 2-1B1 showed hemagglutination inhibiting activity and titers of its hemagglutination inhibiting were 1:640 and 1:320 respectively.10 Monoclonal antibody 2-7F9 and 2-1B1 had the neutralization activity against ICHV, the titer were 1:640 and 1:320 respectively. Monoclonal antibody 1-7G3 and 1-8E6 haven’t.11 Epitope analysis showed that monoclonal antibody of 2-7F9 and 2-1B1 recognize different antigenic epitope.12 Western blotting demonstrated that McAb could specifically combine with a single protein band with molecular weights of 36kDa of recombiant Loop protein and 101kDa of hexon protein of ICHV. There is no positive band in control.13 Ascites purification: 1ml Ascites were used to purify, SDS-PAGE analysis that two bands of antibody were seen under reducing conditions. One is the light chain with molecular weights of 25kDa, the other is the heavy chain with molecular weights of 50kDa. There were also two bands in addition, one lies in between the bands 97.4kDa and 66.2kDa, the other lies in greater than 97.4kDa. But there are more bands in the unpurified Ascites.Conclusions:1 In present study, the recombinant Loop protein was successfully induced, expressed, purified and identificated, which provide valuable materials for further studying the gene of Loop1 and Loop2 of ICHV and the encoding protein function. At the same time it laid a good foundation to construct new genetic engineering product of infectious canine hepatitis.2 After immune BALB/c mice using the recombinant Loop protein, serum with high titer against ICHV was generated, this indicated that the recombinant Loop protein possesses a high immunogenicity.3 Two hybridoma cell lines with neutralization activity secreting monoclonal antibody against ICHV were successfully obtained, it might lay a foundation for further utilizing it to treat the disease and applying it to construct diagnostic method, preparing protein vaccine of infectious canine hepatitis.更多还原