摘要
目的 利用 pGEX 4T 2表达系统表达制备重组人B型钠尿肽 (BNP)抗原。 方法 根据NCBI核苷酸数据库编码为 gi :2 172 6 39人BNPDNA序列 ,设计合成编码BNP 32蛋白的DNA ,并分别在 5′端和 3′端设计BamHI及XhoI位点 ,经两步聚合酶链反应 (PCR)、T A克隆后 ,亚克隆至经BamHI及XhoI酶切的 pGEX 4T 2载体 ,把重组质粒转化大肠杆菌DH5α ,经IPTG诱导表达 ,Glutathionesepharose 4B亲和层析制备GST BNP。通过DNA测序、融合蛋白的分子量分析和Western blot来证实GST BNP质粒及表达的B型钠尿肽 (BNP 32 )抗原的正确性。结果 限制性内切酶和DNA测序结果表明 ,编码BNP 32的DNA准确插入 pGEX 4T 2多克隆位点 ,成功构建了GST BNP的表达质粒 ;诱导表达超声破碎后 ,GST BNP蛋白主要存在于上清液中 ,经亲和层析回收融合蛋白 ,每升诱导菌可得融合蛋白 10 6mg ;Western blot证实制备的融合蛋白是人BNP 32。 结论 通过基因工程技术制备的人BNP抗原 ,既具有免疫原性又具有反应原性 ,能很好地解决酶免分析中的基本要素抗原 (被检测物 )和相应的抗体。
Objective To prepare human B type natriuretic peptide (hBNP 32) with GST fusion protein by pGEX 4T 2 expression system Methods A synthesized DNA segment coding for hBNP 32 was ligated to pGEX 4T 2 vector The recombined plasmid was transferred into E coli DH5α, and GST BNP fusion protein were expressed by induction with IPTG The fusion protein were purified by Glutathione sepharose 4B affinity chromatography in one step DNA sequencing, SDS PAGE and Western Blot were used to verify the plasmids and fusion proteins Results Restriction analysis and DNA sequencing confirmed that the target DNA segments were correct in sequence and insertion direction Expressed fusion proteins were mainly in soluble supernatant The yield of GST BNP fusion protein was 10 6 mg per liter bacteria culture Western Blot result indicated that fusion proteins were recombinant of human B type natriuretic peptide Conclusions A fusion protein plasmid of GST BNP was successfully constructed and the recombinant hBNP 32 obtained by this way contains immunogenicity and reactivity of antigen The fusion protein may be used as quality control substance
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2004年第12期853-856,共4页
Chinese Journal of Laboratory Medicine
基金
浙江省教育厅基金资助项目 (2 0 0 3 0 3 2 8)
