摘要
选用大肠杆菌Ｌ－门冬酰胺酶（ＡＳＰｓⅡ）高效表达质粒ｐＫＡ作为融合表达载体，将水蛙素Ⅲ（ＨＶ３）人工合成编码基因插入 ｐＫＡ质粒的 ＡＳＰｓⅡ编码基因 ａｎｓＢ中，使 ＡＳＰｓ Ⅱ１Ｎ端的 ８９个氨基酸残基通过甲硫氨酸残基与 ＨＶ３形成融合蛋白，从而构建成 ＡＳＰｓⅡ－ＨＶ３融合蛋白表达质粒 ｐＫＡＨ，通过基因操作将该质粒的低拷贝数复制原长更换成 ｐＵＣ高拷贝数复制原点，进而构建成ＡＳＰｓⅡ－ＨＶ３融合蛋白高效表达载体 ｐＵＫＡＨ。该质粒在大肠杆菌 ＪＭ１０５宿主细胞中表达后，融合蛋白（１５． ６ ｋＤ）占细菌总蛋白 １０ ５％，该融合蛋白经 ＣＮＢｒ切割释放出活性水蛭素分子，抗凝血活力达约 ２０ ＡＴＵ／ｍｌ培养液，为以后进一步研究打下了基础。
E. coil ASPs Ⅱ expression plasmid pKA was chosen as the fusion expression vector for HV3 gene. HV3 gene was fused to the truncated ansB gene for ASPs Ⅱ to construct expression plasmid pKAH and pUKAH. Upon induction pUKAH/JM105 expressed a hybrid protein of 15 kD, which was in good agreement with the expected value of 15. 6 kD calculated from its amino acid composition. Densitometric analysis of the gel indicated that this protein represented 10. 5% of total cellular proteins. The fusion protein was cleaved by CNBr, and the yield of biologically active HV3 was about 20 ATU/ml culture.
出处
《中国生化药物杂志》
CAS
CSCD
1999年第1期10-12,共3页
Chinese Journal of Biochemical Pharmaceutics
