CN102649816B - Hmgb蛋白及抗体在制备近江牡蛎抗感染免疫制剂中的应用 - Google Patents
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Abstract
本发明涉及生物技术领域,旨在提供HMGB蛋白及抗体在制备近江牡蛎抗感染免疫制剂中的应用。该蛋白质的氨基酸序列如SEQ ID NO:2 所示;该基因的编码框核苷酸序列如SEQ ID NO.1所示;所述蛋白质在制备近江牡蛎抗感染免疫制剂中的应用,是将所述蛋白质免疫新西兰大白兔进而获得多克隆抗血清。本发明获得了近江牡蛎HMGB基因编码框全序列,并通过构建原核表达载体,表达并纯化了可溶性的HMGB蛋白质产品,并制备了其多克隆兔抗血清。其多克隆兔抗血清具有抑制近江牡蛎病原RLO和革兰氏阴性细菌LPS引起的炎症反应及细胞坏死和细胞凋亡的作用。
Description
技术领域
本发明属于生物技术领域,涉及HMGB基因、蛋白及在制备牡蛎抗感染免疫制剂中的作用。
背景技术
高迁移率族蛋白B(high mobility group box, 简称HMGB)是高迁移率族蛋白家族的成员,是一族广泛存在于真核生物细胞体内,富含电荷的染色体非组蛋白。在结构上HMGB包含三个特征性结构域,即两个具有相似结构及较高同源性的HMG box(A box和B box),以及一个富含天冬氨酸和谷氨酸的酸性C末端。在细胞核内HMGB行使 “DNA伴侣”的功能,能通过与多种转录因子、复制蛋白和甾体受体作用,参与DNA的重组、修复、基因转录调控、细胞复制及分化成熟等生命活动。此外,目前的研究表明,HMGB能在特定条件下释放到细胞外,介导多项炎症反应,是一种重要的前炎症细胞因子和趋化因子,可作为一种“预警信号”调控机体免疫,是细胞内核酸介导的天然免疫反应的普适性哨兵。并且HMGB与多项炎症疾病如脓毒症、关节炎、胰腺炎、动脉样硬化(Kalinina et al., 2004)等发生发展密切相关,是治疗脓毒症等炎症疾病的重要‘靶分子’,而其抗体可以显著降低患脓毒症等炎性疾病的小鼠死亡率。
牡蛎是重要的海水养殖经济贝类,具有“海洋牛奶”之美誉,其肉质细嫩、肉味鲜美、营养价值高,是世界性的水产佳肴。我国是水产大国,牡蛎等贝类养殖是我国海水养殖的支柱产业之一,其中我国牡蛎养殖年产量居世界首位,达到300多万吨(湿重),占我国海水养殖总产量(约1000万吨)的约三分之一(联合国粮农组织(FAO)统计数据)。牡蛎养殖业的迅速发展为我国农业经济作出了重要的贡献,也极大的改善了沿海渔民的生活水平,但是近年来,随着牡蛎种质退化,养殖规模的不断扩大,养殖环境的恶化和养殖模式的局限性,牡蛎养殖病害频发,造成了巨大的经济损失,极大的损害了当地渔民养殖积极性,限制了牡蛎产业进一步的发展。其中研究证实类立克次体(rickettsia-like organism,简称RLO) 是其主要病原之一,因此研究牡蛎抗RLO感染相关细胞因子及其抗感染机制显得尤为重要和迫切,将有助于牡蛎抗感染免疫制剂的开发。
发明内容
本发明要解决的技术问题是,克服现有技术中存在的不足之处,提供HMGB基因、蛋白及其在制备近江牡蛎抗感染免疫制剂中的作用
为解决技术问题,本发明是通过这样的技术方案来实现的:
提供一种近江牡蛎HMGB基因编码的蛋白质,该蛋白质的氨基酸序列如SEQ ID NO:2 所示。
本发明还提供了一种编码前述蛋白质的基因编码框,该基因的编码框核苷酸序列如SEQ ID NO.1所示。
本发明还提供了所述蛋白质在制备近江牡蛎抗感染免疫制剂中的应用,是将所述蛋白质免疫新西兰大白兔进而获得多克隆抗血清。
