【摘要】 目的:利用PCR技术从含有目的基因的质粒pGEX- 4T- 1 -VBMDMP获得带有SnabI和NotI酶切位点并融合有GST的目的基因片段并测序鉴定,将PCR产物纯化、酶切、回收,克隆到pPIC9K载体,转化入大肠杆菌DH5a扩增,用琼脂糖凝胶电泳,限制性内切酶酶切和DNA测序鉴定。方法:BglⅡ线性新构建的表达载体pPIC9K- GST- VBMDMP ,然后用原生质体转化法转化毕赤酵母细胞,并用pPIC9K空质粒也转入酵母细胞进行对照。用煮 冻 煮法裂解已转化有目的基因的毕赤酵母细胞提取DNA并行PCR鉴定,获得阳性重组子,行多克隆筛选和表型鉴定。筛选His+ Muts 表型的转化子,在MGY液体培养基中30℃摇床培养,每2 4h从培养基中转移1ml培养基上清于- 70℃保存,并保持培养瓶中培养基的量和培养基中甲醇0 .5 %的浓度不变,8d后行SDS -PAGE检测表达的蛋白,用GlutathioneSepharose 4B亲和层析柱纯化获得GST- VBMDMP融合蛋白;用凝血酶切割GST- VBMDMP并分离获得VB MDMP。结果:发现VBMDMP能够抑制鼠主动脉内皮细胞管状结构的形成;同时(VBMDMP 10mg/kg ,6mg/kg ,2mg/kg)对小鼠Lewis肺癌原发瘤具有显著抑制作用(瘤重抑制率分别为96 .6 % ,82 .1% ,6 1.2 % )。VBMDMP(GST -VB- MDMP 10mg/kg ,6mg/kg ,2mg/kg)对小鼠Lewis肺癌自发性肺转移具有显著抑制作用(自发?更多还原

【Abstract】 Objective To construct eukaryotic expression v ector of vascular basement membrane-derived multifunctional peptide(VBMDMP) and to measure the anti-angio genesis activity of VBMDMP in MAE and anti-tumor activity of its fusion protein i n Human prostate adenocarcinoma cells (PC-3) transplanted into male SCID mice. Methods GST-VBMDMP containing Snab I and Not I enzyme cutt ing si tus was obtained by polymerase chain reaction(PCR),then purified,digested,recovered and cloned into pPIC9K eukaryotic expression vector. The recombinant pPIC9K-GST-VBMDMP w as transformed into E.coli DH5a and amplified, which was confirmed by restriction e n donuclease digestion and sequencing,then transformed into Pichia pastoris. T he control of pPIC9K was transformed into Pichia pastoristoo. Using a single c olon y, inoculated in MGY liquid medium and growed at 30 ℃ in a shaking incubator. A t each of the time points: 24 h? 48 h?72 h?96 h?120 h?144 h?168 h and 192 h , transf ered 1ml of the supernatants to 1.5ml EP tube and freezed in -70 ℃. At the mean t ime, kept the volume of culture and added methanol to a final concentration of 0 .5% every 24 h to maintain the induction. After 8 d, the supernatants were analyzed by SDS-PAGE for protein expression with Coomassie brilliant blue. The anti-tumor activity of GST-VBMDMP was measured which was purified by GST 4B Col u mn in Human prostate adenocarcinoma cells (PC-3) transplanted into male SCID mi ce and Endothelial Tube Assay was produced.Results The expre ssion vector of VBMDMP gene were constructed successfully.We obtained purified GST-VBMDMP fusion prot ein with GST 4B Column.The primary tumor inhibition rate were 96.6%, 82.1%, 61 .2% with GST-VBMDMP 10mg/kg, 6 mg/kg,2 mg/kg respectively. The tumor metastasi s inhibition rate of PC-3 were 96.8 %,87.9 %,75.3% with GST-VBMDMP 10 mg/kg , 6 mg/kg,2 mg/kg respectively. The data were significant difference in contras t to control group. Conclusion VBMDMP have significant anti- angiogenesis activity and have biological activity of anti-tumor growth and anti-metastasis in PC-3 transplanted into male SCID mice.更多还原