摘要
目的： 探讨诱导时间对目的蛋白表达量的影响， 为大量获得抗ＴＮＦα单链抗体提供实验依据。方法：将Ｅ６ＳｃＦｖ 基因分别克隆入表达载体ｐＥＴ１５ｂＥｔａｇ 和ｐＢＶ２２０ 中， 构建重组表达质粒ｐＥＴＥ６ＳｃＦｖ 和ｐＢＶＥ６ＳｃＦｖ。在化学诱导和温度诱导两种条件下， 于诱导后相同时间点等量取菌体， 用ＳＤＳＰＡＧＥ 对各时间点表达产物扫描定量。结果： ｐＥＴＥ６ＳｃＦｖ （ＢＬ２１） 的阳性克隆过夜菌经ＩＰＴＧ 化学诱导２ ｈ， 就有新生蛋白条带出现； 而ｐＢＶＥ６ＳｃＦｖ （ＤＨ５α） 诱导４ ｈ 后才有新生蛋白条带出现。两者均随诱导时间的延长而加深， 但将诱导时间延长至２０ ｈ 表达量并无明显变化， 表达水平反而下降。结论： ｐＥＴＥ６ＳｃＦｖ （ＢＬ２１） 的最佳诱导时间为４ ｈ ～６ ｈ，ｐＢＶＥ６ＳｃＦｖ （ＤＨ５α） 的最佳诱导时间为８ ｈ 。
Aim:To acquire a large amount of anti human TNF alpha single chain antibody,we investigated the effect of induction condition on quantity of expression products. Methods: The E6ScFv gene fragment was cloned into pET15b Etag and pBV220 expression vectors,respectively for constructing the recombinant expression plasmids pETE6ScFv and pBVE6ScFv. After inducing with IPTG or at 42℃, the quantities of the two expression products at same times were identified by scanning bands on their SDS PAGE gel with CS 9000 thin scanner. Results: After inducing with IPTG for 2 hours, a new protein band of E6ScFv Etag fusion protein in the pETE6ScFv(BL21)was found on SDS PAGE gel. And a new protein band in the pBVE6ScFv(DH5α)was found after inducing for 4 hours at 42℃. Their quantities were increased with the induction time prolonged. On the contrary,expression quantities of both recombinants were decreased by 20 hours'induction. Conclusion: The optimal period of induced with IPTG for pETE6ScFv(BL21)was 4～6 hours, while that at 42℃for pBVE6ScFv(DH5α)was 8 hours.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
1999年第4期263-265,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金!资助项目
No. 39670695
