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
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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。
Staphylococcal enterotoxins (SE) belong to a representative group of superantigens secreted by Gram-positive bacteria Staphylococcus aureus. Currently more than seventeen SEs have been identified, including classical SEs and several newly described serological types. Unlike conventional protein antigens, SEs, when intact, could interact simultaneously with MHC-II molecules and T cell receptors. As the most powerful T cell mitogen, SEs can elicit massive T-cell proliferation and cytokine release both in vivo and in vitro even at a concentrations as low as pg-ng/mL, which suggests their use in immunotherapy of human malignant diseases. From the results of several preclinical studies and early-phase clinical trials, engineered antibody-targeted staphylococcal enterotoxins seemed to be desirable candidates for anti-tumor agents.
In China, staphylococcal enterotoxins C2 (SEC2) is claimed as the main effective component in staphylococcin injection, which is prepared from the filtrate of fermentation broth of Staphylococcus aureus and commonly used as biological response modifier in cancer therapy. The immunomodulatory properties of staphylococcin injection were confirmed in many clinical reports. The injection could induce strong increase in CD4~+ cells, CD4~+/CD8~+ T cell ratio and percentage of NK cells in the treated patients. In addition the side effects correlated with chemotherapy or radiation therapy, such as gastrointestinal tract reaction and bone marrow suppression could be significantly reduced in the injection-treated patients. Short-term efficacy and long-term survival benefit provided by the staphylococcin injection are important for the patients with malignant disease. On the other hand, adverse events were encountered in approximately 20-40% of the patients treated with the injection. The most frequent side effect was mild to moderate fever. Local side effects at the injection site such as pain, swelling and redness also commonly occurred in the injection-treated patients.
In this study, 1-D gel electrophoresis coupled with nano-LC-MS/MS was performed to identify the protein components in the staphylococcal injection products from different manufacturing companies ( Shenyang Xiehe Group Co., Ltd., Zhejiang provincial Yaojiang pharmaceutical Co., Ltd. and Hangzhou Guoguang Pharmaceutical Co., Ltd. The names of the manufacturing companies were replaced by A, B or C in the study on behalf of the companies, and there's no relationship between the order of the company names and A, B or C ) . The results showed more than seventy proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company A; eighteen proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company B and twelve proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company C.
In addition, the gene distribution of staphylococcal enterotoxins in one industrial strain of the injection from one manufacturing company (the name of the company was concealed on behalf of the company) was analyzed by PCR method. The results showed that seven enterotoxin genes (seg, sei, sek, sem, sen, seo and seq) were harbored by the genomic DNA of the industrial strain. Further, the genes of those staphylococcal enterotoxins along with sea and seb were cloned and corresponding recombinant staphylococcal enterotoxins were produced and purified. The in vitro superantigenicities of these recombinant staphylococcal enterotoxins were analyzed and the results showed that those recombinant enterotoxins could induce strong stimulatory effect on proliferation and inhibition effect on tumor cells of murine splenocytes.
In this study, the murine monoclonal antibody and rabbit polyclonal antibody against recombinant staphylococcal enterotoxin I were produced by immunizing Balb/c mouse and New Zealand rabbit with recombinant staphylococcal enterotoxin I, respectively. The ELISA method for the detection and quantification for staphylococcal enterotoxin I was established using the newly-developed murine monoclonal antibody and rabbit polyclonal antibody. The operation conditions of the ELISA method were optimized. The detection linear range of the system was 0.2 - 7.8ng/mL. The intra-day precision and accuracy was 5.1-12.5% and 82.0-112.0%, respectively. The inter-day precision and accuracy was 5.7-13.6% and 88.0-96.5%, respectively. No cross reaction was observed when other rSEs were tested by the system.
On the other hand, SEs are the only superantigens possessing emetic properties. Extensive epidemiological studies have demonstrated that SEA, SEB and SED are the most three important toxins responsible for staphylococcal food poisoning (SFP). However, little is known about the fate of these toxins in the digestive tract after ingestion and how SEs cause the symptoms of SFP. In addition, correlation between their emetic and superantigenic activities remains unclear. The potential transcytosis of the recombinant His-SEC2 was evaluated by using Caco-2 cell model and the established ELISA system for detection of SEC2. The results showed that the recombinant His-SEC2 could be transcytosed by Caco-2 cell monolayer in an intact form, and the amount of transcytosed His-SEC2 was significant higher than that of HRP both from apical side to basolateral side or from basolateral side to apical side in 24 hr.
