血管基膜衍生多功能肽在大肠杆菌中的表达及抗肿瘤活性的测定

Prokaryotic expression of vascular basement membrane-derived multifunctional peptide and its anti-tumor activity assay

目的 构建血管基膜衍生多功能肽(VBMDMP)原核表达载体、诱导表达GST-VBMDMP融合蛋白,观察其对Lewis肺癌自发转移瘤模型的影响。方法 人工合成VB-MDMP DNA序列(人源性IgG3上游铰链区连接肽连接的Tumstatin的两个功能区获得性肽基因序列),构建克隆载体PUC19-VBMDMP,亚克隆到pGEX-4T-1原核表达载体上,经测序和酶切分析鉴定获得了未发生移码突变的PGEX-4T-1-VBMDMP融合载体质粒。转化入大肠杆菌后,用 IPTG诱导表达融合蛋白。Glutathione sepharose 4B层析柱纯化蛋白表达产物。采用Lewis肺癌自发转移瘤模型,检测VBMDMP的抗肿瘤作用。结果 成功的构建pUC19-VB-MDMP克隆和pGEX-4T-1-VBMDMP表达载体,在大肠杆菌获得稳定表达,用Glutathione sepharose 4B层析柱获得纯化的GST-VBMDMP融合蛋白。VBMDMP(GST-VBM-DMP 10、6、2 mg·kg^(-1))对小鼠Lewis肺癌原发瘤具有抑制作用(瘤重抑制率分别为95.5％,80.4％,60.05％)。VBMDMP(GST-VBMDMP 10,6,2 mg·kg^(-1))对小鼠Lewis肺癌自发性肺转移具有抑制作用(自发性肺转移瘤结节抑制率分别为94.8％,86.4％,73.9％)。结论 VBM-DMP对小鼠Lewis肺癌原发瘤和肺转移具有抑制作用。

AIM To construct prokaryotic expression vector of vascular basement membrane-derived multifunctional peptide ( VBMDMP) and to measure the anti-tumor activity of its fusion protein in Lewis lung carcinoma transplanted into C57BL/6 mice. METHODS Vascular basement membrane-derived multifunctional peptide (VBMDMP) sequence(fusion gene of human IgG3 upper hinge region and two tumstatin-derived fragments ) obtained by artificial synthesis was cloned into vector pUC19. We also subcloned it into prokaryotic expression vector pGEX-4T-l. The recombi-nant was confirmed by restriction enzyme digestion and auto-equencer. pGEX-4T-l-VBMDMP was transformed into Ecoli JM109. 0. 1 mmol · L-1 IPTG can induce high expression of GST-VBM-DMP fusion protein. We measured the anti-tumor activity of GST-VBMDMP which was purified by GST 4B Column in Lewis lung carcinoma transplanted into mouse C57BL/6. RESULTS The clone and expression vector of VBMDMP gene were constructed successfully. We obtained purified GST-VBMDMP fusion protein with GST 4B Column. The primary tumor inhibition rates were 95. 5%,80. 4%,60. 05% with GST-VBMDMP 10, 6,2 mg · kg-1 respectively. The tumor metastasis inhibition rates of Lewis lung carcinoma were 94. 8 %,86. 4%,73. 9% with GST-VBMDMP 10,6,2 mg· kg-1 respectively. The data have significant difference compared with control group. CONCLUSION VBMDMP has significant biological activity of anti-tumor growth and anti-metastasis in Lewis lung carcinoma transplanted into mouse C57BL/6.