摘要
目的将α防御素1(HNP 1)基因与J链基因重组为一种新的杀菌肽分子J HNP 1,观察J HNP 1在细胞内的表达及分泌情况。方法采用聚合酶链反应(PCR)方法分别从pCH J质粒和pB abeN eo HNP 1质粒中扩增出J链和HNP 1;进行重组PCR,获取J HNP 1 DNA片段,并插入哺乳动物细胞表达载体pcDNA 3.1()/M yc H isC中;用脂质体转染法将此重组真核表达载体导入CO S 7细胞,然后应用蛋白质免疫印迹法(W estern b lot)检测H is tag标签基因。结果用抗H is抗体检测到细胞可溶性蛋白及培养上清在约24 ku处均有强反应条带显示,其大小与预测相符;对照组未见相应条带显示。结论采用W estern b lot检测到新的杀菌肽分子J HNP 1在细胞内得到表达并分泌到细胞外,为进一步研究J HNP 1杀菌肽的抗菌活性奠定了基础。
Objective To recombine the α-defensin - 1 （HNP 1） and J chain cDNA fragment into a new germicidal molecule J - HNP - 1, and investigate its expression in the transfected COS - 7 cells. Methods J chain and HNP - 1 cDNA were amplified from the plasmids pCH - J and pBabeNeo HNP - 1 respectively, then the J -HNP - 1 DNA fragment was obtained by recombinant polymerase chain reaction （PCR）. The J - HNP - 1 fragments were inserted into pcDNA3.1 （-）/Myc - HisC, which subsequently was made to transfect the COS - 7 cell by lipid transfection method. The expression was confirmed by Western blot. Results Determination with His antibody, a dense band at 24 ku was displayed by the cell lysate and the cell culture supernatant in concordance with that of the predicted extent, but not in the control. Conclusion The new germicidal peptide J - HNP - 1 is found to be expressed intracellularly and could be secreted. The results lay the foundation for further study on the germicidal activity of J - HNP - 1.
出处
《中国危重病急救医学》
CAS
CSCD
北大核心
2006年第2期71-73,共3页
Chinese Critical Care Medicine
基金
国家自然科学基金面上项目(30100197)
关键词
重组聚合酶链反应
杀菌肽
J链
α-防御素-1
转染
recombinant polymerase chain reaction
antimicrobial peptide
J chain
α - defensin - 1
transfection
