金葡液有效成分鉴定及重组金葡菌肠毒素生物学活性研究

发布时间：2018-06-01 06:15

本文选题：金葡菌肠毒素 + 金葡素注射液　；参考：《浙江大学》2009年博士论文

【摘要】： 金葡菌肠毒素由金黄色葡萄球菌分泌产生,是一种典型的超抗原。目前已有超过17种金葡菌肠毒素获得了鉴定,其中包括经典的肠毒素以及新近发现的多种金葡菌肠毒素。和普通抗原分子不同的是,金葡菌肠毒素分子可以以完整形式和MHC-Ⅱ分子和T细胞受体分子结合。作为目前已知的最强大的T细胞超抗原,纳克甚至皮克数量级浓度的金葡菌肠毒素分子在体内和体外均可以导致大量T细胞的增殖并促使其分泌产生多种细胞因子。金葡菌肠毒素的上述特征提示其可以作为一种免疫调节制剂用于人肿瘤以及相关恶性疾病的免疫治疗。目前,相关临床前研究以及临床研究均显示了利用基因工程制备的靶向性的金葡菌肠毒素分子极有望成为新型抗肿瘤制剂。在国内,由金葡菌发酵滤液制备获得的金葡素注射液作为生物反应调节剂已广泛地应用于肿瘤疾病的治疗,而相关生产企业声称其有效成分是金葡菌肠毒素C2(SEC2)。临床报道显示金葡素注射液具有明显的免疫调节作用,患者在进行放化疗过程中联合使用金葡素注射液可使得CD4~+T细胞数量、CD4~+/CD8~+T细胞比例以及NK细胞数量获得显著提高。同时,因放化疗引起的相关毒副作用如胃肠道反应、骨髓抑制等可获得明显的缓解。金葡素注射液的使用对肿瘤患者短期疗效的提高以及生存时间的延长均具有积极的意义。但另一方面,有约20-40%的患者在使用金葡素注射液的过程中会产生以轻度以及中度发热为主的不同程度的副反应,其中也包括在注射部位产生的红肿、疼痛等等。本研究利用了纳升液质联用技术结合一向凝胶电泳技术对不同生产企业(沈阳协和集团有限公司,浙江省耀江药业有限公司,杭州国光药业有限公司,注:因涉及生产企业相关利益,本论文中提及的三家企业分别用企业A、企业B以及企业C代替,但文中出现的企业名称在排列顺序上与A、B、C无对应关系)生产的金葡素注射液产品中的蛋白质成分进行了鉴定。结果显示,由企业A生产的金葡素注射液中,有超过70种金葡菌属以及来源于其他种类革兰氏阳性菌的蛋白质获得了鉴定;由企业B生产的金葡素注射液中有18种金葡菌属以及来源于其他种类革兰氏阳性菌的蛋白质获得了鉴定;由企业C生产的金葡素注射液中有12种金葡菌属以及来源于其他种类革兰氏阳性菌的蛋白质获得了鉴定。本研究同时利用了PCR方法对某企业(注:因涉及生产企业相关利益,企业名称隐去)提供的一株金葡素注射液生产菌株基因组中含有的金葡菌肠毒素基因分布进行了检测。结果显示在该生产菌株基因组中携带有七种肠毒素基因(seg,sei,sek,sem,sen,seo和seq)。我们分别克隆获得了包括上述各种肠毒素基因以及sea、seb在内的多种金葡菌肠毒素基因,并进行了相关多种重组金葡菌肠毒素的表达及纯化。体外研究表明上述获得的重组金葡菌肠毒素具有典型的超抗原性质,可以显著地刺激小鼠脾淋巴细胞的增殖并增强其对肿瘤细胞生长抑制的作用。本研究将重组金葡菌肠毒素Ⅰ免疫Balb/c小鼠以及新西兰白兔,制备获得了抗SEI小鼠单克隆抗体以及兔多克隆抗体。进而利用上述抗体建立了用于检测SEI的ELISA方法并对该方法进行了优化,结果显示所建立的双抗体夹心ELISA法的线性检测范围为0.2-7.8ng/mL,其日间精密度以及准确率分别为5.1-12.5%和82.0-112.0%,其日内精密度以及准确率分别为5.7-13.6%和88.0-96.5%,与其他种类重组肠毒素不存在交叉反应。