1. Identification of active components in the staphylococcin injection products
1.1 SDS-PAGE analysis and in-gel digestion
The staphylococcal injection solution from three manufacturing companies was concentrated approximately 40-fold by ultrafiltration and analyzed by SDS-PAGE. After visualization of the gel, the gel lane was divided into several sections based on band intensity and then cut into small pieces. All gel pieces were placed into small eppendorf tubes for in-gel digestion and manual extraction.
1.2 Nano-LC-MS/MS analysis
Samples were reconstituted in loading buffer and sonicated prior to nano-LC-MS/MS analysis. The MS/MS spectra generated by the hybrid triple quadrupole linear ion trap mass spectrometer were submitted to Mascot 1.9 for database searching against the NCBI firmicutes database.
2. Detection of staphylococcal enterotoxins genes in the genomic DNA of industrial strain of staphylococcin injection and production and superantigenicity of several recombinant staphylococcal enterotoxins
2.1 Detection of staphylococcal enterotoxins genes and production of recombinant staphylococcal enterotoxins
The gene distribution of staphylococcal enterotoxins in the genomic DNA of one industrial strain from one manufacturing company was analyzed by PCR method. The expression vectors for recombinant staphylococcal enterotoxins were constructed by cloning the gene fragments amplified by PCR reactions into pGEX-4T-1 vectors. The recombinant GST-tagged staphylococcal enterotoxins were produced by Escherichia coli BL21 and purified by affinity chromatography. The GST-tag was then released by thrombin digestion from the fusion protein and the recombinant staphylococcal enterotoxin was purified by anion ion-exchange chromatography.
2.2 The superantigenicities of recombinant staphylococcal enterotoxins
Staphylococcal enterotoxin-induced proliferation of murine lymphocytes was measured by MTT assay. The degree of proliferation rates of the lymphocytes induced by the recombinant proteins were presented as stimulation index. Staphylococcal enterotoxin-induced cellular cytotoxicity of murine lymphocytes was also assessed by MTT method. K562-ADM cells were served as target cells and murine splenic lymphocytes were used as effector cells. In addition, rSEI-mediated celluar cytotoxicity of murine lymphocytes was also measured by the RT-CES system. A549 cells were served as target cells and murine splenic lymphocytes were used as effector cells.
3. Establishment of immunoassay for SEI
3.1 Production of rabbit polyclonal antibodies and murine monoclonal antibodies against SEI
New Zealand rabbits were immunized subcutaneously on the back with purified recombinant SEI over a period of approximate 8 weeks and the anti-sera was collected. Balb/c mice were injected subcutaneously on the back with purified recombinant SEI and the animals were boosted with recombinant SEI three days before cell fusion. The splenic lymphocytes obtained from the immunized mice were fused with murine SP2/0 myeloma cells using PEG 4000. The hybridoma cells capable of producing antibodies against recombinant SEI were screened by indirect ELISA and cloned by limiting dilution. Positive hybridoma cells were further cultured injected intraperitoneally to Balb/c mice pretreated with paraffin oil. Ascite fluid was collected from the mice 10-14 days later and the monoclonal antibodies against recombinant SEI were purified with caprylic acid - ammonium sulfate method.
3.2 Establishment of ELISA system
Two procedures were investigated: (1) use of plate coated with rabbit polyclonal antibodies and murine monoclonal antibodies as secondary antibody and (2) use of plate coated with murine monoclonal antibodies and rabbit polyclonal antibodies as secondary antibody. The ELISA assay was optimized by choosing various dilutions of coating antibody, secondary antibody and other operation conditions.
4. Transcytosis of His-tagged SEC2
The ability of Caco-2 cells to transcytose recombinant His-tagged SEC2 was tested using confluent monolayers grown on Transwell filters. His-SEC2 was added to either the apical or basal chamber, horseradish peroxidase was added as an internal control for nonspecific transcytosis. After incubation, apical or basal media were collected depending on whether His-SEC2 was added to the basal or apical media. The established ABS-ELISA system was used to determine amounts of His-SEC2 present in sample solution and the protein was also detected using western-blotting method.
