交叉反应性抗脂多糖单克隆抗体的制备及鉴定
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摘要
脓毒症是ICU病人最常见的死亡原因,尽管其医疗投入巨大,严重脓毒症的死亡率还是居高不下,可达25%-30%,而脓毒症休克的死亡率则更高,达到了40%-70%。
脓毒症可由多种病原微生物感染引起,包括革兰氏阳性(G~+)菌、革兰氏阴性(G~-)菌和真菌等等,而其中G~-菌感染是最重要的一种,占总发病人数的40%以上。脂多糖(LPS)是G~-菌细胞壁的重要组成成分,也是G~-菌感染脓毒症中最主要的毒力因子和抗原成分。所以在G~-菌感染引起的脓毒症的诊断与治疗中,LPS是一个重要的靶点。目前尚无LPS拮抗剂用于临床应用或进入临床观察,但有证据显示,预存抗LPS抗体与败血症的发生、大样本住院肿瘤患者、烧伤、外科手术等因感染死亡的病人数呈负相关,即对LPS诱发的病理进程有明确的保护作用。动物实验显示针对LPS的单克隆抗体(mAb)对实验性脓毒症动物模型具有良好的保护作用。所以,抗LPS的mAb有可能成为治疗G~-菌感染诱发的脓毒症的一种新制剂。
LPS由三部分组成:O特异性抗原,也叫O抗原或体抗原,位于G~-菌细胞壁的最外面,由一长链多糖构成;核心寡糖,是一由大约10个左右的单糖组成的寡糖;类脂A。所以,脂多糖是一结构十分复杂、具有多种异质性的生物大分子,而且属于胸腺非依赖抗原(TI)抗原。与蛋白质等胸腺依赖(TD)抗原不同,TI抗原不能诱导抗体亲和成熟,也不能诱导免疫记忆,所以按常规的免疫方法很难产生高亲和力、交叉反应性抗LPS的mAb。为解决此难题,一个可行的方法就是用分子模拟技术,即用蛋白质、多肽来模拟LPS的抗原结构,从而将多糖类TI抗原转变为多肽类TD抗原,提高抗原的免疫原性。那么,筛选出能够模拟LPS抗原结构的多肽就成为其中关键的一步。
噬菌体展示技术是一种用于筛选和改造功能性短肽和蛋白质强有力的生物技术,广泛应用于免疫学和分子生物学领域。事实上,噬菌体展示技术早已成功的应用于多糖表位的模拟肽筛选,如对脑膜炎奈瑟球菌A、B、C三种血清型的荚膜多糖的模拟肽筛选。这些结果说明,用噬菌体展示技术可以筛选出模拟多糖抗原表位的模拟肽,从而将TI抗原转变为TD抗原,诱导强而有力的记忆性免疫应答,制备出高亲和力、交叉反应性抗LPS的mAb。
所以,本课题的主要研究思路是:利用噬菌体展示技术筛选并合成模拟LPS保守抗原表位的多肽,将其抗原表位的化学性质由脂多糖改变为短肽,由此使TI抗原改变为TD抗原;然后将多肽与适当的载体进行交联,用交联物作为免疫原免疫BALB/c小鼠,制备具有高亲和力、广泛交叉反应性的抗LPS的mAb。最后对其在动物体内、外的保护活性进行初步探讨。
本课题组在前期工作中,以具有交叉保护活性的抗鼠伤寒沙门氏菌多克隆抗体为配基成功的从噬菌体环七肽库中筛选到四个可模拟LPS表位的保守序列,其免疫原性和抗原性已通过一系列实验得以证实。其中一个模拟肽经加入延长序列被命名为13L,我们的前期实验显示,用13L交联蓝载体(BC)作为免疫原免疫BALB/c小鼠后,可以诱导小鼠产生针对G~-菌感染和内毒素休克的保护作用。以这些实验结果为依据,本实验选择13L作为候选模拟肽,交联BC后免疫BALB/c小鼠,制备抗LPS的单克隆抗体。本研究包括以下两个部分:
一、抗脂多糖单克隆抗体的制备及鉴定
模拟肽13L的筛选与合成按照本课题组以前建立的方法进行,现简述如下:以大肠杆菌LPS L2630为配体用亲和层析法从抗鼠伤寒沙门氏菌兔血清中纯化出抗LPS的多克隆抗体;以纯化的多克隆抗体为配体从环七肽库中筛选出模拟LPS抗原表位的多肽,其中的一个亲和力较强的多肽被命名为L7;为了增加L7的结构稳定性,我们在其氨基酸序列两侧各加入两个额外的氨基酸,并命名为13L,然后交由生物公司进行合成。
由于短肽分子量过低而不具备免疫原性,遂将其与蓝载体通过交联剂EDC的作用而进行交联,将交联物13L-BC作为本次实验的免疫原。
用13L-BC免疫6-8周龄的BALB/c小鼠,共免疫7次,免疫程序如下:第1次免疫,将13L-BC与福氏完全佐剂(CFA)充分乳化后,小鼠足垫、皮下多点注射,1mg/ml,100μl/只,3周后进行第二次免疫;第2-5次免疫,将13L-BC与福氏不完全佐剂(IFA)充分乳化后,小鼠足垫、皮下多点注射,1mg/ml,100μl/只,每次间隔2周;第6次免疫,将煮沸处理过的大肠杆菌O111:B4和鼠伤寒杆菌直接行小鼠腹腔注射,5×10~6CFU/只;第七次免疫,2周后,小鼠眼眶静脉采血并包被L2630和L7261用间接ELISA检测血清中抗LPS抗体的滴度,抗体滴度高的小鼠再尾静脉注射10μg L2630加强免疫一次。
3天后,处死小鼠,取其脾细胞与NS-1细胞融合制备杂交瘤,用L2630和L7261进行复合筛选,对双阳性孔进行克隆化。连续三次克隆化100%阳性后,扩大培养杂交瘤细胞,收集杂交瘤细胞行小鼠腹腔注射制备腹水,用亲和层析法从制备的腹水中纯化单克隆抗体。
