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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~(TM)Competent 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。
OBJECTIVE: To clone the two heparin binding domain genes of N-terminal and C-terminal of fibronectin into insect expression system for expression, purify the expressed polypeptide and perform polypeptide binding heparin, FN antigenicity and anti-thrombocytopenic endotoxemia activity Identification. METHODS: The target gene fragment was amplified from FN cDNA by high-fidelity PCR. The target gene fragment was cloned into Bac-to-Bac HT Vector of insect expression system, selected for resistance to ampicillin in E. coli DH5α, and positive plaques were selected by PCR , Restriction enzyme digestion and DNA sequence determination were carried out to determine the correctness. The plasmid was further amplified and further transfected into MAX Efficiency DHIOBac (TM) Competent E.coli. The three antibiotics (Cana, Qingda, tetracycline) combined with IPTG, X -gal blue white spot selection, picked positive white plaque extract plasmid, PCR, restriction enzyme digestion and DNA sequencing was determined, correct transfected Sf 9 insect cells to obtain recombinant baculovirus recombinant baculovirus again transfected Sf9 insect cells Expression of the polypeptide. The Sf9 insect cells were lysed, the supernatant was lysed by SDS-PAGE, the expressed and purified peptides were separated by nickel ion affinity resin, and the heparin and FN antigenic activity were detected by Western blot. Animal experiments to explore rhFNHC-36 polypeptide anti-platelet endotoxemia role. Results: The two heparin binding domain polypeptides of N-terminal and C-terminal of fibronectin were all expressed in insect expression system. The expressed polypeptides had the ability of binding to heparin without FN antigen. Animal experiments preliminary showed rhFNHC-36 polypeptide has anti-platelet endotoxemia role. CONCLUSION: It is feasible to express N-terminal and C-terminal two heparin binding domain polypeptides in insect expression system. The expressed polypeptides are biologically active but their expression levels are still low, and further optimization of expression is needed to improve the expression level.