另一方面,金葡菌肠毒素是唯一一种具有致呕吐活性的超抗原。目前有相当数量的流行病学研究结果显示在金葡菌引起的食物中毒中,SEA、SEB以及SED是最主要的几种致病因子。但是,目前对于金葡菌肠毒素在被摄入至胃肠道后的命运以及金葡菌肠毒素如何引起食物中毒症的机理状仍不甚清楚。同时,金葡菌肠毒素致呕吐活性和超抗原活性之间的关系仍然未知。本研究利用Caco-2细胞模型对重组His-SEC2分子的可能存在的跨膜作用进行了检测。结果显示重组His-SEC2分子可以以完整分子形式透过Caco-2单层细胞膜,且在24小时以内,无论从A侧到B侧或从B侧到A侧透过Caco-2单层细胞膜的His-SEC2的量均明显高于阴性对照HRP的量。 1.金葡素注射液有效成分鉴定 1.1电泳分析以及胶内酶切将来源于三个不同企业的金葡素注射液用超滤浓缩约40倍后进行电泳分析。将凝胶染色后根据条带浓度分割并切成小片。而后将胶碎片置于eppendorf管中进行胶内酶切和肽段提取。 1.2纳升液质联用分析将样品用上样缓冲液溶解、超声,而后进行纳升液质联用分析。利用三重四级杆线性离子阱质谱获得的质谱数据用Mascot 1.9软件针对革兰氏阳性菌数据库进行搜索。 2.金葡素注射液生产菌株中金葡菌肠毒素基因检测和多种重组金葡菌肠毒素的制备及其超抗原活性检测 2.1金葡菌肠毒素基因检测以及重组金葡菌肠毒素的制备利用PCR方法对某企业提供的一株金葡素注射液生产菌株基因组中的肠毒素基因分布进行检测。而后将由PCR获得的多种编码金葡菌肠毒素的基因克隆至pGEX-4T-1质粒构建重组金葡菌肠毒素表达质粒。而后利用大肠杆菌BL21表达带有GST标签的重组金葡菌肠毒素。利用亲和层析纯化带有GST标签的重组金葡菌肠毒素,用凝血酶将GST标签去除后利用阴离子层析对重组金葡菌肠毒素进行纯化。 2.2重组金葡菌肠毒素的超抗原活性检测利用MTT法对金葡菌肠毒素刺激小鼠淋巴细胞增殖的作用进行检测。通过增殖指数计算重组金葡菌肠毒素对小鼠淋巴细胞的刺激强度。同样利用MTT法,以K562-ADM细胞作为靶细胞,对受重组金葡菌肠毒素刺激的小鼠淋巴细胞抑制肿瘤细胞生长的作用进行检测。另外,以A549作为靶细胞,利用实时细胞电子分析系统对受重组SEI刺激的小鼠淋巴细胞抑制肿瘤细胞生长的作用进行检测。 3.SEI免疫检测方法的建立 3.1兔抗SEI单克隆抗体和鼠抗SEI单克隆抗体的制备新西兰大白兔背部皮下注射纯化的重组SEI进行多次免疫,约八周后收集抗SEI血清。Balb/c小鼠背部皮下注射纯化的重组SEI进行免疫,在细胞融合前三天进行冲击免疫。而后利用聚乙二醇4000将免疫小鼠的脾淋巴细胞与小鼠SP2/0骨髓瘤细胞进行融合。用间接ELISA和有限稀释法对可产生抗SEI抗体的杂交瘤细胞进行筛选和克隆扩增。将获得的阳性杂交瘤细胞株腹腔注射经石蜡油致敏的Balb/c小鼠,10-14天后收集腹水并用辛酸-硫酸铵法纯化抗SEI单克隆抗体。 3.2 ELISA检测方法的建立对两种ELISA方法进行了考察:(1)兔抗SEI多克隆抗体作为包被抗体,鼠抗SEI单克隆抗体作为第二抗体;(2)鼠抗SEI单克隆抗体作为包被抗体,兔抗SEI多克隆抗体作为第二抗体。同时,对包被抗体、第二抗体浓度以及其他工作条件进行了优化。 4.His-SEC2的跨膜作用以辣根过氧化物酶作为非特异性跨膜的阴性对照,利用生长在Transwell~(TM)板上的单层Caco-2细胞对重组His-SEC2的跨膜作用进行检测。