【作者】 曹丙蕾；

【导师】 赵宏坤；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2010， 博士

【摘要】 IL-18是一种属于IL-1家族且分布广泛的细胞因子,具有多种生物学功能,其中对机体的免疫增强调节和抗肿瘤作用已受到研究者们的极大关注。人类医学研究证明IL-18在抗微生物感染,尤其是在抗肿瘤免疫方面具有重要的潜在应用价值。且单克隆抗体比多克隆抗体具有较好的优越性,广泛应用到各种疾病的检测、诊断及防治中,并为各种病原微生物的流行病学的分析提供了强有力的工具,极大地提高了临床疾病诊断和治疗的效率。本课题对山羊IL-18(gIL-18)的单克隆抗体进行了研究。将山羊IL-18的成熟蛋白基因经扩增后克隆入原核表达载体pET-28a(+)中,经鉴定和测序后,构建了重组质粒pET-28a-gIL-18。将该质粒转化入表达菌Rosetta(DE3)的感受态细胞中,经诱导表达后获得重组蛋白的表达,并利用载体上所带有的His标签蛋白这一特点,采用Ni-NTA亲和层析法纯化重组蛋白gIL-18,从而获得了纯度较高的蛋白,为下一步进行小鼠免疫及单抗的筛选鉴定做好准备。利用纯化的重组gIL-18蛋白对BALB/c小鼠进行免疫,并建立了检测小鼠抗体水平和单抗鉴定的间接ELISA检测方法,融合前抗体效价达到1:107,满足融合时对小鼠抗体的要求。SP2/0骨髓瘤细胞与加强免疫后的小鼠脾细胞在PEG作用下进行融合,经间接ELISA方法检测融合细胞,对于阳性杂交瘤细胞,通过有限稀释法进行多次克隆化,以筛选出能稳定分泌抗体的杂交瘤细胞,并最终确定下两株单抗2E8和4C4作为试验研究对象,并进行了单抗腹水的大量制备,利用饱和硫酸铵法对腹水进行粗提,再进一步利用Protein G亲和层析柱进行纯化,通过SDS-PAGE电泳分析,得到了纯度较高的IgG。通过对此两种单抗的亚型鉴定表明,此两种单抗均属于IgG1亚型。两种单抗2E8和4C4的效价分别为1:105和1:104。经ELISA叠加试验分析表明2E8和4C4可分别结合到IL-18的不同的抗原位点上,两者具有叠加性。经Western blot鉴定表明,单抗2E8和4C4能够与重组gIL-18蛋白发生特异性的反应,且anti-His单抗也可以与重组gIL-18蛋白发生特异反应,而与hIFN-γ蛋白不发生特异反应,与多抗相比,单抗特异性较强,蛋白印迹条带清晰。经IFA鉴定表明,对于gIL-18的重组真核质粒pcDNA3.1-gIL-18转染293FT细胞表达情况的检测,可以利用单抗的特异性,试验证实单抗可以特异性的检测真核质粒在细胞上的表达,产生特异明显的荧光,且对重组Bacmid-gIL-18转染sf9昆虫细胞的表达进行IFA检测也获得相似的结果。通过对山羊IL-18单抗的研究旨在建立山羊IL-18定量抗原捕获ELISA检测方法,利用对其中一种单抗2E8进行辣根过氧化物酶(HRP)的标记并对其特性及效价(1:6000)进行测定,另一种单抗4C4作为捕获抗体,通过对所需包被抗体(单抗4C4)最佳工作浓度、HRP-2E8-IgG二抗最佳稀释比例和ELISA最佳反应条件等进行研究,经方阵滴定试验最终确立了检测山羊IL-18的定量抗原捕获ELISA的检测方法,并依次建立了检测山羊IL-18的标准曲线,检测灵敏度为16pg/mL,且对其他细胞因子如hIFN-γ和hIL-18应用此检测方法进行反应,结果表明无交叉反应,检测值均低于16pg/mL的最小检出限,此检测方法对于山羊IL-18的检测具有一定的特异性。应用此方法检测体外山羊PBMC在LPS刺激下产生的IL-18的水平,及其健康奶牛和奶牛乳腺炎患牛(山羊IL-18和牛IL-18氨基酸同源性为98%)的血清及乳清进行检测,结果表明,山羊PBMC在LPS刺激下IL-18检测含量为85~267 pg/mL,与细胞阴性对照相比,gIL-18含量有显著提高;健康奶牛血清中IL-18含量为44~135 pg/mL,相比之下,病患牛血清中IL-18含量有明显升高,在111~534 pg/mL之间;健康奶牛乳清中IL-18含量为83~167 pg/mL,病患牛乳清中IL-18含量有明显升高,在171~658 pg/mL之间。此检测方法可以对机体体内和体外在炎症反应时IL-18的水平进行定量的检测,从而为各种临床疾病的诊断和治疗奠定基础。同时,针对IL-18具有免疫治疗及免疫增强作用这一功能,将山羊具有生物学活性IL-18蛋白(由本实验室构建、表达并保存的重组杆状病毒表达的rBgIL-18蛋白)作为免疫调节剂,联合口蹄疫O型、亚洲1型二价灭活疫苗对实验动物山羊分别进行rBgIL-18蛋白与疫苗共免疫组(Ⅰ组)、rBgIL-18蛋白预免疫+疫苗组(Ⅱ组)、单用疫苗组(Ⅲ组)和生理盐水阴性对照组(Ⅳ组)等分组试验,以期对IL-18的免疫增强作用更好地进行诠释和应用,从体液免疫和细胞免疫水平上分析试验结果。通过使用口蹄疫O型和亚洲1型检测试剂盒检测各试验期血清中的抗体滴度,从统计学意义上分析处理数据,结果表明,与免疫前相比,Ⅰ、Ⅱ、Ⅲ组都有较好的免疫效果,抗体水平都较高且维持时间也较长,Ⅰ组可以较快(42d)达到高抗体水平;与阴性对照组相比,Ⅰ组效果最好,抗体效价最高,维持时间也最长,Ⅱ组次之。而且,利用MTT法从细胞免疫水平上分析淋巴细胞的增殖能力,结果表明,免疫前后相比,PI指数明显有所升高,淋巴细胞在免疫后免疫物的刺激下进行增殖分化。同时,通过对全血细胞的分析也可知,与免疫前相比,免疫后各试验组山羊的淋巴细胞数明显增殖,且所占比例也得到提高。因此,IL-18作为免疫增强佐剂有利于提高机体免疫力,提高疫苗的免疫效力,较好地提高机体抵抗力,为以后在生产实践中的应用打下良好的基础。更多还原

【Abstract】 Interleukin-18 is a member of the IL-1 cytokine superfamily and distributed widely and possesses variety of bioactive functions. The immunity regulation and tumor-resistant system to the organisms were payed close attention by the researchers. The human medical researches show that IL-18 plays an important role in the resistance to a variety of diseases, especially in tumor-resistant immunity. The Mabs have more superiority