Conclusion:
In this study, LC-MS coupled with SDS-PAGE was performed to identify protein components in three staphylococcin injection products. The results showed that many types of proteins from S.aureus and other Gram-positive bacteria were confidently identified. Investigation of the relationship between those identified proteins and the clinical efficacy or toxicities of the staphylococcin injection is required in future. In addition, the manufacturing process as well as the standard quality control of injection preparation should be improved to avoid bacteria contamination, reduce impurities and increase clinical efficacies. In this study, the gene distribution of staphylococcal enterotoxins of one industrial strain from one manufacturing company was analyzed, and the results demonstrated that seven se genes were harbored by the strain, indicating that one or a plurality of them may be present in the related staphylococcin injection products. Several recombinant staphylococcal enterotoxins, including those seven s
taphylococcal enterotoxins, were produced using gene engineering approaches. Results showed that the superantigenicity of those recombinant staphylococcal enterotoxins were close to that of native staphylococcal enterotoxin C2. Thus, recombinant staphylococcal enterotoxins provide a new approach for the production of next-generation staphylococcin injection. In this study, murine monoclonal and rabbit polyclonal antibodies against SEI were produced. Using the newly-developed antibodies, sandwich ELISA system for the detection of SEI at concentration of ng/mL was established, which could be used in the clinical diagnosis of SEI-related diseases and development of SEI-related drugs in future.
The fate and mechanism of enterotoxic activities of SEs in human gastrointestinal tract remains to be determined. In this study, the transcytosis of recombinant His-SEC2 was preliminarily investigated. The results indicated that the recombinant His-SEC2 could specifically cross the Caco-2 cell monolayer in an intact form, indicating that SEC2 and other serological types of staphylococcal enterotoxins could cross the intestinal epithelial cells and then cause local or systemic symptoms. In addition, the results also have implications for the development of oral drugs in which staphylococcal enterotoxins are used as the main effective components.
In conclusion, in this study, the protein components in the staphylococcin injection products and the superantigenic activities of recombinant staphylococcal enterotoxins were preliminarily investigated. In addition, the immunoassay for SEI was also established and the transcytosis of recombinant His-SEC2 was preliminarily investigated. The study would shed light into the future research on the staphylococcal enterotoxins
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。
Staphylococcal enterotoxins (SE) belong to a representative group of superantigens secreted by Gram-positive bacteria Staphylococcus aureus. Currently more than seventeen SEs have been identified, including classical SEs and several newly described serological types. Unlike conventional protein antigens, SEs, when intact, could interact simultaneously with MHC-II molecules and T cell receptors. As the most powerful T cell mitogen, SEs can elicit massive T-cell proliferation and cytokine release both in vivo and in vitro even at a concentrations as low as pg-ng/mL, which suggests their use in immunotherapy of human malignant diseases. From the results of several preclinical studies and early-phase clinical trials, engineered antibody-targeted staphylococcal enterotoxins seemed to be desirable candidates for anti-tumor agents.
In China, staphylococcal enterotoxins C2 (SEC2) is claimed as the main effective component in staphylococcin injection, which is prepared from the filtrate of fermentation broth of Staphylococcus aureus and commonly used as biological response modifier in cancer therapy. The immunomodulatory properties of staphylococcin injection were confirmed in many clinical reports. The injection could induce strong increase in CD4~+ cells, CD4~+/CD8~+ T cell ratio and percentage of NK cells in the treated patients. In addition the side effects correlated with chemotherapy or radiation therapy, such as gastrointestinal tract reaction and bone marrow suppression could be significantly reduced in the injection-treated patients. Short-term efficacy and long-term survival benefit provided by the staphylococcin injection are important for the patients with malignant disease. On the other hand, adverse events were encountered in approximately 20-40% of the patients treated with the injection. The most frequent side effect was mild to moderate fever. Local side effects at the injection site such as pain, swelling and redness also commonly occurred in the injection-treated patients.
In this study, 1-D gel electrophoresis coupled with nano-LC-MS/MS was performed to identify the protein components in the staphylococcal injection products from different manufacturing companies ( Shenyang Xiehe Group Co., Ltd., Zhejiang provincial Yaojiang pharmaceutical Co., Ltd. and Hangzhou Guoguang Pharmaceutical Co., Ltd. The names of the manufacturing companies were replaced by A, B or C in the study on behalf of the companies, and there's no relationship between the order of the company names and A, B or C ) . The results showed more than seventy proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company A; eighteen proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company B and twelve proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company C.
