【摘要】 目的:将纤维连接蛋白N端和C端两个肝素结合域基因片断克隆至昆虫表达系统中进行表达,分离纯化所表达多肽并进行多肽结合肝素、FN抗原性和抗血小板减少内毒素血症活性鉴定。方法:用高保真PCR技术从FNcDNA中扩增目的基因片段,将目的基因片段克隆至昆虫表达系的Bac-to-Bac HT Vector,在E.coliDH5α中抗氨苄青霉素选择,挑选阳性菌斑通过PCR、酶切鉴定和DNA序列测定,确定无误,再扩增提取质粒,进一步转染MAX Efficiency DHIOBac^TMCompetent E.coli,经三种抗菌素（卡那、庆大、四环素）联合IPTG、X-gal蓝白斑选择,挑取阳性白色菌斑提取质粒,PCR、酶切鉴定和DNA序列测定,确定无误,转染Sf 9昆虫细胞获取重组杆状病毒,重组杆状病毒再次转染Sf9昆虫细胞表达多肽。裂解Sf9昆虫细胞,SDS-PAGE分析裂解上清,镍离子亲和树脂分离纯化表达的多肽,Western blot鉴定多肽结合肝素和FN抗原性活性。动物实验探讨rhFNHC-36多肽抗血小板减少内毒素血症的作用。结果:纤维连接蛋白N端和C端两个肝素结合域多肽均在昆虫表达系统中得到表达,所表达的多肽具有与肝素结合的能力而不具FN抗原性。动物实验初步显示rhFNHC-36多肽具有抗血小板减少内毒素血症的作用。结论:在昆虫表达系统中表达纤维连接蛋白N端和C端两个肝素结合域多肽是可行的,所表达多肽具生物活性,但表达量尚较低,有待进一步优化表达以提高表达量。更多还原

【Abstract】 Objective:Cloning two genes of heparin-binding domain of fibronectin into vector of insect expression and expressing two polypeptide in insect cell Sf9.Purifing polypeptide with Ni-NTA and testing biological activity of polypeptide binding with heparin and antygen of FN with Western blot.Methods:The gene of two heparin-binding domain of fibronectin were amplified from FNcDNA by high fidelity PCR.The aim DNA sequences were cloned into Bac-to-Bac ? HT Vector（AorB） to get recombinant plasmid.The recombinant plasmid was transformed into E.coli DH5α and the positive plasmid was selected throuth ampicillin resistance to get positive recombinant donor.After being sequenceed and determined right read frame,positive donor was transformed into MAX Efficiency? DH10Bac^TM Competent E.coli to get positive bacmid（white colony） by gentamicin,kanamycin and tetracycline resistance with IPTG and X-gal.Recombinant bacmid was transformed into Sf9 cell.Isolated baclovirus from supernatant of culture Sf9 cell and performed a viral plaque assay,then recombinant baclovirus were transformed into Sf9 cell again,polypeptide was expressed in it.Polypeptide in supernatant of insect cell lysate were purified with Ni-NTA colummn.Binding heparin and FN antygen activity of polypeptides were detected by Western blot assay.It was determined in mice that rhFNHC-36 polypeptide antagonizes endotoxemia with thrombocytopenia.Results;Two heparin-binding polypeptide of fibronectin were expressed in insect cell sf9.Expressed polypeptides can bind with heparin in vitro and have losted antygen of FN The rhFNHC-36 polypeptide has function of antagonizing endotoxemia with thrombocytopenia in mice.Conclusion:Two heparin-binding polypeptide of fibronectin can be expressed in insect expression system.Expressed polypeptide can bind with heparin in vivo,not with antygen of FN and have function of antagonizing endotoxemia with thrombocytopenia in mice.更多还原