本发明还提供了前述蛋白质制备的多克隆抗血清(即HMGB蛋白的多克隆兔抗体)在抑制近江牡蛎病原RLO和革兰氏阴性细菌细胞壁成分——脂多糖(Lipopolysaccharide,简称LPS)引起的炎症反应及细胞坏死和细胞凋亡中的应用。
本发明的有益效果在于:
本发明获得了近江牡蛎HMGB基因编码框全序列,并通过构建原核表达载体,表达并纯化了可溶性的HMGB蛋白质产品,并制备了其多克隆兔抗血清。其多克隆兔抗血清具有抑制近江牡蛎病原RLO和革兰氏阴性细菌LPS引起的炎症反应及细胞坏死和细胞凋亡的作用。
具体实施方式
本发明的下述实施例所使用分子生物学的方法均为已知的技术。
本发明中的近江牡蛎HMGB基因编码的蛋白质,该蛋白质的氨基酸序列如SEQ ID NO:2 所示。编码前述蛋白质的基因编码框的核苷酸序列如SEQ ID NO.1所示。
本发明所述蛋白质在制备近江牡蛎抗感染免疫制剂中的应用,是将所述蛋白质免疫新西兰大白兔进而获得多克隆抗血清。该多克隆抗血清(即HMGB蛋白的多克隆兔抗体)应用在抑制近江牡蛎病原RLO和革兰氏阴性细菌细胞壁成分——脂多糖(Lipopolysaccharide,简称LPS)引起的炎症反应及细胞坏死和细胞凋亡。
本发明的实现步骤包括:
1、以近江牡蛎血淋巴细胞总RNA为模板,构建cDNA文库,获得HMGB的基因编码框核苷酸序列。
2、利用DNA重组技术将HMGB基因编码框的序列片段克隆到合适的pMD19-T载体(Takara公司,日本)中,再经限制性内切酶酶切,与经同样酶切的pET-32(a) 原核表达载体(Novagen公司,德国)连接,获得重组表达质粒pET-32(a)-HMGB。
3、将阳性重组质粒PET-32(a)-HMGB转化到表达宿主菌E. coli Rossetta(DE3 )(Novagen公司,德国),异丙基硫代半乳糖苷(IPTG)(Sangon公司,加拿大)诱导表达,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测。
4、将阳性表达菌液扩大培养,利用蛋白质纯化试剂盒分离纯化重组蛋白质。
5、培养近江牡蛎单层血淋巴细胞,并分别用LPS/RLO刺激,其中部分刺激添加HMGB多克隆兔抗血清,部分刺激添加HMGB免疫前的兔血清。
6、选择不同的时间点收集处理过的细胞,用荧光定量PCR检测炎症相关细胞因子LITAF(LPS-induced TNF-α factor,脂多糖诱导的肿瘤坏死因子α)的表达,用流式细胞仪检测血淋巴细胞坏死和凋亡。
具体的操作步骤如下:
1、近江牡蛎血淋巴细胞cDNA文库的构建及筛选
提取近江牡蛎血淋巴细胞总RNA,根据In-Fusion® SMARTTM cDNA library construction kit (BD Clotech 公司)构建cDNA文库。利用M13引物对文库阳性质粒进行PCR扩增并测序。
2、近江牡蛎HMGB编码框核苷酸全长序列的获得
通过对文库筛选,我们得到一个和哺乳动物HMGB有较高同源性的基因,我们将其命名为Ca-HMGB(Ca代表近江牡蛎)。该基因的编码框核苷酸全长序列为SEQ ID NO:1。
3、PET-32(a)-HMGB表达载体的构建
以近江牡蛎cDNA为模板扩增目的片段,PCR 扩增Ca-HMGB的反应条件为:94℃预变性5 分钟,然后35个循环(94℃变性30 秒, 51℃退火30秒, 72℃延伸45秒),72℃延伸10 分钟。PCR产物分别连入PMD-19T 载体,转化大肠杆菌DH5α(Takara公司,日本),涂布LBA筛选平板,挑取若干个克隆进行PCR鉴定及进一步的测序鉴定,将PCR阳性克隆产物用BamHI、 XhoI限制性内切酶(Takara公司,日本)双酶切,连接入经过同样双酶切的PET-32a载体的相应位点上,转化大肠杆菌DH5α ( 天根公司,中国)。PCR筛选阳性克隆,经测序鉴定获得编码框正确的表达载体PET-32(a)-HMGB。
