【摘要】 以鼻咽组织cDNA为模板,PCR扩增NAP1基因编码区,PCR产物克隆入pUCm-T载体,筛选阳性克隆.构建NAP1基因编码区的原核表达载体pEGX-4T-2/NAP1,转化大肠杆菌BL21.经IPTG诱导表达后,在不同时段收集菌体,提取细菌的全部蛋白.经12%SDS-聚丙烯酰胺凝胶电泳检测,发现细菌全蛋白中在35KD～36KD处多出一条明显条带.用兔抗人NAP1多克隆抗体,利用WesternBlotting对该融合蛋白的表达进行了鉴定,NAP1基因的融合蛋白表达成功,为制备NAP1单克隆抗体和获得有生物活性的NAP1蛋白,进一步研究NAP1蛋白的功能奠定了基础.更多还原

【Abstract】 The gene coding for protein NAP1 was amplified from nasopharynx biopsies cDNA by using PCR technique. PCR product was cloned initially into pUCm-T vector, then transformed into E.coil DH5α. Clones containing the vectors were selected on LB-plus ampicillin(100μg/ml) plates, and plasmid DNA was extracted and digested with enzymes. Recombinant expression plasmids pEGX-4T-2/ NAP1 were constructed from pUCm-T / NAP1 vector and pEGX-4T-2 expression vector. Plasmids of containing the right insertion were sequenced to confirm its identify and retransformed into E.coil BL21.Bacteria lysates prepared from 1mmol/L IPTG induced cultures were loaded directly onto SDS-PAGE. Upon IPTG induction, the recombinants pEGX-4T-2/ NAP1 produced indeed a new protein with an apparent MW of 35 KD(containing GST). Western blot results by using rabbit Anti-NAP1 polyclonal antibody showed that the right recombinant protein was obtained. In conclusion, we obtained recombinant pEGX-4T-2 expression vector containing NAP1.The protein has been expressed successfully. It will establish the obtaining of biological activity NAP1 protein and preparation of the NAP1 antigen and monoclonal antibodies for further functional study of NAP1 protein .更多还原