【作者】 甘雨耕；

【导师】 蔡绍皙； 王红兵；

【作者基本信息】 重庆大学 ， 生物医学工程， 2003， 硕士

【摘要】 中性白细胞抑制因子（Neutrophil inhibitory factor,NIF）是一种犬的钩虫（Ancylostoma caninum）分泌的糖蛋白，理论分子量为41 kDa，其cDNA编码的蛋白质由274 个氨基酸残基组成，N端有17个氨基酸残基的信号肽，成熟蛋白有257个氨基酸残基。NIF是一种新型抗炎因子，它能专一地结合人中性白细胞膜上的整合素CD11b/CD18，从而抑制活化的中性白细胞释放H2O2以及粘附血管内皮细胞，以此减轻中性白细胞介导的组织损伤。目的：美国辉瑞公司将rNIF用于治疗与局部出血性中风相关的再灌注损伤，现已进入Ⅱ期临床。仅美国每年就大约有750 000人患上局部出血性中风，如果rNIF顺利通过临床实验，无疑具有极大的商业价值。在基因工程中，rNIF的表达一般采用真核表达系统，美国Corvas公司的Moyle等人就利用巴斯德毕赤酵母成功表达出rNIF，但尚未见利用原核表达系统表达rNIF的报道，本研究初步探讨了如何利用大肠杆菌表达rNIF并进行纯化。方法：根据有关文献报道的编码NIF成熟肽的cDNA序列并参照引物设计的基本原则，设计出一对引物，以含目的cDNA的质粒PUC57为模板，利用PCR技术扩增目的cDNA。用限制性内切酶NdeⅠ和BamHⅠ双酶切扩增产物和表达质粒pET-21a(+)，连接目的cDNA和表达质粒的大片段，将连接产物用CaCl2 法转化大肠杆菌DH5α。抽提重组质粒pET-21a(+)/NIF，用NdeⅠ和BamHⅠ小量双酶切，确定目的cDNA与表达质粒的大片段是否正确连接，然后由上海生工用Sanger双脱氧链终止法-PUC体系对目的cDNA测序，确定目的cDNA的基因序列是否正确。大量抽提重组质粒，转化宿主菌BL21（DE3）plysS，经IPTG诱导，确定rNIF在宿主菌BL21（DE3）plysS中以可溶形式或包涵体形式高效表达，通过发酵大量制备rNIF。离心分离表达菌体和培养基，收获的菌体用Tris-HCl 缓冲液（pH8.0）+超声破菌处理，再离心收集包涵体沉淀，经过洗涤、溶解和复性，rNIF复性溶液经Q-Sepharose层析、羟基磷灰石（HTP）层析、Sephacryl S-100凝胶过滤层析，得到纯化的rNIF。在纯化过程中，用Bradford法测定rNIF浓度。凝胶扫描15％SDS-PAGE胶条，测定rNIF在全菌蛋白中的百分含量。HPLC分析rNIF纯品的纯度。通过中性白细胞与96孔细胞培养板的粘附性实验，测定rNIF抑制中性白细胞粘附的活性。结果：本研究成功利用PCR技术扩增出目的cDNA并克隆于表达质粒pET-21a(+)。对目的cDNA的序列分析结果表明，扩增的目的cDNA序列与文献报道一致。经IPTG诱导3 h，rNIF在宿主菌BL21（DE3）plysS中以包涵体形式实现高效表达。凝胶扫描的分析结果表明，目的蛋白（28.9 kDa）约占全菌蛋白的20％。HPLC的分析结果表明，rNIF纯品的纯度在98％以上。活性测定结果表明，rNIF能有效抑制中性白细胞的粘附。结论：本研究在大肠杆菌中高效表达出具有生物活性的rNIF,而且摸索出一套相应的纯化工艺，该工艺成本低、工艺衔接合理、分离效率高，可以方便的放大至中试和生产规模,这些结果为利用大肠杆菌制备rNIF奠定了基础。更多还原

【Abstract】 Neutrophil inhibitory factor（NIF）is a 41-kilodalton glycoprotein from the canine hookworm (Ancylostoma caninum) that binds CD11b/CD18 on neutrophil membrane. NIF comprises a mature polypeptide of 257 amino acids, preceded by a 17-amino acid leader. NIF is a novel anti-inflammatory agent, which blocks the adhesion of activated human neutrophils to vascular endothelial cells as well as the release of H2O2 from activated neutrophils . NIF can alleviate neutrophils-mediated tissue trauma.Objective: Pfizer Inc. in Amercia is conducting the studies on rNIF(recombinant neutrophil inhibitory factor) for treating reperfusion injury associated with ischemic stroke in a PhaseⅡclinical trial. Although Moyle et al. of Corvas International Inc in Amercia have reported that rNIF can be stably expressed in Pichia pastoris, it has not been reported that rNIF can be expressed in E.coli. The present study discussed expression in E.coli BL21(DE3)plysS and purification of rNIF. Methods: The cDNA encoding mature protein of NIF was amplified by PCR. After PCR product and expression vector pET-21a（+）were digested by NdeⅠand BamHⅠ, the cDNA was cloned into expression vector pET-21a(+) between the sites of NdeⅠand BamHⅠ, which was transformed into E.coli DH5α. The recombinant plasmid was determined, before it was transformed into E.coli BL21(DE3)plysS. After being induced with IPTG , E.coli BL21(DE3)plysS cell was collected and broken by ultrasonic processor. In the supernatant or precipitate of E.coli BL21(DE3)plysS lysates, a specific 28.9 kDa protein band appeared in 15％SDS-PAGE. Cell that expressed rNIF and medium were separated by centrifugation. The cell was in Tris-HCl buffer(pH8.0) and broken by ultrasonic processor. rNIF was purified by three step chromatography comprising of Q-Sepharose FF anion exchange , Hydroxyapatite ion exchange and Sephacryl S-100 gel filtration. HPLC analysis can determine purification of purified rNIF. The biological activity of rNIF was determined by neutrophil-plastic adhesion assay. Gel scanning of 15％SDS-PAGE can determine the rNIF percentage in the total cell protein. Results:The cDNA encoding mature protein of NIF was successfully amplified by PCR and cloned into expression vector pET-21a（+） between the sites of NdeⅠand BamHⅠ. Its cDNA sequence was determined. After three hour induction with IPTG, rNIF was over-expressed in E.coli BL21(DE3)plysS as inclusion bodies . After inclusion bodies of rNIF was renatured,renaturation solution was used to purify. It is indicated by Gel<WP=7>scanning of 15％SDS-PAGE that the expression level of rNIF was about 20％ of the total protein. It is indicated by HPLC analysis that rNIF was purified to approximately 98％. It is indicated by neutrophil-plastic adhesion assay that rNIF can efficiently inhibit the adhesion of neutrophils. Conclusion: rNIF can be over-expressed in E.coli BL21(DE3)plysS that has biological acivity in the present study.Moreover, a suit of corresponding purification methods for rNIF was obtained. These results show possibility that rNIF can be produced on a large scale using E.coli.更多还原