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金黄色葡萄球菌凝固酶coa基因的克隆、表达及其生物活性分析
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作者 郭海勇 赵雪 +6 位作者 朱小雨 赵研彤 林依丹 施爽 李铄 计银铎 宋勤叶 《中国人兽共患病学报》 CAS CSCD 北大核心 2020年第9期696-702,共7页
目的体外表达金黄色葡萄球菌(Staphylococcus aureus,S.aureus)凝固酶Coa,评价其生物学活性。方法根据GenBank中金黄色葡萄球菌凝固酶coa基因序列设计特异性引物,采用PCR方法从S.aureus USA300 CA-MRSA 923株基因组中扩增coa基因片段,... 目的体外表达金黄色葡萄球菌(Staphylococcus aureus,S.aureus)凝固酶Coa,评价其生物学活性。方法根据GenBank中金黄色葡萄球菌凝固酶coa基因序列设计特异性引物,采用PCR方法从S.aureus USA300 CA-MRSA 923株基因组中扩增coa基因片段,将其克隆至pET-24b(+)表达载体,构建重组表达质粒pET-24b-coa;将经双酶切及测序鉴定正确的重组质粒转化感受态细胞E.coli BL21(DE3)中进行IPTG诱导表达;通过SDS-PAGE和Western blot对表达的重组Coa(rCoa)进行鉴定,测定rCoa对人血液和兔血浆的凝固活性;最后用纯化的重组蛋白rCoa免疫BALB/c小鼠,经间接ELISA和Western blot分析其免疫原性。结果构建的重组质粒pET-24b-coa在E.coli BL21(DE3)中以包涵体形式表达rCoa,分子量约74.8 ku,与预期蛋白大小相符;rCoa在体外具有凝固人全血和兔血浆活性,能刺激小鼠产生高效价的抗Coa特异性抗体。结论成功地获得了具有良好凝固酶活性和抗原性的金黄色葡萄球菌凝固酶重组蛋白rCoa,为进一步深入研究S.aureus凝固酶Coa的功能及以Coa为靶点的抗金黄色葡萄球菌抑制剂和疫苗的研制奠定基础。 展开更多
关键词 金黄色葡萄球菌 凝固酶 生物活性 克隆表达
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猪圆环病毒3型Cap蛋白的原核表达及其免疫原性分析
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作者 腾召剑 林依丹 +4 位作者 周双海 李潭清 张义明 宋勤叶 郑士学 《中国兽医杂志》 CAS 北大核心 2021年第5期47-51,I0003,共6页
为了表达猪圆环病毒3型(PCV3)衣壳(Cap)蛋白,分析其免疫原性,本试验构建了不包含Cap蛋白核定位序列的原核表达质粒pET-28a-PCV3-Cap,应用大肠杆菌Escherichia coli表达系统诱导表达了重组Cap蛋白;通过SDS-PAGE、Western Blot及液相色谱... 为了表达猪圆环病毒3型(PCV3)衣壳(Cap)蛋白,分析其免疫原性,本试验构建了不包含Cap蛋白核定位序列的原核表达质粒pET-28a-PCV3-Cap,应用大肠杆菌Escherichia coli表达系统诱导表达了重组Cap蛋白;通过SDS-PAGE、Western Blot及液相色谱-质谱联用(LC-MS/MS)技术对表达蛋白进行了鉴定;分析预测了蛋白的抗原表位,并通过动物试验分析蛋白的免疫原性。结果显示,在31℃下经0.5 mmol/L的IPTG诱导4 h,蛋白(25.4 kDa)表达量最高,其主要以包涵体形式表达;表达蛋白与PCV3参考毒株Cap蛋白氨基酸序列的一致性为88.4%(152/172 aa),并且预测的抗原表位与参考毒株的相符;重组蛋白能够刺激小鼠产生特异性抗体,平均抗体水平≥1∶12800。上述结果表明,表达的蛋白具有良好的免疫原性。该试验为深入解析PCV3-Cap蛋白抗原表位的免疫活性以及相关亚单位疫苗和特异性抗体检测技术的研究提供了科学依据。 展开更多
关键词 PCV3 CAP蛋白 原核表达 抗原表位 免疫原性
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Perioperative and long-term outcome of thymectomy for myasthenia gravis: comparison of surgical approaches and prognostic analysis 被引量:8
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作者 LIU Cheng-wu LUO Meng +9 位作者 MEI Jian-dong ZHU Yun-ke PU Qiang MA lin CHE Guo-wei lin yi-dan WU Zhu WANG Yun KOU Ying-li LIU Lun-xu 《Chinese Medical Journal》 SCIE CAS CSCD 2013年第1期34-40,共7页
Background Thymectomy is an established treatment for myasthenia gravis (MG), and video-assisted thoracoscopic surgery (VATS) thymectomy has become an acceptable surgical procedure. This study aimed to compare the... Background Thymectomy is an established treatment for myasthenia gravis (MG), and video-assisted thoracoscopic surgery (VATS) thymectomy has become an acceptable surgical procedure. This study aimed to compare the results of VATS thymectomy and open thymectomy and to identify the prognostic factors after thymectomy. Methods The clinical data of 187 consecutive thymectomies performed between July 2000 and December 2009 were retrospectively reviewed; 75 open thymectomies and 112 VATS thymectomies. Clinical efficacy and variables influencing outcome were assessed by Kaplan-Meier survival curves and Cox proportional hazards regression analysis. Results The operative blood loss in the VATS group was significantly less than that in the open group ((62.14+55.43) ml vs. (137.87+165.25) ml, P 〈0.05). The postoperative crisis rate increased with the severity of preoperative MG and the prescription dose of anticholinesterase. Complete follow-up information of patients more than 12 months after the thymectomy was obtained on 151 cases, 89 cases from the VATS group and 62 cases from the open group, with a mean follow-up period of 59.3 months, range from 12 to 117 months. Complete stable remission (CSR) was the end point for evaluation of the treatment results. The overall five-year CSR rate was 57.5%. Two good prognostic factors were identified; preoperative prescription of anticholinesterase alone (P=0.035) and non-thymomatous MG (P=0.003). The five-year CSR rate of the ocular type of MG reached a high level of 67.4%. Conclusions Thymectomy can achieve good long-term CSR in MG, and VATS is an ideal alternative method. High-dose prescription of anticholinesterase and the advanced stage by Myasthenia Gravis Foundation of America (MGFA) classification have higher risks of postoperative crisis. Preoperative prescription of anticholinesterase alone and non-thymomatous MG are good prognostic factors. Thymectomy should also be considered for the ocular tvioe of MG. 展开更多
关键词 myasthenia gravis THYMECTOMY video-assisted thoracoscopic surgery transsternal thymectomy PROGNOSIS
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