摘要
目的 构建编码人Ⅱ型胶原蛋白 2 5 0 2 70多肽片段(HuCⅡ 2 5 0 2 70 )的多聚基因 ,以获得高效表达、高度可溶和便于纯化的重组多肽基因。方法 选用大肠杆菌 (E .coli)偏爱密码子优化HuCⅡ 2 5 0 2 70的基因组成。通过化学合成和PCR法 ,获得HuCⅡ 2 5 0 2 70的基因 ,并利用BamHI和BglⅡ同裂酶进行酶切位点串联获得 6聚体。对影响重组HuCⅡ 2 5 0 2 70表达的各种因素进行系统研究 ,以获得优化表达的条件。对表达产物进行分离纯化。结果 酶切分析和DNA测序证实 ,6聚目的基因的构建正确。融合表达的量明显高于非融合表达 ,在优化条件下 ,可达 30 %。表达产物的可溶性明显提高 (在 90 %以上 ) ,其相对分子质量 (Mr)与预期的相符 ,纯化后的纯度达 90 %以上。结论 实现了HuCⅡ 2 5 0 2 70的高效表达 ,为研究类风湿性关节炎的发病机制和治疗提供了实验依据。为在原核细胞中高效表达的胶原蛋白的功能性小片段 。
Aim To construct the recombinant polymerized gene encoding human collagen type Ⅱ polypeptide 250~270(HuC Ⅱ 250~270), so as to obtain the recombinant polypeptide gene with high expression, high solubility and convenient for purification. Methods Using chemical synthesis and PCR techniques based on the known mRNA sequence of the fragment(HuCⅡ 250~270), composition of HuCⅡ 250~270 gene was optimized by adopting the preference codon in E.coli . A hexamerized gene was formed by several monogene polymerization to each other through isoschizomer cleavage sites with the same sticky end. In order to obtain optimal expression condition, systematic investigation on the factors of influencing expression in procaryotic cells was made. Then expressed fusion peeptide was isolated and purified. Results By endonuclease digesition analysis and DNA sequencing we proved that the constructed hexamerized HuCⅡ 250~270 gene was identical with the expection. The expressed level and solubility of fusion protein were much higher than those of nonfusion expression protein. Under optimal condition, the fusion protein accounted for 30 per cent of total bacterial proteins and the solubitlity of expressed fusion protein amounted to 90 per cent. The relative molecular mass( M r)of expressed product was in accord with prediction. The purity of purified fusion protein reached 90 per cent. Conclusion The high level expression of HuCⅡ250~270 gene may provide a condition for further researching the pathogenesis and therapy of rheumatoid arthritis, simultaneously it is also a useful reference material exploring the high expression of functional collagen peptide in prokaryotic cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2002年第4期383-386,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点科技攻关项目基金资助
No .9690 10 5 2 62
