本研究旨在获得产气荚膜梭菌α毒素(CPA)C末端(第247-370位氨基酸,CPA_C)三拷贝串联融合蛋白,并评价其免疫原性。对已知的A型产气荚膜梭菌CPAC编码基因(GenBank登录号:AY823400.1)进行优化设计,以同向串连的方式串联成三拷贝基因(GCPA_(...本研究旨在获得产气荚膜梭菌α毒素(CPA)C末端(第247-370位氨基酸,CPA_C)三拷贝串联融合蛋白,并评价其免疫原性。对已知的A型产气荚膜梭菌CPAC编码基因(GenBank登录号:AY823400.1)进行优化设计,以同向串连的方式串联成三拷贝基因(GCPA_(C3)),片段之间用柔性氨基酸linker(GGGS)连接,经人工合成后克隆至原核表达载体pET-30a(+)中进行表达与纯化,获得CPA_C的三拷贝重组蛋白(rCPA_(C3))。利用Western blotting方法检测rCPA_(C3)与A型产气荚膜梭菌毒素抗血清的反应性。将rCPA_(C3)与Montanide ISA 201佐剂混合乳化制备疫苗,免疫4只健康家兔,检测一免及二免后兔血清的中和抗体效价。在二免21d后,对家兔经耳缘静脉注射1个家兔最低致死剂量(MLD)的A型产气荚膜梭菌毒素,检测rCPA_(C3)对家兔的免疫保护效果。结果显示,rCPA_(C3)主要以包涵体的形式表达,且能与A型产气荚膜梭菌毒素抗血清反应;每毫升的一免抗血清可中和30～50个、二免抗血清可中和70～100个小鼠MLD的A型产气荚膜梭菌毒素。采用1个家兔MLD的A型产气荚膜梭菌毒素攻毒后,对照组家兔100%(4/4)死亡,免疫组得到了100%(4/4)的保护。以上结果说明,rCPA_(C3)具有良好的免疫原性,为A型产气荚膜梭菌病基因工程疫苗的研制提供了重要的试验数据。展开更多
In order to select an optimum extraction method for the target glycoprotein （TGP） from jellyfish （Rhopilema esculentum） oral-arms, a high performance liquid chromatography （HPLC）-assay for the determination of t...In order to select an optimum extraction method for the target glycoprotein （TGP） from jellyfish （Rhopilema esculentum） oral-arms, a high performance liquid chromatography （HPLC）-assay for the determination of the TGP was developed Purified target glycoprotein was taken as a standard glycoprotein. The results showed that the calibration curves for peak area plotted against concentration for TGP were linear （r= 0.9984, y=4.5895x＋47.601） over concentrations ranging from 50 to 400mgL^-1. The mean extraction recovery was 97.84% （CV2.60%）. The fractions containing TGP were isolated from jellyfish （R esculentum） oral-arms by four extraction methods： 1） water extraction （WE）, 2） phosphate buffer solution （PBS） extraction （PE）, 3） ultrasound-assisted water extraction （UA-WE）, 4） ultrasound-assisted P/3S extraction （UA-PE）. The lyophilized extract was dissolved in Milli-Q water and analyzed directly on a short TSK-GEL G4000PWXL （7.8 mm×300 ram） column. Our results indicated that the UA-PE method was the optimum extraction method selected by HPLC.展开更多
文摘本研究旨在获得产气荚膜梭菌α毒素(CPA)C末端(第247-370位氨基酸,CPA_C)三拷贝串联融合蛋白,并评价其免疫原性。对已知的A型产气荚膜梭菌CPAC编码基因(GenBank登录号:AY823400.1)进行优化设计,以同向串连的方式串联成三拷贝基因(GCPA_(C3)),片段之间用柔性氨基酸linker(GGGS)连接,经人工合成后克隆至原核表达载体pET-30a(+)中进行表达与纯化,获得CPA_C的三拷贝重组蛋白(rCPA_(C3))。利用Western blotting方法检测rCPA_(C3)与A型产气荚膜梭菌毒素抗血清的反应性。将rCPA_(C3)与Montanide ISA 201佐剂混合乳化制备疫苗,免疫4只健康家兔,检测一免及二免后兔血清的中和抗体效价。在二免21d后,对家兔经耳缘静脉注射1个家兔最低致死剂量(MLD)的A型产气荚膜梭菌毒素,检测rCPA_(C3)对家兔的免疫保护效果。结果显示,rCPA_(C3)主要以包涵体的形式表达,且能与A型产气荚膜梭菌毒素抗血清反应;每毫升的一免抗血清可中和30～50个、二免抗血清可中和70～100个小鼠MLD的A型产气荚膜梭菌毒素。采用1个家兔MLD的A型产气荚膜梭菌毒素攻毒后,对照组家兔100%(4/4)死亡,免疫组得到了100%(4/4)的保护。以上结果说明,rCPA_(C3)具有良好的免疫原性,为A型产气荚膜梭菌病基因工程疫苗的研制提供了重要的试验数据。
基金support from the National High Technology Research and Development Program of China (No.2006AA09Z438)
文摘In order to select an optimum extraction method for the target glycoprotein （TGP） from jellyfish （Rhopilema esculentum） oral-arms, a high performance liquid chromatography （HPLC）-assay for the determination of the TGP was developed Purified target glycoprotein was taken as a standard glycoprotein. The results showed that the calibration curves for peak area plotted against concentration for TGP were linear （r= 0.9984, y=4.5895x＋47.601） over concentrations ranging from 50 to 400mgL^-1. The mean extraction recovery was 97.84% （CV2.60%）. The fractions containing TGP were isolated from jellyfish （R esculentum） oral-arms by four extraction methods： 1） water extraction （WE）, 2） phosphate buffer solution （PBS） extraction （PE）, 3） ultrasound-assisted water extraction （UA-WE）, 4） ultrasound-assisted P/3S extraction （UA-PE）. The lyophilized extract was dissolved in Milli-Q water and analyzed directly on a short TSK-GEL G4000PWXL （7.8 mm×300 ram） column. Our results indicated that the UA-PE method was the optimum extraction method selected by HPLC.
