摘要
从pro-UK全长cDNA中切下包括信号肽序列的N端371bp片段;经Fnu4HI不完全酶切将信号肽序列去掉,回收304bp片段;该片段再与人工合成的含Hind Ⅲ、EcoRV内切酶位点和起始密码ATG的寡核苷酸片段连接、转化得一重组质粒,该中间质粒经酶切和序列分析证明构建正确后再与pro-UK cDNA其余片段相连接,得到全长pro-UK cDNA。我们将所得不含信号肽序列的pro-UK cDNA重组到带有P_RPL启动子的原核表达载体pBV220中,转化大肠杆菌JM101,42℃诱导6h,菌体超声破碎后其上清及不溶性成份均可测出尿激酶活性。用ELISA法检测尿激酶抗原性表现为强阳性;纤维蛋白溶解平板法(FAPA)测定活性可达500—1000IU/L,经复性活性可达60000IU/L,且表达产物可被抗尿激酶血清特异中和,经相差显微镜及电镜观察均证明表达蛋白绝大部分以包涵体形式存在。Western blot分析证明表达产物为单链pro-UK,分子量约为47kDa,与国外文献报导一致。
Non-singnal peptide human pro-urokinase cDNA was obtained using synthetic oligonucleotide and DNA recombination methods, and was successfully expressed in E.coli. The plasmid pMMUK which contains pro-UK cDNA (including both the entire coding sequence and the sequence for signal peptide) was digested with HindⅢ and Pst Ⅰ, so that the N-terminal 371bp fragment could be recovered. A 304bp fragment was collected from the 371bp fragment partially digested with Fnu4HI in order to remove the signal peptide sequeoce. A intermediate plasmid was formed after this 304 fragment and the synthetic oligonucleotide was ligated with pUC18. Correctness of the ligation was confirmed by enzyme digestions and sequencing. Joining the Pst Ⅰ -Pst Ⅰ fragment of pro-UK to the intermediate plasmid, we got the final plasmid which contains the entire coding sequence of pro-UK without signal peptide. The suitable coding sequence was inserted into pBV220 just under the control of temperature-induced promoter P_RP_L, mature prourokinase was expressed in E.coli at 42℃. Both sonicated supernatant and inclusion body of the bacterial host JM101 showed positive results by ELISA and FAPA essay. After renaturation, the biological activity of the expressed product was increased from 500—1000IU/L to about 60000IU/L. The bacterial pro-UK gives a molecular weigbt of about 47000 daltons by Western blot analysis and it can be completely inhibited by UK antiserum but not by t-PA antiserum and normal rabbit serum.
出处
《生物工程学报》
CAS
CSCD
北大核心
1993年第2期122-127,共6页
Chinese Journal of Biotechnology
关键词
大肠杆菌
表达
尿激酶原
CDNA
Human pro-urokinase cDNA
E.coli
gene expression
renaturation