共融合3次,融合率在60%-100%之间,阳性率在1%-5%之间。通过筛选、克隆化最后获得一株能稳定分泌抗LPS抗体的杂交瘤细胞,命名为SMU-3A8。包被L2630检测SMU-3A8的腹水效价和纯化抗体效价,结果分别为1:1×10~6和8ng/ml。经IgG亚类鉴定试剂盒鉴定,SMU-3A8的亚类为IgG2a。用间接ELISA、WesternBlot、Dot-ElISA、免疫荧光和流式细胞术显示SMU-3A8可以和四种商品化的LPS反应,即大肠杆菌O111∶B4型LPS(L2630)、大肠杆菌O127∶B8型LPS(L3880)、鼠伤寒沙门氏菌LPS(L7261)和肠炎沙门氏菌LPS(L6761),其亲和力分为4.9×10~8L/mol、1.6×10~7L/mol、4.2×10~8L/mol和1.5×10~7L/mol。并可以和七种G~-菌(大肠杆菌O111∶B4,大肠杆菌O127∶B8,鼠伤寒沙门氏菌,福氏痢疾杆菌,绿脓假单胞菌,结肠炎耶尔森菌,鼠疫耶尔森菌)的超声上清及细菌全菌反应,但不能和金黄色葡萄球菌、肺炎链球菌、变形杆菌等G~+菌反应。说明SMU-3A8是具有广泛交叉反应活性的抗LPS单克隆抗体。
二、抗脂多糖单克隆抗体的体内、外保护活性
在第一部分实验中,我们已成功的制备了一株能与多种商品化LPS和G~-菌具有交叉反应性的单克隆抗体SMU-3A8。我们用补体依赖细胞毒试验和SMU-3A8对LPS刺激单核细胞产生NO的影响来检测其体外活性;用SMU-3A8对致死量G~-菌感染小鼠存活率的影响以及对内毒素休克小鼠存活率的影响来检测其体内活性,具体过程如下:
1.补体介导的细胞毒试验细菌37℃振荡培养5小时后,室温8000rpm,离心10分钟,用无菌PBS洗涤三次并调整细菌浓度为10~4CFU/ml。然后按体积1∶1的比例加入倍比稀释的SMU-3A8(初始浓度10μg/ml),转入到96孔培养板中,180μl/孔,加入豚鼠血清,20μl/孔,设不加抗体及不加补体的空白对照组。37℃反应1小时后,每孔取出10μl用无菌PBS稀释20倍至终体积200μl,然后均匀涂布于LB平板上,37℃过夜培养,计数形成的细菌集落。
结果显示,在补体存在的情况下,SMU-3A8并不能对金葡菌、大肠杆菌、鼠伤寒菌和痢疾杆菌构成杀伤作用。
2.SMU-3A8对LPS刺激单核巨噬细胞产生NO的影响小鼠单核巨噬细胞系RAW264.7为本实验的靶细胞。用完全培养基调整细胞浓度为10~6个/ml,加入到24孔培养板中,500μl/孔。然后同时加入终浓度为10μg/ml的L2630和倍比稀释的SMU-3A8,设同型对照和空白对照。37℃培养24小时后,收集上清,用NO检测试剂盒检测上清中NO的浓度。
结果显示,在较高浓度下(≥10μg/ml)SMU-3A8对L2630刺激RAW264.7产生NO有一定的抑制作用,如在10μg/ml时,其抑制率为20%左右。
3.SMU-3A8对细菌感染和内毒素休克小鼠模型的保护作用参照本课题组以前建立的G~-菌感染和内毒素休克动物模型,并做了适当调整,作为本次实验的动物模型。G~-菌过夜培养后用无菌PBS调整浓度,BABL/c小鼠腹腔注射,使其在4-7天时,死亡率为100%。BALB/c小鼠尾静脉直接注射LPS来制备内毒素休克模型,200μg/只,使其在12-36小时死亡率为100%。细菌或LPS注射2小时前,于小鼠尾静脉注射不同浓度的SMU-3A8,设同型对照和空白对照。观察并记录小鼠的存活情况。
结果显示,SMU-3A8在高浓度时能将鼠伤寒感染小鼠的死亡率从100%降低到75%左右,对大肠杆菌,痢疾杆菌感染小鼠则没有保护作用。对本实验中的内毒素休克模型也没有保护作用。
结论
在本实验中,我们用模拟肽模拟LPS的抗原表位并将模拟肽与蓝载体交联作为免疫原,成功制备了一株具有交叉反应活性的抗LPS mAb SMU-3A8。ELISA,Dot-ELISA,Western Blotting,免疫荧光,流式细胞检测结果显示,SMU-3A8可以和四种商品化的LPS以及七种G~-菌反应,但不和G~+菌反应。体内、外保护实验显示,SMU-3A8可以减少L2630刺激小鼠单核巨噬细胞系RAW264.7产生NO的量,并可以提高鼠伤寒菌感染小鼠的存活率,具有一定的保护作用。
据我们所知,还未有用模拟肽制备抗LPS mAb的报道,我们的实验为多糖等TI抗原的mAb制备提供了一个新策略。SMU-3A8具有交叉反应性和一定的保护活性,有可能成为LPS研究的一个重要的工具,在临床G~-菌感染的诊断和治疗中也可能会发挥一定作用。
Sepsis is the most common cause of death in many intensive care units.Despite extensive research on the pathophysiology of sepsis and the technical advances,the mortality associated with severe sepsis and sepsis shock are 25%to 30%and 40%to 70%,respectively.