4、重组蛋白质的表达
将阳性重组质粒PET-32(a)-HMGB转化表达宿主菌E. coli Rossetta(DE3 )感受态,涂布LBA平板,37℃培养过夜。挑取一个单克隆菌落,转入LBA培养液,37℃振摇培养过夜。取适量菌液,按1:100扩大培养至OD600为 0.4-0.5时,将菌液分为若干等份,不加或分别加入IPTG,使其终浓度在0-1.0 mmol/L,继续培养6小时,离心收集宿主菌。细菌经超声波裂解后(300W,20分钟,超声2秒钟,间隔3秒钟)离心分离上清液和沉淀,分别上样,进行SDS-PAGE电泳。
5、重组蛋白质的纯化
将阳性表达质粒扩大培养,按上述方法超声处理,利用Ni-NTA亲和层析柱(Novagen公司,德国)对表达产物进行分离纯化,并采用12%的SDS-PAGE进行鉴定。
挑选体重约1.5 kg的健康新西兰白雄兔进行免疫。经过一次初始免疫和三次加强免疫后,颈动脉取血,分离血清,存储于-80℃。
6、蛋白质定量
将纯化鉴定过的蛋白质按Brad-ford法进行定量(Bradford MM. Arapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal Biochem. 1976, 72: 248-254)。
7、Ca-HMGB多克隆兔抗血清制备
蛋白质定量后,取适量蛋白,挑选2只体重约2 kg的健康新西兰大白兔(雄兔),进行多克隆抗体制备,用纯化所得的PET32a-HMGB为抗原加入等体积的佐剂充分乳化后免疫兔子,采用多点背部皮下注射,共免疫四次。
(1)免疫前,从耳静脉处收集3-5ml正常血清作为检测抗体时的阴性对照。
(2)用剪刀剪去兔子背部部分兔毛,酒精消毒后进行背部皮下多点注射。
(3)初次免疫:取约1 mg抗原,加入等体积的完全弗氏佐剂充分乳化后注射,背部选取8-10个点,每个点注射约 0.1 ml;
(4) 第二次免疫:间隔14天后进行,抗原量为0.5 mg,加入等体积不完全弗氏佐剂充分乳化后注射,方法同上;
(5)第三、四次免疫:间隔7天后按同样方法进行,第四次免疫后,从耳静脉采血1ml分离血清,用双相琼脂扩散法进行免疫血清的抗体效价检测,效价应达到1:16以上才能放血;
(6)分离血清:第四次免疫后第4天,待抗体效价达到要求后,进行颈动脉采血并分离血清。分装后,-80℃保存。
8、Western blot(免疫印迹)检测抗体效价
参照Sambrook等的方法进行(Sambrook J,Russell D W. Molecular Cloning: A Laboratory Manual, 3rd ed. New York:Cold Spring Harbor Laboratory Press, 2001),1:5000稀释HMGB多克隆兔抗血清检测抗体效价,具体步骤如下:
9、近江牡蛎单层血淋巴细胞培养及处理
参考Lacoste 和Canesi 等的报道(Lacoste A., Cueff A. and Poulet S. A., 2002. P35-sensitive caspases, MAP kinases and Rho modulate beta-adrenergic induction of apoptosis in mollusc immune cells. Journal of cell science 115, 761-8;Canesi L, Lorusso LC, Ciacci C,Betti M, Zampini M, Gallo G. Environmental estrogens can affect the function of mussel hemocytes through rapid modulation of kinasepathways. Gen Comp Endocrinol. 2004 138:58-69.),具体步骤如下:
(1) 取10-15只健康、活性良好的牡蛎,实验室暂养后自来水冲洗外壳;
(2) 用一次性注射器从围心腔中抽取10-15 ml 血淋巴液;
(3) 取适量血淋巴液,800 xg,离心5 分钟,上清液用0.22 µm孔径的滤膜过滤后得到血清;
(4) 向每个培养皿中加入1 ml血淋巴液,15℃孵育30 分钟;
(5) 吸去未贴壁血淋巴细胞,向每个培养皿中加入2 ml血清,置于15℃无菌培养箱中备用。
(6) 培养24小时后,更换血清,分别做以下处理:
a)在血淋巴细胞中加入RLO(1ul/106 cells),部分添加抗HMGB多克隆血清(1:1000),分别培养0小时、1.5小时、4小时、8小时和12小时,提取总RNA,用于real-time RT-PCR分析LITAF 表达变化;
b)在血淋巴细胞中加入LPS(100ng/ml),部分添加抗HMGB多克隆血清(1:1000),分别培养0小时、1.5小时、4小时、8小时和12小时,提取总RNA,用于real-time RT-PCR分析LITAF 表达变化;
c)在血淋巴细胞中加入RLO(1ul/106 cells),部分添加抗HMGB多克隆血清(1:1000),部分添加免疫前血清(1:1000),培养12小时后流式细胞仪检测血淋巴细胞凋亡和坏死;
d)在血淋巴细胞中加入LPS(100ng/ml),部分添加抗HMGB多克隆血清(1:1000),部分添加免疫前血清(1:1000),培养12小时后流式细胞仪检测血淋巴细胞凋亡和坏死。
10、荧光定量PCR
按Takara公司(日本)荧光定量PCR说明书进行,采用28s rDNA序列为内参,每个样品做3个重复样本,反应条件为:95oC 3分钟变性;40个循环扩增(95oC 20秒,55oC 40秒)。反应结束后,使用Bio-Rad iCycle IQ5 荧光定量PCR仪自带软件包进行溶解曲线分析,得到的数据应用相对CT法(2-ΔΔCT)分析,在使用CT方法前,确定目的基因与看家基因扩增效率基本一致,数据分析采用学生t检验方法。
11、流式细胞仪检测细胞凋亡分析
凋亡分析按照Annexin V-FITC细胞凋亡检测试剂盒对细胞进行,Annexin V-FITC的绿色荧光通过FITC通道(FLI)检测,PI红色荧光通过PI(FL2或FL3)检测。
具体的实施例子:
1、近江牡蛎血淋巴细胞cDNA文库的构建和筛选
提取近江牡蛎血淋巴细胞总RNA,根据In-Fusion® SMARTTMcDNA libraryconstruction kit(BD Clotech 公司)构建cDNA文库。利用M13引物对文库阳性质粒进行PCR扩增并测序。筛选出近江牡蛎高迁移率族蛋白(HMGB)。
2、HMGB的原核表达、纯化和鉴定
将阳性重组质粒PET-32 (a)-HMGB转化到表达宿主菌E. coli Rossetta (DE3 )感受态,涂布LBA平板,37℃培养过夜。挑取一个单克隆菌落,转入LBA培养液,37℃振摇培养过夜。取适量菌液,按1:100扩大培养至OD600为 0.4-0.5时,将菌液分为若干等份,不加或分别加入IPTG,使其终浓度在0-1.0 mmol/L,继续培养6小时,离心收集细菌。细菌经超声波裂解后(300W,20分钟,超声2秒钟,间隔3秒钟)离心分离上清液和沉淀,分别上样,进行SDS-PAGE电泳。将阳性表达质粒扩大培养,利用Ni-NTA亲和层析柱对表达产物进行分离纯化,进行12% 的SDS-PAGE电泳鉴定纯化的蛋白质产物。
