摘要
利用加端PCR技术构建asnB-RGD工程菌,获得C末端带有RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)三肽的门冬酰胺酶(AnsB-RGD),以改进L-门冬酰胺酶的作用。以pUA18为模板,利用pUC18上游普适引物和根据L-门冬酰胺酶Ⅱ基因(ansB)C末端下游序列设计的引物来扩增目标基因。将目标基因连于pMD18-T载体,转化宿主菌JM109,筛选出的阳性克隆通过HindⅢ、Nco I双酶切下目标基因连到含T7启动子的高效表达载体pET28a上,再转化BL21宿主菌。高效表达的工程菌摇瓶发酵,渗透休克法提取AnsB-RGD融合蛋白,12％的SDS-PAGE检测酶蛋白并考察酶的活力、特异性及其对血液系统的影响。2％琼脂糖凝胶电泳、DNA测序鉴定构建的pET28a-ansB-RGD工程菌,奈氏法、SDS-PAGE及免疫学方法检测AnsB-RGD融合蛋白。DNA序列分析证明RGD三肽已经连接在L-门冬酰胺酶的C末端,带有RGD三肽的融合蛋白仍然能与抗L-门冬酰胺酶的抗体结合,初步发现AnsB-RGD融合蛋白有促进血液凝固作用,但酶活力下降。
The purpose is to construct genetic engineering strain expressing L-Asparaginase with C terminal Arg-Gly-Asp-extension (AsnB-RGD), and its function research. The gene encoding AsnB-RGD was obtained by add-PCR using recombnant plasmid pUA18 as template. The amplified fragment was cloned into the pET28a to construct the recombinant express vector. The recombinants were selected by enzyme activity assay. Genetic engineering strain was cultured by fermentation and method of osmotic shock extract AsnB-RGD fusion protein. Specity of purified enzyme and effecton on blood coagulation were detected. DNA sequencing shows RGD was engineeried into C-termi-nus of L-ASP, and the chimeric enzyme was purified to approximate homogeneity with ammonium sulfate precipitation and DEAE-cellulose chromatography. The purified protein has pharmacological impaction blood system. RGD peptides have been conjected to C of L-ASP and AsnB-RGD have effect on step-up hemagglutination.
出处
《药物生物技术》
CAS
CSCD
2003年第6期353-357,共5页
Pharmaceutical Biotechnology