Sepsis is triggered by gram-negative and gram-positive bacteria,fungi,and other microorganisms in which gram-negative bacteria are responsible for approximately 40%of total episodes.Lipopolysaccharide(LPS),the major constituent of the outer membrane of gram-negative bacteria,is the predominant antigenic and toxic component,It thus has been qualified as a target for diagnostic and therapeutic treatment for infections associated with gram-negative bacteria.At present,there is no ideal antagonist against LPS and lipid A for clinical prophylaxis and therapy.But there were some evidences that the pre-existed of antibodies against LPS are protective and the amount of antibody against core glycolipid of LPS in vivo is correlated with the survival in sepsis patients.Monoclonal antibodies(mAbs) to the LPS have been evaluated in a variety of experimental models and used in therapeutic trials in human,and some were found to improve survival in patients with gram-negative sepsis.
LPS is consisted of three distinct parts:the O-specific antigen,also called O-antigen or somatic antigen,which is a long chain polysaccharide;the oligosaccharide core,composed of approximately 10 monosaccharides;the lipid A. LPS is structurally complex,heterogeneous macromolecule,and belongs to typeⅠthymus-independent(TI) antigen.The respone to TI antigen is strikingly different from the response to most proteins.TI antigens generally fail to elicit a memory response and usually do not show affinity maturation,making it difficult to produce mAbs against LPS with high affinity and broadly cross-reactivity if using the traditional immunization schedual.A reasonable alternative may derive from the use of molecular mimicry technology,and the searching for effective formulations that mediate reactive immune responses to TI antigen has consequently focused on identifying peptides mimic of TI antigens.Peptides,which are intrinsical thymus-dependent(TD) antigen and mimic the epitope structure of LPS,have the potential to overcome the limitations of TI antigen and obtain high affinity, cross-reactive mAbs to LPS.
Phage display is a simple methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping and infectious disease research. Actually,phage display has successfully identified peptides mimic of polysaccharides, for instance,peptides mimic of the capsular polysaccharide of three of the Neisseria meningitides serotypes A,B and C have been identified using phage display.The results of these studies are encouraging that the peptides identified in this way do elicit immune responses in murine models and are suitable for generating high affinity mAbs.
Our objective is to screen and synthesize the peptide that mimic the conservative epitopes of LPS,conjugate the peptide with adequate carrier,then use the conjugation as immunogen to generate some high affinity cross reactive mAbs against lipopolysaccharide and investigate the potential protective activity of the mAbs against G~- bacteria infection in animal models.
In our previous research,four peptides mimetic of the conservative structure of S.typhi-LPS were screened from phage display cyclic7-peptide library by using purified polyclonal anti- S.typhi LPS antibodies with cross protection against G~-bacteria infection.The antigenicity and immunogenicity of these peptides were identified by serials of experiment in vivo and in vitro,and one of the peptides mimicry to LPS epitope was synthesized and named as 13L.Our previous results suggested that the conjugation of 13L with Blue Carrier(BC) could be used as immunogen and the BALB/c mice immunized with peptide-BC conjugation were protected against G~- bacteria infection and endotoxic shock induced by injection of LPS.Based on these research,13L was chosen as the candidate to immunize the mice and generate high affinity cross reactive mAbs against lipopolysaccharide.
This research can be divided into the following two parts.
PartⅠGeneration and characterization of cross reactive mAbs To Lipopolysaccharide
The peptide 13L was screened and synthesized as described previously.Briefly,the LPS mimotopes were screened from c7c phage display peptide library by using polyclonal antibody against E.coli LPS 2630(L2630) purified with affinity chromatograph.One of the peptides mimicry to LPS epitope was selected and named as L7.the origin sequence of L7 was added four additional amino acids on the two sides to stabilize the conformation and named as 13L,which was synthesized with 90%purity and stored at -20℃until use.2 mg 13L and 4 mg BC were added to 1 ml MES(0.1 M,pH4.5) followed by adding 1 mg EDC,subsequently,the mixed solution was agitated gently at 25℃for 2 hours to generate conjugation of 13L with BC.After that,the conjugation was dialysed against PBS to remove free EDC and labeled as 13L-BC,which is the immunogen for following immunization procedure.