than the polyclonal antibodies and were applied to the detection, diagnosis and treatment of variety of diseases. The Mabs can provide powerful tools for the analysis of variety of micrograms’epidemiology and greatly enhance the diagnosis and treatment of clinical diseases.In this study, the Mabs were prepared and studied. The gene of goat mature IL-18 proteins was amplified and cloned into prokaryotic expression vector pET-28a(+). By the identification and sequencing, the recombinant plasmid pET-28a-gIL-18 was constructed. The recombinant plasmid pET-28a-gIL-18 was transformed into Rosetta (DE3) competent cells and induced and obtain the expression of the recombinant proteins. Using the feature of the His tags in the vector, the recombinant gIL-18 proteins were purified by Ni-NTA Resin affinity chromatography. The purified proteins were used for the immunity of the mice, screening and identification of the Mabs.The mice were immunized using purified gIL-18 proteins. And, the indirect ELISA detection was constructed for detection of mice’antibodies and identification of Mabs. The titers of mice were up to 1:107 before the fusion at the satisfaction of the conditions of the fusion. The splenocytes were fused with SP2/0 myeloma cells under the effect of PEG. The fusion cells were detected by indirect ELISA. The positive hybridoma cells were sub-cloned twice by limited dilution methods to obtain the hybridoma cells secreting antibodies stably. Finally, the two Mabs (2E8 and 4C4) were obtained. The ascites fluids were harvested and purified by ammonium sulfate precipitation followed by Protein G affinity chromatography. The purified IgG was obtained and analyzed by SDS-PAGE.The identification of isotype of Mabs showed that the two Mabs were IgG1 isotype. The titers of 2E8 and 4C4 were 1:10~5 and 1:10~4, respectively. ELISA additity test showed that the binding of two Mabs, 2E8 and 4C4, was additive and both bound to the distinct epitopes. Western blot showed that two Mabs, 2E8 and 4C4, could react with recombinant gIL-18 proteins specifically and anti-His Mabs could also react with recombinant gIL-18 proteins specifically and could not react with hIFN-γproteins. Moreover, compared with the polyclonal antibody, the Mabs had high specificial and clear bands. The Mabs could be used to detect the expression of recombinant eukaryotic plasmid pcDNA3.1-gIL-18 transfected into the 293FT cells specifically by IFA. IFA displayed intense green fluorescence. Also, the expression of recombinant Bacmid-gIL-18 transfected into sf9 was detected specifically by IFA.The goat IL-18 quantitative sandwich ELISA detection was constructed using two Mabs. The Mab 2E8 was marked with HRP and the titer of HRP-2E8-IgG was identified as 1:6000.The other Mab 4C4 was used as the capture antibody. By the phalanx trial to optimize the concentration of coated