In addition, the gene distribution of staphylococcal enterotoxins in one industrial strain of the injection from one manufacturing company (the name of the company was concealed on behalf of the company) was analyzed by PCR method. The results showed that seven enterotoxin genes (seg, sei, sek, sem, sen, seo and seq) were harbored by the genomic DNA of the industrial strain. Further, the genes of those staphylococcal enterotoxins along with sea and seb were cloned and corresponding recombinant staphylococcal enterotoxins were produced and purified. The in vitro superantigenicities of these recombinant staphylococcal enterotoxins were analyzed and the results showed that those recombinant enterotoxins could induce strong stimulatory effect on proliferation and inhibition effect on tumor cells of murine splenocytes.
In this study, the murine monoclonal antibody and rabbit polyclonal antibody against recombinant staphylococcal enterotoxin I were produced by immunizing Balb/c mouse and New Zealand rabbit with recombinant staphylococcal enterotoxin I, respectively. The ELISA method for the detection and quantification for staphylococcal enterotoxin I was established using the newly-developed murine monoclonal antibody and rabbit polyclonal antibody. The operation conditions of the ELISA method were optimized. The detection linear range of the system was 0.2 - 7.8ng/mL. The intra-day precision and accuracy was 5.1-12.5% and 82.0-112.0%, respectively. The inter-day precision and accuracy was 5.7-13.6% and 88.0-96.5%, respectively. No cross reaction was observed when other rSEs were tested by the system.
On the other hand, SEs are the only superantigens possessing emetic properties. Extensive epidemiological studies have demonstrated that SEA, SEB and SED are the most three important toxins responsible for staphylococcal food poisoning (SFP). However, little is known about the fate of these toxins in the digestive tract after ingestion and how SEs cause the symptoms of SFP. In addition, correlation between their emetic and superantigenic activities remains unclear. The potential transcytosis of the recombinant His-SEC2 was evaluated by using Caco-2 cell model and the established ELISA system for detection of SEC2. The results showed that the recombinant His-SEC2 could be transcytosed by Caco-2 cell monolayer in an intact form, and the amount of transcytosed His-SEC2 was significant higher than that of HRP both from apical side to basolateral side or from basolateral side to apical side in 24 hr.
1. Identification of active components in the staphylococcin injection products
1.1 SDS-PAGE analysis and in-gel digestion
The staphylococcal injection solution from three manufacturing companies was concentrated approximately 40-fold by ultrafiltration and analyzed by SDS-PAGE. After visualization of the gel, the gel lane was divided into several sections based on band intensity and then cut into small pieces. All gel pieces were placed into small eppendorf tubes for in-gel digestion and manual extraction.
1.2 Nano-LC-MS/MS analysis
Samples were reconstituted in loading buffer and sonicated prior to nano-LC-MS/MS analysis. The MS/MS spectra generated by the hybrid triple quadrupole linear ion trap mass spectrometer were submitted to Mascot 1.9 for database searching against the NCBI firmicutes database.
2. Detection of staphylococcal enterotoxins genes in the genomic DNA of industrial strain of staphylococcin injection and production and superantigenicity of several recombinant staphylococcal enterotoxins
2.1 Detection of staphylococcal enterotoxins genes and production of recombinant staphylococcal enterotoxins
The gene distribution of staphylococcal enterotoxins in the genomic DNA of one industrial strain from one manufacturing company was analyzed by PCR method. The expression vectors for recombinant staphylococcal enterotoxins were constructed by cloning the gene fragments amplified by PCR reactions into pGEX-4T-1 vectors. The recombinant GST-tagged staphylococcal enterotoxins were produced by Escherichia coli BL21 and purified by affinity chromatography. The GST-tag was then released by thrombin digestion from the fusion protein and the recombinant staphylococcal enterotoxin was purified by anion ion-exchange chromatography.
2.2 The superantigenicities of recombinant staphylococcal enterotoxins
Staphylococcal enterotoxin-induced proliferation of murine lymphocytes was measured by MTT assay. The degree of proliferation rates of the lymphocytes induced by the recombinant proteins were presented as stimulation index. Staphylococcal enterotoxin-induced cellular cytotoxicity of murine lymphocytes was also assessed by MTT method. K562-ADM cells were served as target cells and murine splenic lymphocytes were used as effector cells. In addition, rSEI-mediated celluar cytotoxicity of murine lymphocytes was also measured by the RT-CES system. A549 cells were served as target cells and murine splenic lymphocytes were used as effector cells.