3、HMGB多克隆抗血清制备及效价检测
用纯化的HMGB蛋白免疫健康新西兰大白兔,取免疫前血清为阴性对照,经过一次初始免疫和三次加强免疫后,颈动脉取血,分离血清,存储于-80℃。
Western-blot 1:5000检测抗体效价确定获得具有较高特异性的HMGB多克隆兔抗血清。
4、HMGB多克隆兔抗血清抑制LPS/RLO刺激引起的炎症相关因子LITAF的表达
近江牡蛎单层血淋巴细胞体外培养24小时后,更换血清后将细胞分为LPS刺激组;LPS + anti-Ca-HMGB刺激组;RLO刺激组;RLO + anti-Ca-HMGB刺激组。荧光定量PCR检测0小时、1.5小时、4小时、8小时和12小时的LITAF mRNA表达量变化。其中LITAF是一个新发现的重要转录因子,被认为可以调控肿瘤坏死因子TNFα的表达,而LPS是革兰氏阴性菌主要致病成分脂多糖。结果表明:刺激后4小时,Ca-HMGB多克隆兔抗血清能显著抑制LPS和RLO刺激引起的LITAF表达量上调(LPS+ anti-Ca-HMGB,4-12小时分别抑制37.71%, 89.99%和86.62%;RLO + anti-Ca-HMGB,4-12小时分别抑制74%,62.57%和56.23%)。Ca-HMGB多克隆抗血清具有有效抑制革兰氏阴性菌和RLO感染引起的炎症反应的作用。因此,Ca-HMGB多克隆抗血清可有效应用于近江牡蛎抗RLO和革兰氏阴性菌等微生物感染的免疫制剂相关产品的开发中。
5、HMGB多克隆兔抗血清抑制LPS/RLO刺激引起的细胞凋亡和坏死
近江牡蛎单层血淋巴细胞体外培养24小时后,更换血清后将细胞分为LPS刺激组;LPS + anti-Ca-HMGB刺激组;LPS + 免疫前血清组;RLO刺激组;RLO + anti-Ca-HMGB刺激组;RLO + 免疫前血清组。12小时后采用Annexin V-FITC/PI 双染流式细胞技术检测血淋巴细胞凋亡和坏死率,结果表明:添加Ca-HMGB兔抗血清较未添加和添加免疫前血清组,LPS/RLO诱导的细胞坏死和晚期细胞凋亡均显著降低,细胞存活率显著增加(较未添加组和添加免疫前血清组,添加Ca-HMGB多克隆抗血清组,RLO刺激晚期细胞凋亡率均降低了约26%,坏死细胞率均降低了约35%。活细胞数分别增加了7%和5%;LPS刺激组,晚期凋亡细胞分别减少了约50%和18%,坏死细胞分别约减少了22%和48%,活细胞数分别增加了约34%和30%)。Ca-HMGB抗血清能够显著抑制革兰氏阴性菌和RLO感染引起的细胞凋亡和细胞坏死,显著增加细胞存活率,可有效应用于近江牡蛎抗革兰氏阴性菌和RLO等微生物感染的免疫制剂相关产品的开发中。
最后,还需要注意的是,以上列举的仅是本发明的若干具体实施例框架。显然,本发明不限于以上实施例,本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (1)
1.HMGB蛋白的多克隆兔抗血清在制备抑制RLO刺激引起的近江牡蛎炎症相关因子LITAF表达的制剂中的应用,其特征在于,所述HMGB蛋白的氨基酸序列如SEQ ID NO.2所示;该蛋白的基因编码序列如SEQ ID NO.1所示。
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accession HM749973.1;Xu T et al;《genbank》;20120406;1-2 * |
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