Expression and anticancer activity analysis of recombinant human uPA1‑43-melittin.
Expression and anticancer activity analysis of recombinant human uPA1‑43-melittin.
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DOI：
10.3892/ijo.2014.2750
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发表时间：
2015-02
影响因子：
5.2
通讯作者：
Man-man Su;Weiqin Chang;M. Cui;Yang Lin;Shuying Wu;Tianmin Xu
中科院分区：
文献类型：
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作者：
Man-man Su;Weiqin Chang;M. Cui;Yang Lin;Shuying Wu;Tianmin Xu
本研究的目的是表达一种可用于卵巢癌靶向治疗的融合蛋白。目的构建人尿激酶型纤溶酶原激活剂（uPA）（1-43）-蜂毒肽真核表达载体，在毕赤酵母中表达重组人uPA（1 -43）-蜂毒肽（rhuPA（1-43）-melittin），并检测其对卵巢癌细胞的抗肿瘤作用。根据uPA 1 -43的天然氨基酸序列合成编码uPA 1 -43氨基酸和蜂毒肽的DNA序列，并将其插入pPICZαC载体中。将uPA（1 -43）-melittin -pPICZαC转化毕赤酵母X-33，甲醇诱导表达rhuPA（1-43）-melittin。通过对卵巢癌细胞生长的抑制作用、细胞周期检测和TUNEL法检测重组融合蛋白的生物学活性。重组质粒uPA（1-43）-蜂毒肽-pPICZαC的DNA序列分析结果表明，编码人uPA 1-43氨基酸和蜂毒肽1-26氨基酸的DNA已正确插入pPICZαC载体中。经甲醇诱导后，SDS-PAGE和westernblot分析显示融合蛋白分子量约为7.6kDa。重组蛋白能抑制SKOV 3细胞的生长，诱导SKOV 3细胞周期阻滞和凋亡。该融合蛋白对正常组织无明显毒性。rhuPA（1-43）-蜂毒素在毕赤酵母中得到了高效表达。以uPAR特异性结合的uPA（1-43）氨基酸作为融合蛋白的靶向部分，利用蜂毒肽的抗肿瘤活性，重组融合蛋白能够抑制卵巢肿瘤的生长，可用于有效的靶向治疗。
The present study is focused on expression of a target fusion protein which can be used in ovarian cancer target therapy. It aimed to construct human urokinase-type plasminogen activator (uPA)(1-43)-melittin eukaryotic expression vector to express recombinant human uPA(1-43)-melittin (rhuPA(1-43)-melittin) in P. pastoris and to detect its anticancer effects on ovarian cancer cells. The DNA sequences that encode uPA1-43 amino acids and melittin were synthesized according to its native amino acid sequences and consequently inserted into pPICZαC vector. Then uPA1-43-melittin -pPICZαC was transformed into P. pastoris X-33, and rhuPA(1-43)-melittin was expressed by methonal inducing. The bioactivities of recombinant fusion protein were detected with inhibition effects on growth of ovarian cancer cells, cell cycle detection and TUNEL assay. The results of DNA sequence analysis of the recombinant vector uPA(1-43)-melittin -pPICZαC demonstrated that the DNA encoding human uPA 1-43 amino acids and 1-26 amino acids of melittin was correctly inserted into the pPICZαC vector. After being induced by methonal, fusion protein with molecular weight 7.6 kDa was observed on the basis of SDS-PAGE and western blot analysis. The recombinant protein was able to suppress growth of SKOV3, induce cell cycle arrest and apoptosis of SKOV3 cells. The fusion protein does not have any obvious toxicity on normal tissues. RhuPA(1-43)-melittin was successfully expressed in P. pastoris. Taking uPA(1-43) amino acids specifically binding to uPAR as targeted part of fusion protein, and making use of antitumor activity of melittin, the recombinant fusion protein it was able to inhibit growth of ovarian tumors and to be applied for effective targeted treatment.