Six femal BALB/c mice,6-8 weeks old,each was immunized with 200μg 13L-BC emulsified in equal volum of complete freund's adjuvant(CFA) by footpad inoculation and subcutaneous(s.c.) injection.Subsequently,immunization were carried out with 100μg 13L-BC emulsified in equal volum of incomplete freund's adjuvant(IFA) by footpad inoculation and s.c.injection at two weeks intervals.After the 5th immunization,5×10~6 CFU of hot-killed E.coli O111:B4 and Salmonella typhimuria were injected intraperitoneally.Two weeks later,mice were bled from orbital vein and serum titers were screened for reactivity to L2630 and L7261,the LPS of E.coli O111:B4 add Salmonella typhimurium,using Enzyme-linked immunosorbent assay(ELISA).Mice were given the final booster of 10μg /body L2630 in 100μl phosphate-buffered saline(PBS,pH 7.4) by caudal vein injection. Three days after the final booster,mice were sacrificed and splenocytes were generated and fused with NS-1 myeloma cells which cutured in RPMI-1640 medium containing 20%fetal bovine serum in the presence of PEG4000 according to established protocols.Hybridomas were selected by using HAT medium,and positive wells were screened by indirect ELISAs coated with L2630 and L7261 separately.Sub-cloning was performed five times using the limiting dilution method. Monoclonal antibodys were harvested from ascites fluid of BABL/c mice in which hybridoma had been injected intraperitoneal(i.p.).Monoclonal antibodys were purified from ascites using HiTrap protein G HP column according to the manufacture's instructions.The concentration and isotypes of the mAbs produced were determined using BCA protein assay kit and a mouse monoclonal antibody isotyping kit respectively,according to the manufacture's instructions.After three fusions and cloning,one hybridoma cell line which secrete monoclonal antibodie against LPS was obtained and named as SMU-3A8,of which the isotype belongs to IgG2a.
The binding characteristic of SMU-3A8 to different bacteria and LPSs was carried out by indirect ELISA,Dot-ELISA,western blotting,immunofluorescence, and flow cytometry,and the results indicated that SMU-3A8 can react with four commercial LPSs and seven G~- negative bacterial with high affinity.
PartⅡThe characterizing protective activitys of SMU-3A8 in vitro and vivo
1.Test for bactericidal activity.The bacteria were grown overnight in Luria-Bertani(LB) Medium,centrifuged for 10 mins at room temperature,8000rpm. The precipitation was washed three times,and resuspended to 10~4 CFU/ml in sterile PBS.SMU-3A8(10μg/ml) was serially diluted(2-fold dilutions) in PBS,mixed 1:1 (100μl/100μl) with bacteria,and placed inU-bottom 96-well plates.Fresh guinea pig complement was added to a 20%final concentration and the plates were incubated for 1h at 37℃.Complement alone was used as control.After the incubation,samples were diluted(20-fold dilutions in PBS) and plated on LB agar.The CFU were counted after overnight incubation at 37℃.The results suggested that SMU-3A8 has no batericidal activity against E.coli O111:B4,E.coli O128,S.syphimurium and dysentery bacilli in the presence of active complement.
2.LPS-induced NO secretion.The murine macrophage cell line RAW264.7 were cultured in 0.5 ml of pyrogen-free medium in the presence of L2630 and in the presence or absence of increasing concentrations of SMU-3A8 or of an mAb belongs to IgG2a as control.Culture supernatants were collected after 24h for NO determination by using a Nitric Oxide Assay Kit.The data demenstrated that SMU-3A8 could inhibit the secretion of NO induced by LPS stimulation to the murine macrophage cell line RAW264.7 at high concentration.
3.Effect of SMU-3A8 upon bacterial challenge and endotoxic shock.the G~-bacteria infection animal model was established by i.p injection of S.typhi to female BABL/c mice,which could lead to 100%mortality in 4-7 days.Endotoxin shock model was induced by i.v.injection of a 100%lethal dose of L7261 or of L2630 to female BALB/c mice within 12-36 h.SMU-3A8(range 10 to 1,000μg per mouse), diluted in pyrogen-free NaCl solution,was administered i.v.2 h prior to bacteria or LPS challenging.Survival rate was recorded.The results indicated that SMU-3A8 could not improve the survival rate of endotoxin shock mice induced by i.v.LPS injection,but could decrease the death rate of S.typhi infection mice from 100%to 75%.
