摘要
目的 :克隆含tPA中 355个氨基酸密码子 ( 1- 3和 176- 52 7氨基酸 )的cDNA序列(tPA355) ,将其在大肠杆菌融合蛋白表达系统中表达 ,并在体外复性使其具有激活纤溶酶原的生物活性。方法 :采用RT -PCR技术从人黑色素瘤细胞Bowes中克隆出tPA355cDNA ,然后在 pET32a( +)BL2 1(DE3)大肠杆菌表达系统中表达 ,将表达出的融合蛋白Trx -tPA355(Thioredoxin ,Trx)包涵体在体外进行变性、复性和纯化以使其具有激活纤溶酶原的生物活性。结果 :测序结果表明本研究克隆的编码tPA中 355个氨基酸密码子的cDNA序列与美国专利 (公开号 :5,587,159)中对应的序列完全一致 ,将其在 pET32a( +) /BL2 1(DE3)大肠杆菌表达系统中表达可获得稳定表达的融合蛋白Trx -tPA355包涵体 ,该包涵体占菌体总蛋白的 30 %左右 ,此融合蛋白经变性、复性后具有激活纤溶酶原的生物活性。结论 :含tPA中 355个氨基酸密码子 ( 1- 3和 176- 52 7氨基酸 )的cDNA在大肠杆菌Trx融合蛋白表达系统中可获得稳定表达 ,表达的融合蛋白产物在体外经变性、复性后具有激活纤溶酶原的生物活性。
Clone human tPA cDNA which contains 355 amino acid(1-3 and 176-527 amino acid)codons(tPA 355 ),express them in fusion protein(thioredoxin,Trx)expression system of E.coli after their sequences have been determined,refold the fusion protein Trx tPA 355 and detect its bioactivity in vitro. Methods:human tPA cDNA which contains 355 amino acids was cloned by RT-PCR from human melanoma cell line Bowes,and expressed in fusion protein expression system pET32a(+)/BL21(DE3).The fusion protein Trx-tPA 355 was refolded and bioassayed in vitro. Results:The corresponding sequence of tPA 355 and the patent cDNA(U.S.No.5,587,159)was identical.The expression of Trx tPA 355 were expressed effectively in pET32a(+)/BL21(DE3)system,about 30% of total cell protein.And Trx tPA 355 was refolded and could activate plasminogen in vitro. Conclusions:The human tPA 355 cDNA which fused with Trx cDNA,could be expressed stably,and the fusion protein Trx tPA 355 could refolded effectively and exhibited plasminogen stimulating activity in vitro.
出处
《生物工程进展》
CSCD
2000年第6期63-67,共5页
Progress in Biotechnology
