铜绿微囊藻生物钟蛋白的表达纯化与抗体制备

为了获得融合表达的铜绿微囊藻(Microcystic aeruginosa)生物钟蛋白KaiA、KaiB、KaiC并制备其相应的多克隆抗体,将kaiA、kaiB、kaiC基因分别克隆到原核表达质粒pET-His中.重组质粒pET-His-KaiA,pET-His-KaiB和pET-His-KaiC经酶切和测序鉴定后,分别转化E.coli BL21(DE3)进行融合表达.经SDS-PAGE分析可知,融合表达的KaiA、KaiB和KaiC蛋白表达量可分别达到菌体总蛋白的25%、40%和20%.经亲和层析后融合蛋白KaiA和KaiB的纯度分别达95%和92%,而KaiC经胶回收纯化后纯度也可达93%.将纯化后的三种Kai蛋白作为抗原分别免疫小鼠制备多克隆抗体,经ELISA检测抗体滴度表明,制备的抗KaiA、抗KaiB和抗KaiC的多克隆抗体效价高,分别可达到1:50000、1:60000和1:100000.Western blotting结果表明:获得的多克隆抗体具有较高的效价,抗体能识别相应的Kai蛋白,具有较高的特异性,能用于铜绿微囊藻生物钟蛋白KaiA、KaiB和KaiC的表达节律检测.     

Immunodetection of legume proteins resistant to small intestinal digestion in weaned piglets

Experiments were conducted to investigate the biochemistry of digestion of the major storage proteins from soya bean, pea, faba bean, blue lupin, and chickpea seeds in the ileum of piglets. Hyperimmune plasmas against the crude protein extracts and the purified 11S and 7S globulin fractions of each legume seed and an anti‐pea albumin PA2 and lectin antibody were used. They served to probe immunoblots of feed protein extracts and ileal digesta samples. Globally, the recognition by plasmas of intact or partially digested proteins in ileal digesta was rather faint, in agreement with the fairly high in vivo digestibility data obtained with these legume seed proteins. Nevertheless, immunoreactive polypeptides found in digesta of piglets fed pea, faba bean and chickpea belonged mainly to proteins of the 7S family, and to other proteins including low‐molecular weight components such as PA2 albumin and lectin in the case of pea. In piglets fed lupin, nearly intact polypeptides from the 11S family were detected. To conclude, the present immunochemical study conducted on ileal digesta of piglets revealed a few dietary legume proteins of the vicilin and albumin families. Legumin proteins were demonstrated unequivocally in the case of lupin and white chickpea. Copyright © 2003 Society of Chemical Industry     

人抗氧化蛋白-1在大肠杆菌中的表达、纯化及活性鉴定

为得到较纯的具有活性的人抗氧化蛋白Peroxiredoxin1(Prx1),本文先用PCR的方法扩增了人Prx1的cDNA序列,然后将其连接到pET28表达载体上,从而构建了编码全长的人抗氧化蛋白-1基因的pET28-Prx1原核表达质粒。经双酶切及测序鉴定后将表达质粒转入大肠杆菌BL21(DE3)进行表达,并通过SDS-PAGE和western blot来鉴定表达产物。随后用Ni-NTA螯合亲和层析的方法纯化重组蛋白,并测定该酶的米氏常数,用质粒保护实验鉴定该蛋白其活性。最终表达载体经酶切和测序鉴定证实构建成功,表达产物在SDS-PAGE和western blot图的25 kD左右,与prx1真实的蛋白分子量相符,说明Prx1蛋白成功表达。Prx1纯化后产量达到7.59 mg/g菌体,纯度达到88.50%。纯化后的Prx1在37℃和pH 7.4的条件下催化H2O2反应的米氏常数Km为2.06×10-4 mol/L,质粒保护实验表明Prx1可以保护pUC18质粒免受氧化损伤。因此,Prx1在大肠杆菌中成功表达并得到较纯的活性蛋白。     

人甲胎蛋白AFP在大肠杆菌中表达和鉴定

本研究通过应用基因工程技术,重组构建编码人全长甲胎蛋白AFP基因的原核表达载体pET22b-AFP,并将其转化到大肠杆菌宿主菌BL21（DE3）中进行诱导表达目的蛋白AFP。采用PCR法扩增编码AFP蛋白的cDNA序列片段,并将其克隆到含有His-tag的pET22b原核表达载体上;重组质粒经双酶切及测序鉴定正确后转化到大肠杆菌BL21(DE3)中进行IPTG诱导表达;对诱导过程中不同浓度的IPTG和不同的温度进行优化;选定最佳的优化条件进行诱导表达目的蛋白;SDS-PAGE电泳和Western Blot法鉴定表达产物。结果表明,通过PCR扩增技术获得了编码AFP蛋白的基因片段;重组质粒经双酶切和基因测序等方法鉴定后,确认了重组质粒已经构建成功;表达产物通过利用SDS-PAGE和Western Blot法检测到在66 kDa附近出现条带,与预期值相符,表明重组AFP蛋白已成功表达。     

重组小分子多肽的SDS-PAGE分析方法

比较了小分子多肽SDS PAGE的各种显色方法 ,结果表明 ,考马斯亮蓝R 2 5 0染色需要 3μg以上的样品 ,相对分子质量 30 0 0左右的肽才能得到清晰条带 ,而对含量低的基因工程产物不能显色 .银染可用于检测微量的分子多肽 ,银染过程中用戊二醛和甲醛进行敏化而减少固定时间 ,可显著提高检测灵敏度 ,是检测微量基因工程产物的好方法