Conclusively,in this study,we successfully generated an mAb specific to LPS using a novel immunization procedure:peptide mimics of the epitope of LPS was screened by phage display and conjugated to protein carrier as the immunogen followed by boosting with bacteria ans LPS.The mAb can recognize seven different strains of gram-negative bacteria and four kinds of commercial LPSs with high affinity,and shows some protective activities against S.typhi infection in mouse model.It may provide a useful tool for research on LPS.Moreover,the immunization procedure may offer another consideration to generate mAbs for therapeutic treatment with sepsis,
脓毒症可由多种病原微生物感染引起,包括革兰氏阳性(G~+)菌、革兰氏阴性(G~-)菌和真菌等等,而其中G~-菌感染是最重要的一种,占总发病人数的40%以上。脂多糖(LPS)是G~-菌细胞壁的重要组成成分,也是G~-菌感染脓毒症中最主要的毒力因子和抗原成分。所以在G~-菌感染引起的脓毒症的诊断与治疗中,LPS是一个重要的靶点。目前尚无LPS拮抗剂用于临床应用或进入临床观察,但有证据显示,预存抗LPS抗体与败血症的发生、大样本住院肿瘤患者、烧伤、外科手术等因感染死亡的病人数呈负相关,即对LPS诱发的病理进程有明确的保护作用。动物实验显示针对LPS的单克隆抗体(mAb)对实验性脓毒症动物模型具有良好的保护作用。所以,抗LPS的mAb有可能成为治疗G~-菌感染诱发的脓毒症的一种新制剂。
LPS由三部分组成:O特异性抗原,也叫O抗原或体抗原,位于G~-菌细胞壁的最外面,由一长链多糖构成;核心寡糖,是一由大约10个左右的单糖组成的寡糖;类脂A。所以,脂多糖是一结构十分复杂、具有多种异质性的生物大分子,而且属于胸腺非依赖抗原(TI)抗原。与蛋白质等胸腺依赖(TD)抗原不同,TI抗原不能诱导抗体亲和成熟,也不能诱导免疫记忆,所以按常规的免疫方法很难产生高亲和力、交叉反应性抗LPS的mAb。为解决此难题,一个可行的方法就是用分子模拟技术,即用蛋白质、多肽来模拟LPS的抗原结构,从而将多糖类TI抗原转变为多肽类TD抗原,提高抗原的免疫原性。那么,筛选出能够模拟LPS抗原结构的多肽就成为其中关键的一步。
噬菌体展示技术是一种用于筛选和改造功能性短肽和蛋白质强有力的生物技术,广泛应用于免疫学和分子生物学领域。事实上,噬菌体展示技术早已成功的应用于多糖表位的模拟肽筛选,如对脑膜炎奈瑟球菌A、B、C三种血清型的荚膜多糖的模拟肽筛选。这些结果说明,用噬菌体展示技术可以筛选出模拟多糖抗原表位的模拟肽,从而将TI抗原转变为TD抗原,诱导强而有力的记忆性免疫应答,制备出高亲和力、交叉反应性抗LPS的mAb。
所以,本课题的主要研究思路是:利用噬菌体展示技术筛选并合成模拟LPS保守抗原表位的多肽,将其抗原表位的化学性质由脂多糖改变为短肽,由此使TI抗原改变为TD抗原;然后将多肽与适当的载体进行交联,用交联物作为免疫原免疫BALB/c小鼠,制备具有高亲和力、广泛交叉反应性的抗LPS的mAb。最后对其在动物体内、外的保护活性进行初步探讨。
本课题组在前期工作中,以具有交叉保护活性的抗鼠伤寒沙门氏菌多克隆抗体为配基成功的从噬菌体环七肽库中筛选到四个可模拟LPS表位的保守序列,其免疫原性和抗原性已通过一系列实验得以证实。其中一个模拟肽经加入延长序列被命名为13L,我们的前期实验显示,用13L交联蓝载体(BC)作为免疫原免疫BALB/c小鼠后,可以诱导小鼠产生针对G~-菌感染和内毒素休克的保护作用。以这些实验结果为依据,本实验选择13L作为候选模拟肽,交联BC后免疫BALB/c小鼠,制备抗LPS的单克隆抗体。本研究包括以下两个部分:
一、抗脂多糖单克隆抗体的制备及鉴定
模拟肽13L的筛选与合成按照本课题组以前建立的方法进行,现简述如下:以大肠杆菌LPS L2630为配体用亲和层析法从抗鼠伤寒沙门氏菌兔血清中纯化出抗LPS的多克隆抗体;以纯化的多克隆抗体为配体从环七肽库中筛选出模拟LPS抗原表位的多肽,其中的一个亲和力较强的多肽被命名为L7;为了增加L7的结构稳定性,我们在其氨基酸序列两侧各加入两个额外的氨基酸,并命名为13L,然后交由生物公司进行合成。
由于短肽分子量过低而不具备免疫原性,遂将其与蓝载体通过交联剂EDC的作用而进行交联,将交联物13L-BC作为本次实验的免疫原。
用13L-BC免疫6-8周龄的BALB/c小鼠,共免疫7次,免疫程序如下:第1次免疫,将13L-BC与福氏完全佐剂(CFA)充分乳化后,小鼠足垫、皮下多点注射,1mg/ml,100μl/只,3周后进行第二次免疫;第2-5次免疫,将13L-BC与福氏不完全佐剂(IFA)充分乳化后,小鼠足垫、皮下多点注射,1mg/ml,100μl/只,每次间隔2周;第6次免疫,将煮沸处理过的大肠杆菌O111:B4和鼠伤寒杆菌直接行小鼠腹腔注射,5×10~6CFU/只;第七次免疫,2周后,小鼠眼眶静脉采血并包被L2630和L7261用间接ELISA检测血清中抗LPS抗体的滴度,抗体滴度高的小鼠再尾静脉注射10μg L2630加强免疫一次。
3天后,处死小鼠,取其脾细胞与NS-1细胞融合制备杂交瘤,用L2630和L7261进行复合筛选,对双阳性孔进行克隆化。连续三次克隆化100%阳性后,扩大培养杂交瘤细胞,收集杂交瘤细胞行小鼠腹腔注射制备腹水,用亲和层析法从制备的腹水中纯化单克隆抗体。
共融合3次,融合率在60%-100%之间,阳性率在1%-5%之间。通过筛选、克隆化最后获得一株能稳定分泌抗LPS抗体的杂交瘤细胞,命名为SMU-3A8。包被L2630检测SMU-3A8的腹水效价和纯化抗体效价,结果分别为1:1×10~6和8ng/ml。经IgG亚类鉴定试剂盒鉴定,SMU-3A8的亚类为IgG2a。用间接ELISA、WesternBlot、Dot-ElISA、免疫荧光和流式细胞术显示SMU-3A8可以和四种商品化的LPS反应,即大肠杆菌O111∶B4型LPS(L2630)、大肠杆菌O127∶B8型LPS(L3880)、鼠伤寒沙门氏菌LPS(L7261)和肠炎沙门氏菌LPS(L6761),其亲和力分为4.9×10~8L/mol、1.6×10~7L/mol、4.2×10~8L/mol和1.5×10~7L/mol。并可以和七种G~-菌(大肠杆菌O111∶B4,大肠杆菌O127∶B8,鼠伤寒沙门氏菌,福氏痢疾杆菌,绿脓假单胞菌,结肠炎耶尔森菌,鼠疫耶尔森菌)的超声上清及细菌全菌反应,但不能和金黄色葡萄球菌、肺炎链球菌、变形杆菌等G~+菌反应。说明SMU-3A8是具有广泛交叉反应活性的抗LPS单克隆抗体。