3. Establishment of immunoassay for SEI
3.1 Production of rabbit polyclonal antibodies and murine monoclonal antibodies against SEI
New Zealand rabbits were immunized subcutaneously on the back with purified recombinant SEI over a period of approximate 8 weeks and the anti-sera was collected. Balb/c mice were injected subcutaneously on the back with purified recombinant SEI and the animals were boosted with recombinant SEI three days before cell fusion. The splenic lymphocytes obtained from the immunized mice were fused with murine SP2/0 myeloma cells using PEG 4000. The hybridoma cells capable of producing antibodies against recombinant SEI were screened by indirect ELISA and cloned by limiting dilution. Positive hybridoma cells were further cultured injected intraperitoneally to Balb/c mice pretreated with paraffin oil. Ascite fluid was collected from the mice 10-14 days later and the monoclonal antibodies against recombinant SEI were purified with caprylic acid - ammonium sulfate method.
3.2 Establishment of ELISA system
Two procedures were investigated: (1) use of plate coated with rabbit polyclonal antibodies and murine monoclonal antibodies as secondary antibody and (2) use of plate coated with murine monoclonal antibodies and rabbit polyclonal antibodies as secondary antibody. The ELISA assay was optimized by choosing various dilutions of coating antibody, secondary antibody and other operation conditions.
4. Transcytosis of His-tagged SEC2
The ability of Caco-2 cells to transcytose recombinant His-tagged SEC2 was tested using confluent monolayers grown on Transwell filters. His-SEC2 was added to either the apical or basal chamber, horseradish peroxidase was added as an internal control for nonspecific transcytosis. After incubation, apical or basal media were collected depending on whether His-SEC2 was added to the basal or apical media. The established ABS-ELISA system was used to determine amounts of His-SEC2 present in sample solution and the protein was also detected using western-blotting method.
Conclusion:
In this study, LC-MS coupled with SDS-PAGE was performed to identify protein components in three staphylococcin injection products. The results showed that many types of proteins from S.aureus and other Gram-positive bacteria were confidently identified. Investigation of the relationship between those identified proteins and the clinical efficacy or toxicities of the staphylococcin injection is required in future. In addition, the manufacturing process as well as the standard quality control of injection preparation should be improved to avoid bacteria contamination, reduce impurities and increase clinical efficacies. In this study, the gene distribution of staphylococcal enterotoxins of one industrial strain from one manufacturing company was analyzed, and the results demonstrated that seven se genes were harbored by the strain, indicating that one or a plurality of them may be present in the related staphylococcin injection products. Several recombinant staphylococcal enterotoxins, including those seven s
taphylococcal enterotoxins, were produced using gene engineering approaches. Results showed that the superantigenicity of those recombinant staphylococcal enterotoxins were close to that of native staphylococcal enterotoxin C2. Thus, recombinant staphylococcal enterotoxins provide a new approach for the production of next-generation staphylococcin injection. In this study, murine monoclonal and rabbit polyclonal antibodies against SEI were produced. Using the newly-developed antibodies, sandwich ELISA system for the detection of SEI at concentration of ng/mL was established, which could be used in the clinical diagnosis of SEI-related diseases and development of SEI-related drugs in future.
The fate and mechanism of enterotoxic activities of SEs in human gastrointestinal tract remains to be determined. In this study, the transcytosis of recombinant His-SEC2 was preliminarily investigated. The results indicated that the recombinant His-SEC2 could specifically cross the Caco-2 cell monolayer in an intact form, indicating that SEC2 and other serological types of staphylococcal enterotoxins could cross the intestinal epithelial cells and then cause local or systemic symptoms. In addition, the results also have implications for the development of oral drugs in which staphylococcal enterotoxins are used as the main effective components.
In conclusion, in this study, the protein components in the staphylococcin injection products and the superantigenic activities of recombinant staphylococcal enterotoxins were preliminarily investigated. In addition, the immunoassay for SEI was also established and the transcytosis of recombinant His-SEC2 was preliminarily investigated. The study would shed light into the future research on the staphylococcal enterotoxins
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