组织因子途径抑制物-2基因原核表达及其重组蛋白生物活性研究

Study on prokaryotic expression and partial biological activities for tissue factor pathway inhibitor-2

目的构建人组织因子途径抑制物2(TFPI2)基因的原核表达载体并在大肠杆菌中有效表达;探索重组TFPI2抑制纤溶酶活性及其对人卵巢癌细胞(A2780)迁徙浸润能力的影响。方法1.以TFPI2cDNA为模板,PCR扩增得到645bpDNA片段,克隆入表达载体pET28a,并转化大肠杆菌BL21株表达。2.限制性内切酶和菌落PCR法鉴定TFPI2基因DNA片段,Westernblot法鉴定TFPI2融合蛋白。3.建立TFPI2抑制纤溶酶的动力学方法并测定重组TFPI2对纤溶酶的抑制作用。4.在体外用Boyden小室,以不同浓度TFPI2作用A2780细胞进行迁徙和浸润实验。结果1.成功构建重组TFPI2的原核表达载体pET28a并在BL21株中表达、纯化,获得大量TFPI2蛋白。2.Westernblot证实表达的融合蛋白为TFPI2。3.重组TFPI2对液相和固相中的纤溶酶具有显著抑制作用,且呈剂量效应关系。4.在迁徙实验中,将A2780TFPI2不同浓度组与A2780对照组细胞穿过人工膜的细胞数进行比较,经t检验,无统计学意义(P>0.05);在浸润实验中,将A2780TFPI2不同浓度组与A2780对照组细胞穿过人工膜的细胞数进行比较及TFPI2不同浓度组间的细胞数进行比较,经t检验,具有显著差异(P<0.01)。结论1.人TFPI2基因的表达,为TFPI2病理生理的作用和临床研究提供了丰富材料。2.重组TFPI2抑制纤溶酶活性的研究,为探讨TFPI2对人卵巢癌细胞浸润转移能力的影响等提供了实验依据。3.重组TFPI2对人卵巢癌细胞体外自身运动能力无影响,但可显著抑制人卵巢癌细胞的体外浸润能力,为将来卵巢癌用蛋白酶抑制剂治疗提供一可能的靶向依据。

Objectives To express human TFPI-2 gene in E. coli and get the recombinant protein; to investigate the recombinant activities of antiplasmin and antitumor. Methods （ 1 ） Encoding region of mature protein of human TFPI-2 was amplified by PCR. The 645 bp PCR product was cloned into expression vector pET28a and transformed into BI21 strain to get expression. （2）Identifying the DNA segment of TFPI- 2 with enzyme digestion and colony PCR and the TFPI-2 fusion protein with western blot respectively. （3） To establish the kinetics assay for detecting the TFPI-2 and test it＇s inhibiting plasmin activities. （4）To assess ovarian tumor cell migratory and invasive behaviors with the Boyden chamber in vitro. Results （1） Expression plasmid of recombinant TFPI-2 was constructed and high-level expression of TFPI-2 was produced as fusion protein. （2） The TFPI-2 fusion protein was identified by western blot. （3） Antiplasmin activity of the TFPI-2 was confirmed in the fluid and on the cell surface. （4） In invasion assay, the number of the A2780 and A2780-TFPI-2 groups transferred the Matriged matrix-coated PVPF membrane was obvious decreased compared with that of A2780 group （ P 〈 0. 001 ）, while in migration assay, no significant difference was observed between A2780-TFPI-2 groups and A2780 group （ P 〉 0. 05 ）. Conclusion （ 1 ） Prokaryotic expression of TFPI-2 gene was got, which can provide the rich experimental material for investigating the role of TFPI-2 in relative fields; （2）Recombinant TFPI-2 has antiplasmin activity, which provides the experiment basis for investigating the role of recombinant TFPI-2 in human ovarian tumor migration and invasion in vitro; （3） The recombinant TFPI-2 inhibits the invasive ability of human ovarian tumor cells in vitro, but has no effect on the migration, which may provide a target basis for treating human ovarian tumor with TFPI-2 protein therapy.