二、抗脂多糖单克隆抗体的体内、外保护活性
在第一部分实验中,我们已成功的制备了一株能与多种商品化LPS和G~-菌具有交叉反应性的单克隆抗体SMU-3A8。我们用补体依赖细胞毒试验和SMU-3A8对LPS刺激单核细胞产生NO的影响来检测其体外活性;用SMU-3A8对致死量G~-菌感染小鼠存活率的影响以及对内毒素休克小鼠存活率的影响来检测其体内活性,具体过程如下:
1.补体介导的细胞毒试验细菌37℃振荡培养5小时后,室温8000rpm,离心10分钟,用无菌PBS洗涤三次并调整细菌浓度为10~4CFU/ml。然后按体积1∶1的比例加入倍比稀释的SMU-3A8(初始浓度10μg/ml),转入到96孔培养板中,180μl/孔,加入豚鼠血清,20μl/孔,设不加抗体及不加补体的空白对照组。37℃反应1小时后,每孔取出10μl用无菌PBS稀释20倍至终体积200μl,然后均匀涂布于LB平板上,37℃过夜培养,计数形成的细菌集落。
结果显示,在补体存在的情况下,SMU-3A8并不能对金葡菌、大肠杆菌、鼠伤寒菌和痢疾杆菌构成杀伤作用。
2.SMU-3A8对LPS刺激单核巨噬细胞产生NO的影响小鼠单核巨噬细胞系RAW264.7为本实验的靶细胞。用完全培养基调整细胞浓度为10~6个/ml,加入到24孔培养板中,500μl/孔。然后同时加入终浓度为10μg/ml的L2630和倍比稀释的SMU-3A8,设同型对照和空白对照。37℃培养24小时后,收集上清,用NO检测试剂盒检测上清中NO的浓度。
结果显示,在较高浓度下(≥10μg/ml)SMU-3A8对L2630刺激RAW264.7产生NO有一定的抑制作用,如在10μg/ml时,其抑制率为20%左右。
3.SMU-3A8对细菌感染和内毒素休克小鼠模型的保护作用参照本课题组以前建立的G~-菌感染和内毒素休克动物模型,并做了适当调整,作为本次实验的动物模型。G~-菌过夜培养后用无菌PBS调整浓度,BABL/c小鼠腹腔注射,使其在4-7天时,死亡率为100%。BALB/c小鼠尾静脉直接注射LPS来制备内毒素休克模型,200μg/只,使其在12-36小时死亡率为100%。细菌或LPS注射2小时前,于小鼠尾静脉注射不同浓度的SMU-3A8,设同型对照和空白对照。观察并记录小鼠的存活情况。
结果显示,SMU-3A8在高浓度时能将鼠伤寒感染小鼠的死亡率从100%降低到75%左右,对大肠杆菌,痢疾杆菌感染小鼠则没有保护作用。对本实验中的内毒素休克模型也没有保护作用。
结论
在本实验中,我们用模拟肽模拟LPS的抗原表位并将模拟肽与蓝载体交联作为免疫原,成功制备了一株具有交叉反应活性的抗LPS mAb SMU-3A8。ELISA,Dot-ELISA,Western Blotting,免疫荧光,流式细胞检测结果显示,SMU-3A8可以和四种商品化的LPS以及七种G~-菌反应,但不和G~+菌反应。体内、外保护实验显示,SMU-3A8可以减少L2630刺激小鼠单核巨噬细胞系RAW264.7产生NO的量,并可以提高鼠伤寒菌感染小鼠的存活率,具有一定的保护作用。
据我们所知,还未有用模拟肽制备抗LPS mAb的报道,我们的实验为多糖等TI抗原的mAb制备提供了一个新策略。SMU-3A8具有交叉反应性和一定的保护活性,有可能成为LPS研究的一个重要的工具,在临床G~-菌感染的诊断和治疗中也可能会发挥一定作用。
Sepsis is the most common cause of death in many intensive care units.Despite extensive research on the pathophysiology of sepsis and the technical advances,the mortality associated with severe sepsis and sepsis shock are 25%to 30%and 40%to 70%,respectively.
Sepsis is triggered by gram-negative and gram-positive bacteria,fungi,and other microorganisms in which gram-negative bacteria are responsible for approximately 40%of total episodes.Lipopolysaccharide(LPS),the major constituent of the outer membrane of gram-negative bacteria,is the predominant antigenic and toxic component,It thus has been qualified as a target for diagnostic and therapeutic treatment for infections associated with gram-negative bacteria.At present,there is no ideal antagonist against LPS and lipid A for clinical prophylaxis and therapy.But there were some evidences that the pre-existed of antibodies against LPS are protective and the amount of antibody against core glycolipid of LPS in vivo is correlated with the survival in sepsis patients.Monoclonal antibodies(mAbs) to the LPS have been evaluated in a variety of experimental models and used in therapeutic trials in human,and some were found to improve survival in patients with gram-negative sepsis.
