摘要
将液氮保存的弓形虫 Ｎ Ｔ 株经小鼠复壮后，取腹腔液提取弓形虫基因组 Ｄ Ｎ Ａ，采用 Ｐ Ｃ Ｒ 方法从弓形虫 Ｎ Ｔ 株中扩增出约 ８００ ｂｐ 的片段。产物经 Ｅｃｏ Ｒ Ｉ和 Ｘｂａ Ｉ酶切后，克隆到 ｐ Ｕ Ｃ１９ 载体中，构建了 ｐ Ｂ Ｖ Ｐ３０ 非融合表达质粒和 ｐ Ｅ Ｔ Ｐ３０ 融合表达质粒。ｐ Ｂ Ｖ Ｐ３０ 转化到宿主菌 Ｄ Ｈ５α、ｐ Ｅ Ｔ Ｐ３０ 转化到宿主菌 Ｂ Ｌ２１ （ Ｄ Ｅ３）后，分别经温控诱导和 Ｉ Ｐ Ｔ Ｇ 诱导，产物经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 分析，ｐ Ｂ Ｖ Ｐ３０ 未发现表达产物，ｐ Ｅ Ｔ Ｐ３０ 出现约 ３０ ０００ 的产物。 Ｗ ｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ 显示，该蛋白与兔抗弓形虫血清发生特异性反应；薄层扫描显示，该蛋白占菌体总蛋白的 ２０％ 以上。
Based on the sequence of the major surface antgen gene of T.gondii published by Burg in 1993, two primers were designed and synthesized. The sequence of primer 1 is: 5′CG GAA TTC ATG TTC ACT CAA GTG CCC T 3′;The sequence of primer 2 is: 5′GC TCT AGA TCA CGC GCG ACA CAG CTG CG 3′. The coding sequence of P30 was obtained from the genomic DNA by PCR. The amplified fragment was digested with Eco RI and Xba I, then cloned into the vector PUC19. Two recombinant prokaryotic expression plasmids, pBV P30 and pET P30, were constructed. Plasmid pBV P30 was used for non fusion protein expression plasmid pET P30 for fusion protein expression. The recombinant plsmids were then transfected intoE.coli. SDS PAGE and Western blot were performed to detect the P30 gene product. No production of P30 was observed in E.coli containing pBV P30. On the other hand, P30 in pET P30 was highly expressed as a fusion protein whose molecular weight was about 30 000, the expressed protein accounted for over 20% of the total cell proteins by densitometric scanning.
出处
《中国兽医学报》
CAS
CSCD
北大核心
1999年第4期356-358,共3页
Chinese Journal of Veterinary Science