His-SEC2加入至Transwell~(TM)板的A侧或B侧,经过孵育后,根据His-SEC2加入的位置收集B侧或者A侧的培养基。而后利用实验室先前建立的ABS-ELISA方法对样品溶液中His-SEC2的含量进行检测,同时利用Western-blotting方法对其中的His-SEC2进行检测。结论本研究利用液质联用偶联SDS凝胶电泳对三种金葡素注射液中的蛋白质成分进行了鉴定。结果显示,从三种针剂中有多种金葡菌属以及其他革兰氏阳性菌属蛋白质获得了鉴定。在今后的工作中,需要对鉴定获得的蛋白质与金葡素注射液的临床疗效或毒副作用之间的关系进行进一步研究。同时,质谱提示现有的金葡素注射液制备生产工艺以及质量控制标准需要进行进一步的改进和提高以规范生产工艺、避免菌株污染、减少共存杂质、提高临床疗效。本研究对其中一家企业提供的生产菌株中肠毒素基因分布进行检测,结果显示其中含有七种肠毒素基因,因此提示上述七种金葡菌肠毒素的一种或者几种可能存在于相关金葡素注射液产品中。本研究利用基因工程的方法制备获得了包括上述七种金葡菌肠毒素在内的多种重组金葡菌肠毒素,结果显示获得的重组金葡菌肠毒素在体外具有与天然SEC2相近的超抗原活性,因此,利用基因工程方法获得的重组金葡菌肠毒素为制备新一代金葡素注射液提供了一种崭新的选择。本研究还制备获得了抗SEI小鼠单克隆抗体和兔多克隆抗体,并在此基础上建立了ELISA夹心法用于微量SEI的检测,可应用于今后相关疾病的临床诊断和相关产品的生产研发。目前对于金葡菌肠毒素在人胃肠道内的命运以及产生肠道活性的作用机制仍然有待进一步研究。本研究对重组His-SEC2的跨膜作用进行了初步研究,结果显示完整的重组His-SEC2分子可以以一种特异的方式透过Caco-2单层细胞膜,该结果提示SEC2以及其他种类的金葡菌肠毒素可能可以透过小肠上皮细胞从而引起局部或全身性症状的产生。同时,上述结果也提示了今后研制以金葡菌肠毒素为有效成分的口服制剂的可能性。综上所述,本研究对现有的金葡素注射液产品中的蛋白类成分以及重组金葡菌肠毒素的超抗原活性进行了初步的探索和研究,建立了针对SEI的免疫学检测方法,对重组His-SEC2的跨膜作用进行了初步的研究,为今后金葡菌肠毒素研究工作的开展建立了一定的基础。
[Abstract]:Staphylococcus aureus enterotoxins are produced by staphylococcus aureus secretion , and is a typical superantigen . More than 17 kinds of staphylococcus aureus enterotoxins have been identified , including classical enterotoxins and recently discovered various kinds of Staphylococcus aureus enterotoxins . Unlike common antigen molecules , the staphylococcus aureus enterotoxins can be combined with MHC - II molecules and T cell receptor molecules .