LPS is consisted of three distinct parts:the O-specific antigen,also called O-antigen or somatic antigen,which is a long chain polysaccharide;the oligosaccharide core,composed of approximately 10 monosaccharides;the lipid A. LPS is structurally complex,heterogeneous macromolecule,and belongs to typeⅠthymus-independent(TI) antigen.The respone to TI antigen is strikingly different from the response to most proteins.TI antigens generally fail to elicit a memory response and usually do not show affinity maturation,making it difficult to produce mAbs against LPS with high affinity and broadly cross-reactivity if using the traditional immunization schedual.A reasonable alternative may derive from the use of molecular mimicry technology,and the searching for effective formulations that mediate reactive immune responses to TI antigen has consequently focused on identifying peptides mimic of TI antigens.Peptides,which are intrinsical thymus-dependent(TD) antigen and mimic the epitope structure of LPS,have the potential to overcome the limitations of TI antigen and obtain high affinity, cross-reactive mAbs to LPS.
Phage display is a simple methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping and infectious disease research. Actually,phage display has successfully identified peptides mimic of polysaccharides, for instance,peptides mimic of the capsular polysaccharide of three of the Neisseria meningitides serotypes A,B and C have been identified using phage display.The results of these studies are encouraging that the peptides identified in this way do elicit immune responses in murine models and are suitable for generating high affinity mAbs.
Our objective is to screen and synthesize the peptide that mimic the conservative epitopes of LPS,conjugate the peptide with adequate carrier,then use the conjugation as immunogen to generate some high affinity cross reactive mAbs against lipopolysaccharide and investigate the potential protective activity of the mAbs against G~- bacteria infection in animal models.
In our previous research,four peptides mimetic of the conservative structure of S.typhi-LPS were screened from phage display cyclic7-peptide library by using purified polyclonal anti- S.typhi LPS antibodies with cross protection against G~-bacteria infection.The antigenicity and immunogenicity of these peptides were identified by serials of experiment in vivo and in vitro,and one of the peptides mimicry to LPS epitope was synthesized and named as 13L.Our previous results suggested that the conjugation of 13L with Blue Carrier(BC) could be used as immunogen and the BALB/c mice immunized with peptide-BC conjugation were protected against G~- bacteria infection and endotoxic shock induced by injection of LPS.Based on these research,13L was chosen as the candidate to immunize the mice and generate high affinity cross reactive mAbs against lipopolysaccharide.
This research can be divided into the following two parts.
PartⅠGeneration and characterization of cross reactive mAbs To Lipopolysaccharide
The peptide 13L was screened and synthesized as described previously.Briefly,the LPS mimotopes were screened from c7c phage display peptide library by using polyclonal antibody against E.coli LPS 2630(L2630) purified with affinity chromatograph.One of the peptides mimicry to LPS epitope was selected and named as L7.the origin sequence of L7 was added four additional amino acids on the two sides to stabilize the conformation and named as 13L,which was synthesized with 90%purity and stored at -20℃until use.2 mg 13L and 4 mg BC were added to 1 ml MES(0.1 M,pH4.5) followed by adding 1 mg EDC,subsequently,the mixed solution was agitated gently at 25℃for 2 hours to generate conjugation of 13L with BC.After that,the conjugation was dialysed against PBS to remove free EDC and labeled as 13L-BC,which is the immunogen for following immunization procedure.
Six femal BALB/c mice,6-8 weeks old,each was immunized with 200μg 13L-BC emulsified in equal volum of complete freund's adjuvant(CFA) by footpad inoculation and subcutaneous(s.c.) injection.Subsequently,immunization were carried out with 100μg 13L-BC emulsified in equal volum of incomplete freund's adjuvant(IFA) by footpad inoculation and s.c.injection at two weeks intervals.After the 5th immunization,5×10~6 CFU of hot-killed E.coli O111:B4 and Salmonella typhimuria were injected intraperitoneally.Two weeks later,mice were bled from orbital vein and serum titers were screened for reactivity to L2630 and L7261,the LPS of E.coli O111:B4 add Salmonella typhimurium,using Enzyme-linked immunosorbent assay(ELISA).Mice were given the final booster of 10μg /body L2630 in 100μl phosphate-buffered saline(PBS,pH 7.4) by caudal vein injection. Three days after the final booster,mice were sacrificed and splenocytes were generated and fused with NS-1 myeloma cells which cutured in RPMI-1640 medium containing 20%fetal bovine serum in the presence of PEG4000 according to established protocols.Hybridomas were selected by using HAT medium,and positive wells were screened by indirect ELISAs coated with L2630 and L7261 separately.Sub-cloning was performed five times using the limiting dilution method. Monoclonal antibodys were harvested from ascites fluid of BABL/c mice in which hybridoma had been injected intraperitoneal(i.p.).Monoclonal antibodys were purified from ascites using HiTrap protein G HP column according to the manufacture's instructions.The concentration and isotypes of the mAbs produced were determined using BCA protein assay kit and a mouse monoclonal antibody isotyping kit respectively,according to the manufacture's instructions.After three fusions and cloning,one hybridoma cell line which secrete monoclonal antibodie against LPS was obtained and named as SMU-3A8,of which the isotype belongs to IgG2a.
