湖北农业科学
湖北農業科學
호북농업과학
2014年
10期
2443-2447
,共5页
胶原蛋白多肽%大肠杆菌(Escherichia coli)%表达%优化
膠原蛋白多肽%大腸桿菌(Escherichia coli)%錶達%優化
효원단백다태%대장간균(Escherichia coli)%표체%우화
collagen peptide%Escherichia coli%expression%optimization
以Ⅵ型人胶原蛋白A2链基因为模板，PCR扩增得到目的基因CP6，构建了重组胶原蛋白表达载体，然后筛选高效表达的宿主菌，检测重组质粒的不稳定性以及对重组菌培养条件进行优化。结果表明，重组胶原蛋白在Rosetta（DE3)中表达量相对最高，重组质粒稳定性也较高。较佳的表达条件为接种量4%或5%，37℃、200 r/min培养至OD600 nm为0.7时，加入0.5 mol/L IPTG，培养5 h，并且纯化的重组蛋白与小鼠抗人COL6A2的单克隆抗体有特异性反应。
以Ⅵ型人膠原蛋白A2鏈基因為模闆，PCR擴增得到目的基因CP6，構建瞭重組膠原蛋白錶達載體，然後篩選高效錶達的宿主菌，檢測重組質粒的不穩定性以及對重組菌培養條件進行優化。結果錶明，重組膠原蛋白在Rosetta（DE3)中錶達量相對最高，重組質粒穩定性也較高。較佳的錶達條件為接種量4%或5%，37℃、200 r/min培養至OD600 nm為0.7時，加入0.5 mol/L IPTG，培養5 h，併且純化的重組蛋白與小鼠抗人COL6A2的單剋隆抗體有特異性反應。
이Ⅵ형인효원단백A2련기인위모판，PCR확증득도목적기인CP6，구건료중조효원단백표체재체，연후사선고효표체적숙주균，검측중조질립적불은정성이급대중조균배양조건진행우화。결과표명，중조효원단백재Rosetta（DE3)중표체량상대최고，중조질립은정성야교고。교가적표체조건위접충량4%혹5%，37℃、200 r/min배양지OD600 nm위0.7시，가입0.5 mol/L IPTG，배양5 h，병차순화적중조단백여소서항인COL6A2적단극륭항체유특이성반응。
Designed primers to obtain the target gene (CP6) fragment through PCR,the template contained the protein polypeptide gene of Ⅵ type human collagen,and constructed the recombinant plasmid,and then screened the host bacteria of high efficient expression,detected the instability of recombinant plasmid,as well as optimized the culture conditions.The results showed that the best host bacterium was Rosetta (DE3) and the recombinant plasmid has higher stability. The optimal expression conditions were inoculum of 4% and 5%,37℃,200 r/min,cultured to OD600 nm 0.7 and added 0.5 mol/L IPTG to the culture medium,and then cultured 5 h. Western blotting showed that the expressed protein could specifically bind with human monoclonal antibody COL6A2.