antibody (4C4), dilution of HRP-2E8-IgG and condition of ELISA, the goat IL-18 quantitative sandwich ELISA detection was constructed. The standard curve of goat IL-18 was determined by the quantitative sandwich ELISA and the minimum detection was 16pg/mL. This ELISA to detect hIFN-γand hIL-18 showed that there were no cross reaction and the minimum detection were below 16pg/mL. Thus, the quantitative sandwich ELISA detection for goat IL-18 was specific.The quantitative sandwich ELISA was applied to detect the IL-18 levels of the goat PBMC at the stimulation of LPS and the sera and whey of the healthy cows and cows infected with mastitis. The results showed that the detection values of goat PBMC were 85~267 pg/mL. Compared with negative control cells, goat IL-18 levels were improved. For detection of sera, the detection values of healthy cows were 44~135 pg/mL.And, the detection values of cows infected with mastitis were 111~534 pg/mL and were improved compared with the forth. For detection of whey, the detection values of healthy cows were 83~167 pg/mL. And, the detection values of cows infected with mastitis were 171~658 pg/mL and the values of IL-18 were improved. The quantitative sandwich ELISA could detect IL-18 levels quantitatively at the inflammation in vitro and in vivo and set the basis for the diagnosis and treatment of variety of diseases.And, to study the immunity enhancement of IL-18, rBgIL-18 proteins were used as adjuvants to co-administrate goats with the vaccine (FMD bivalent inactive vaccine of O type and Asian 1 type). The goats were distributed as four groups:Ⅰgroup: rBgIL-18 + vaccine; Ⅱgroup: pre-rBgIL-18 + vaccine;Ⅲgroup: vaccine;Ⅳgroup: control. The results were analyzed at the level of humoral and cellular immunity to interpret the immunity enhancement of IL-18.The titers at variety of times post-immunization in sera were detected by FMD O type and Asian 1 type ELISA kit. The results showed that compared with pre-immunization,Ⅰ、ⅡandⅢgroup had higher titers of antibodies and maintained high titer levels for longer times andⅠgroup could arrive at higher titer levels faster. Compared with the control, the immunity levels ofⅠgroup were better and had higher titers and maintained high titer levels for longer times. The results ofⅡgroup were next to that ofⅠgroup. Moreover, the cellular immunity was detected by MTT to analyze the proliferation of lymphocytes. The results showed that in contrast with the control, PI values were greatly improved and the lymphocytes could proliferate at the stimulation of vaccine.And, the analysis of the blood cells showed that compared with pre-immunization, the lymphocytes of goats were significantly proliferated and the proportion of the lymphocytes were also improved. Thus, as immunity adjuvant, IL-18 plays an important role in enhancing immunity and immunity effects.更多还原