The binding characteristic of SMU-3A8 to different bacteria and LPSs was carried out by indirect ELISA,Dot-ELISA,western blotting,immunofluorescence, and flow cytometry,and the results indicated that SMU-3A8 can react with four commercial LPSs and seven G~- negative bacterial with high affinity.
PartⅡThe characterizing protective activitys of SMU-3A8 in vitro and vivo
1.Test for bactericidal activity.The bacteria were grown overnight in Luria-Bertani(LB) Medium,centrifuged for 10 mins at room temperature,8000rpm. The precipitation was washed three times,and resuspended to 10~4 CFU/ml in sterile PBS.SMU-3A8(10μg/ml) was serially diluted(2-fold dilutions) in PBS,mixed 1:1 (100μl/100μl) with bacteria,and placed inU-bottom 96-well plates.Fresh guinea pig complement was added to a 20%final concentration and the plates were incubated for 1h at 37℃.Complement alone was used as control.After the incubation,samples were diluted(20-fold dilutions in PBS) and plated on LB agar.The CFU were counted after overnight incubation at 37℃.The results suggested that SMU-3A8 has no batericidal activity against E.coli O111:B4,E.coli O128,S.syphimurium and dysentery bacilli in the presence of active complement.
2.LPS-induced NO secretion.The murine macrophage cell line RAW264.7 were cultured in 0.5 ml of pyrogen-free medium in the presence of L2630 and in the presence or absence of increasing concentrations of SMU-3A8 or of an mAb belongs to IgG2a as control.Culture supernatants were collected after 24h for NO determination by using a Nitric Oxide Assay Kit.The data demenstrated that SMU-3A8 could inhibit the secretion of NO induced by LPS stimulation to the murine macrophage cell line RAW264.7 at high concentration.
3.Effect of SMU-3A8 upon bacterial challenge and endotoxic shock.the G~-bacteria infection animal model was established by i.p injection of S.typhi to female BABL/c mice,which could lead to 100%mortality in 4-7 days.Endotoxin shock model was induced by i.v.injection of a 100%lethal dose of L7261 or of L2630 to female BALB/c mice within 12-36 h.SMU-3A8(range 10 to 1,000μg per mouse), diluted in pyrogen-free NaCl solution,was administered i.v.2 h prior to bacteria or LPS challenging.Survival rate was recorded.The results indicated that SMU-3A8 could not improve the survival rate of endotoxin shock mice induced by i.v.LPS injection,but could decrease the death rate of S.typhi infection mice from 100%to 75%.
Conclusively,in this study,we successfully generated an mAb specific to LPS using a novel immunization procedure:peptide mimics of the epitope of LPS was screened by phage display and conjugated to protein carrier as the immunogen followed by boosting with bacteria ans LPS.The mAb can recognize seven different strains of gram-negative bacteria and four kinds of commercial LPSs with high affinity,and shows some protective activities against S.typhi infection in mouse model.It may provide a useful tool for research on LPS.Moreover,the immunization procedure may offer another consideration to generate mAbs for therapeutic treatment with sepsis,
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[25]Habich,C.,K.Kempe,R.van der Zee,et al.Heat shock protein 60:specific binding of lipopolysaccharide[J].J Immunol,2005,174(3):1298-1305.
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[28]Park,I.,I.H.Choi,S.J.Kim,et al.Peptide mimotopes of Neisseria meningitidis group B capsular polysaccharide[J].Yonsei Med J,2004,45(4):755-758.
[29]Prinz,D.M.,S.L.SmithsonM.A.Westerink.Two different methods result in the selection of peptides that induce a protective antibody response to Neisseria meningitidis serogroup C[J].J Immunol Methods,2004,285(1):1-14.
[30]郑山根,刘.,朱平,富宁.交叉保护性抗LPS多克隆抗体的制备与LPS多表位模拟肽的筛选[J].中华微生物学和免疫学杂志,2003,23(11):903-906.
[31]罗海波,郑.健,朱.平,et al.脂多糖模拟肽13L诱导对小鼠内毒素休克的保护反应[J].生物化学与生物物理进展,2008,35(11):1312-1319.
[32]Beatty,J.D.,B.G.Beatty W.G.Vlahos.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].J Immunol Methods,1987,100(1-2):173-179.
[33]Xu,Y.,N.YamamotoK.D.Janda.Catalytic antibodies:hapten design strategies and screening methods[J].Bioorg Med Chem,2004,12(20):5247-5268.
[34]Manosalva,H.,A.E.De IoannesM.I.Becker.Development of monoclonal antibodies bearing the internal image of the gizzerosine epitope and application in a competitive ELISA for fish meal[J].Hybrid Hybridomics,2004,23(1):45-54.
[35]De Ioannes,P.,B.Moltedo,H.Oliva,et al.Hemocyanin of the molluscan Concholepas concholepas exhibits an unusual heterodecameric array of subunits [J].J Biol Chem,2004,279(25):26134-26142.
[36]曹雪涛.免疫学研究的发展趋势及我国免疫学研究的现状与展望[J].中国免疫学杂志,2009,25(1):10-23.
[37]Scheid,J.F.,H.Mouquet,N.Feldhahn,et al.Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals[J].Nature,2009,458(7238):636-640.