为获得腐败梭菌α毒素和D型产气荚膜梭菌ε毒素的共表达产物,并鉴定其致死活性和反应原性,评价其免疫保护效力,利用PCR扩增腐败梭菌α毒素和产气荚膜梭菌ε毒素基因Gcsa和Gcpe并克隆至pETDuet-1质粒,转化E.coli BL21(DE3),自诱导培养基...为获得腐败梭菌α毒素和D型产气荚膜梭菌ε毒素的共表达产物,并鉴定其致死活性和反应原性,评价其免疫保护效力,利用PCR扩增腐败梭菌α毒素和产气荚膜梭菌ε毒素基因Gcsa和Gcpe并克隆至pETDuet-1质粒,转化E.coli BL21(DE3),自诱导培养基诱导这2段基因共表达;用SDS-PAGE和Western-blot以及小鼠毒性试验、抗毒素血清中和试验对表达产物进行鉴定;将诱导后的大肠杆菌灭活,制成铝佐剂疫苗1 m L/只皮下注射免疫家兔2次,测定免疫血清中和抗体效价,用1 MLD的腐败梭菌毒素和D型产气荚膜梭菌毒素分别攻击。结果显示,所构建的E.coli BL21(pETDuet-Gcsa-Gcpe)可以同时表达Gcsa和Gcpe,表达产物能够与腐败梭菌和D型产气荚膜抗毒素血清反应;且具有小鼠致死毒性,只有用2种抗毒素一同作用,才能将其毒性中和;制成灭活疫苗免疫家兔,免疫血清对腐败梭菌毒素的中和抗体效价为2 MLD/0.1 m L,对D型产气荚膜梭菌毒素的中和抗体效价≥50 MLD/0.1 m L;用2种毒素攻击,免疫组家兔全部保护,对照组全部死亡,达到《中华人民共和国兽药典》(2015版)效力检验标准。结果表明,所构建的表达载体可以实现α毒素和ε毒素的活性共表达,本研究为腐败梭菌和产气荚膜梭菌的多联基因工程灭活疫苗的研究提供了实验基础。展开更多
Clostridium septicum(C.septicum)gas gangrene is well documented in the literature,typically in the setting of trauma or immunosuppression.In this paper,we report a unique case of spontaneous clostridial myonecrosis in...Clostridium septicum(C.septicum)gas gangrene is well documented in the literature,typically in the setting of trauma or immunosuppression.In this paper,we report a unique case of spontaneous clostridial myonecrosis in a patient with Crohn's disease and sulfasalazineinduced neutropenia.The patient presented with left thigh pain,vomiting and diarrhea.Blood tests demonstrated a profound neutropenia,and magnetic resonance imaging of the thigh confirmed extensive myonecrosis.The patient underwent emergency hip disarticulation,followed by hemicolectomy.C.septicum was cultured from the blood.Following completion of antibiotic therapy,the patient developed myonecrosis of the right pectoral muscle necessitating further debridement,and remains on lifelong prophylactic antibiotic therapy.展开更多
文摘本研究旨在获得重组腐败梭菌α毒素,并评价其毒力及免疫原性。对已知的腐败梭菌α毒素编码基因进行优化设计,通过人工合成获得基因片段Gcsa。将Gcsa克隆至原核表达载体pET-30a(+)中进行表达与纯化,从而获得重组蛋白rCSA。利用Western blot方法检测纯化的rCSA与腐败梭菌α毒素抗血清的反应性,并采用犬肾细胞系(MDCK)和小鼠来检测rCSA的毒力。以甲醛脱毒后的rCSA与Montanide ISA 201佐剂以1∶1的比例制备疫苗,免疫家兔,按照《中华人民共和国兽药典》(2015年版)规定的方法检测兔血清的中和抗体效价。结果表明,rCSA为可溶性表达,比例可达50%,且能与腐败梭菌抗血清反应。MDCK细胞毒性试验显示,0.15ng·mL^(-1)的rCSA即可引起明显的细胞病变;小鼠安全性试验显示,rCSA对ICR小鼠的最小致死量是1.5×10~6 ng·kg^(-1);每毫升的一免抗血清可中和100~130个小鼠MLD的腐败梭菌毒素;每毫升的二免抗血清可中和360~480个小鼠MLD的腐败梭菌毒素;1个家兔MLD的腐败梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔得到了4/4的保护。以上结果表明,rCSA具有良好的免疫原性,可作为腐败梭菌病基因工程亚单位疫苗的候选抗原。
文摘为获得腐败梭菌α毒素和D型产气荚膜梭菌ε毒素的共表达产物,并鉴定其致死活性和反应原性,评价其免疫保护效力,利用PCR扩增腐败梭菌α毒素和产气荚膜梭菌ε毒素基因Gcsa和Gcpe并克隆至pETDuet-1质粒,转化E.coli BL21(DE3),自诱导培养基诱导这2段基因共表达;用SDS-PAGE和Western-blot以及小鼠毒性试验、抗毒素血清中和试验对表达产物进行鉴定;将诱导后的大肠杆菌灭活,制成铝佐剂疫苗1 m L/只皮下注射免疫家兔2次,测定免疫血清中和抗体效价,用1 MLD的腐败梭菌毒素和D型产气荚膜梭菌毒素分别攻击。结果显示,所构建的E.coli BL21(pETDuet-Gcsa-Gcpe)可以同时表达Gcsa和Gcpe,表达产物能够与腐败梭菌和D型产气荚膜抗毒素血清反应;且具有小鼠致死毒性,只有用2种抗毒素一同作用,才能将其毒性中和;制成灭活疫苗免疫家兔,免疫血清对腐败梭菌毒素的中和抗体效价为2 MLD/0.1 m L,对D型产气荚膜梭菌毒素的中和抗体效价≥50 MLD/0.1 m L;用2种毒素攻击,免疫组家兔全部保护,对照组全部死亡,达到《中华人民共和国兽药典》(2015版)效力检验标准。结果表明,所构建的表达载体可以实现α毒素和ε毒素的活性共表达,本研究为腐败梭菌和产气荚膜梭菌的多联基因工程灭活疫苗的研究提供了实验基础。
文摘Clostridium septicum(C.septicum)gas gangrene is well documented in the literature,typically in the setting of trauma or immunosuppression.In this paper,we report a unique case of spontaneous clostridial myonecrosis in a patient with Crohn's disease and sulfasalazineinduced neutropenia.The patient presented with left thigh pain,vomiting and diarrhea.Blood tests demonstrated a profound neutropenia,and magnetic resonance imaging of the thigh confirmed extensive myonecrosis.The patient underwent emergency hip disarticulation,followed by hemicolectomy.C.septicum was cultured from the blood.Following completion of antibiotic therapy,the patient developed myonecrosis of the right pectoral muscle necessitating further debridement,and remains on lifelong prophylactic antibiotic therapy.