The clinical report shows that the injection of gold - glucose injection has obvious immunoregulation effect , and the use of gold - glucoside injection can make the number of CD4 ~ + T cells , CD4 ~ + / CD8 ~ + T - cell ratio and NK cell count .

The results showed that there were more than 70 kinds of Staphylococcus aureus and proteins derived from other kinds of Gram - positive bacteria .

This study also used the PCR method to detect the gene distribution of Staphylococcus aureus Enterotoxin contained in the genome of a strain of Staphylococcus aureus , which was supplied by a certain enterprise ( Note : Due to the relevant interests of the production enterprise and the name of the enterprise ) . The results showed that the genome of the production strain was carried with seven kinds of enterotoxin genes . In vitro studies have shown that the above - mentioned recombinant Staphylococcus aureus Enterotoxin has typical superantigen properties , which can significantly stimulate the proliferation of mouse spleen lymphocytes and enhance its effect on tumor cell growth inhibition .

The anti - SEI mouse monoclonal antibody and rabbit polyclonal antibody were prepared by immunizing Balb / c mice and New Zealand white rabbits . The ELISA method for detecting SEI was established and optimized . The results showed that the linear detection range of the established double - antibody sandwich ELISA was 0.2 - 7.8 ng / mL . The precision and accuracy of the antibody were 5.7 - 13.6 % and 88.0 - 96.5 % , respectively , and no cross - reaction with other kinds of recombinant enterotoxins .

The results show that the recombinant His - SEC2 molecule can penetrate Caco - 2 monolayer cell membrane in the form of intact molecule , and the amount of His - SEC2 through Caco - 2 monolayer cell membrane from side A to B side or from side B to side A is significantly higher than that of negative control HRP .

1 . Identification of the effective components of Jinglucoside injection

1.1 electrophoretic analysis and in - gel enzyme digestion

The gold - glucose injection from three different enterprises was subjected to electrophoretic analysis by ultrafiltration concentration of about 40 times . The gel was stained and cut into pieces according to the band concentration . Then , the gel fragments were placed in an epp tube to perform an in - gel enzyme digestion and peptide segment extraction .

1.2 Natto - liquid mass spectrometry

The samples were dissolved in a sample buffer , followed by nanoliter liquid chromatography . The mass spectrum data obtained by using the triple quadrupole linear ion trap mass spectrometry was searched for Gram - positive bacteria databases using the Mascot 1.9 software .

Detection of Staphylococcus aureus Enterotoxin Gene and Preparation of Various Recombinant Staphylococcus aureus Enterotoxin and Detection of Its Superantigen Activity

2.1 Detection of Enterotoxin Gene of Staphylococcus aureus and Preparation of Recombinant Staphylococcus aureus Enterotoxin

A recombinant S . aureus Enterotoxin with GST tag was constructed by affinity chromatography , and the recombinant S . aureus was purified by thrombin .

2.2 Detection of Superantigen Activity of Recombinant Staphylococcus aureus Enterotoxin

In addition , A549 cells were used as target cells to detect the growth of tumor cells in lymphocytes stimulated by recombinant Staphylococcus aureus Enterotoxin . In addition , A549 cells were used as target cells to detect the growth of tumor cells by using real - time cellular electronic analysis system .

3 . Establishment of SEI Immunodetection Method

3.1 Preparation of Rabbit Anti - SEI Monoclonal Antibody and Mouse Anti - SEI Monoclonal Antibody

Balb / c mice were injected into Balb / c mice sensitized by paraffin oil for 10 - 14 days , and the anti - SEI monoclonal antibody was purified by means of an indirect ELISA and a limited dilution method .

3.2 Establishment of ELISA Test Method

Two ELISA methods were investigated : ( 1 ) the rabbit anti - SEI polyclonal antibody was used as the coating antibody , the mouse anti - SEI monoclonal antibody was used as the second antibody ; ( 2 ) the mouse anti - SEI monoclonal antibody was used as the coating antibody and the rabbit anti - SEI polyclonal antibody was used as the second antibody . Meanwhile , the antibody , the second antibody concentration and the other working conditions were optimized .

4 . Transmembrane Effect of His - SEC2

The cross - membrane interaction of His - SEC2 was detected by using a single layer of Caco - 2 cells grown on the Transwell ~ ( TM ) plate . His - SEC2 was added to the A - side or B - side of Transwell ~ ( TM ) plate . After incubation , the contents of His - SEC2 in the sample solution were detected according to the position of His - SEC2 , and the His - SEC2 was detected by Western - blotting .

Conclusion

The results showed that there were seven kinds of recombinant strains of Staphylococcus aureus and other Gram - positive bacteria . The results showed that there were seven kinds of recombinant strains of Staphylococcus aureus and other Gram - positive bacteria .

The results show that the complete recombinant His - SEC2 molecule can penetrate the Caco - 2 monolayer cell membrane in a specific way , suggesting that the intact recombinant His - SEC2 molecule can penetrate the small intestine epithelial cells to induce local or systemic symptoms .

In conclusion , this study has carried out a preliminary exploration and research on the protein components and the superantigen activity of recombinant Staphylococcus aureus Enterotoxin , and established an immunological detection method for SEI . The cross - membrane effect of recombinant His - SEC2 was studied .
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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