抗鼻咽癌质粒pFY转化和菌种筛选的研究

Study on transformation of anti-nasopharyngeal carcinoma plasmid pFY and bacterial strains screening

目的筛选携带抗鼻咽癌质粒pFY的稳定高产菌株。方法以CaCl2法制备大肠杆菌JM109感受态,将抗鼻咽癌质粒pFY转化JM109感受态,对琼脂平板上获得的菌落进行筛选,选出符合标准的单菌落为菌种,进行菌种稳定性实验。用质粒提取试剂盒检测质粒含量。将抗鼻咽癌质粒pFY转染到细胞CNE-2中,四氮唑蓝(MTT)比色法观察转染试剂及质粒载体对细胞生长增殖的影响。结果筛选得到菌株的培养液中质粒DNA含量为30mg/mL,超螺旋DNA比例为92%。经电泳和酶切鉴定,该菌株的50子代所携带质粒与原代一致。质粒pFY对CNE-2细胞株生长有明显的抑制作用。结论成功筛选出携带抗鼻咽癌质粒pFY的稳定高产菌株,为大批量制备临床应用级质粒奠定了基础。

Objective To screen the stable high-producing strains carrying anti-nasopharyngeal carcinoma（NPC） plasmid pFY. Methods Competent E. coli JM109 was prepared by the CaCl2 method and transformed with anti-NPC plasmid pFY. The bacterial colonies obtained from the agar plate were screened for selecting the single colony conforming to the standards as the bac- terial strain and conducting the stability test. The plasmid content was detected by the plasmid extraction reagent kit. Anti-NPC plasmid pFY was transfeeted into nasopharynegal carcinoma cell line CNE-2. The influence of transfection reagent and the plasmid vector on the cell proliferation was detected by MTT. Results The DNA concentration of plasmids in the culture solution of bacte- rial strain obtained by screening was 30 mg/mL. The proportion of supercoiled DNA was 92 %. The identification of electrophoresis and restriction enzymes showed that the plasmids harbored in the 50th progeny of this strain were same as those in the primary. Plasmid pFY had the evident inhibiting effect on the growth of CNE-3 cell line. Conclusion The stable high-producing strains of E. coli carrying anti-NPC plasmid pFY is successfully screened out, which lays the foundation for large-scale preparation of plasmid pFY for clinical utility.