质粒载体pUC19在头孢哌酮抗性基因(CPZ^r)克隆中的应用探讨

Application of vector plasmid pUC19 to cloning of cefoperazone resistance gene carried on pFC

目的 :研究质粒pUC19是否为头孢哌酮抗性基因 (CPZr)分子克隆的理想载体。方法 :氯化钙法将质粒pUC19转化至工程菌E .coliDH5获得pUC19转化菌 ;采用试管二倍液体稀释法进行pUC19转化菌对 2 5种抗生素的敏感性测定 ;鸟枪法克隆耐药质粒pFC片段到载体pUC19获得含有CPZr 的重组质粒。结果 :pUC19转化菌对氨苄青霉素高度耐药 (MIC >2 5 6 μg/ml) ,对第一代头孢菌素中度耐药 ,对第二代头孢菌素中的头孢孟多耐药 ,而对头孢呋新敏感 ,在第三代头孢菌素中只对头孢哌酮耐药 (MIC ,6 4μg/ml) ,对其他第三代头孢菌素及 β 内酰胺酶抑制剂均高度敏感 ,同时敏感性测定结果还发现pUC19转化菌对氟喹诺酮类和氨基糖甙类抗性素均敏感。pUC19作为载体克隆到含有头孢哌酮抗性基因 (CPZr)的重组质粒少 ,克隆效率低 ,未能达到预期的目标。结论 :pUC19不适合作为CPZr 的克隆载体。

E.coliHX88108 strain which demonstrated high level resistance to cefoperazon (CPZ) was isolated from a severely infected patient in 1988. Five plasmids coexisting in the strain were designated pFC, pFT1, pFT2, pFT3 and pFX, respectively. Four plasmids except pFX conferred CPZ resistance. In order to search an ideal plasmid as the vector in cloning of cefoperazone resistance gene (CPZ r) from resistance plasmid pFC, we studied plasmid pUC19 which is used widely in molecular cloning. pUC19 was transformed into E.coliDH 5 prepared using calcium chloride. MICs of 25 antibiotics against E.coli pUC19 transformant were determined by the broth dilution method with Mueller Hintion broth, the results showed that pUC19 transformant is resistant to ampicillin, staphcillin, amoxycillin, cefradine, cefomandole, cefazolin and cefoperazone (MIC, 64μg/ml), but is susceptible to cefuroxime, ceftazidine, ceftriaxone, nofloxacin and aminoglycosides. This suggests that pUC19 could encode CPZ resistance. Cloning of CPZ r from pFC with pUC19 as the vector was performed. The fragments of pFC generated by the partial digestion with Sau3AI were inserted into BamHI site of pUC19 and transformed into E.coliDH 5. Seven clones containing CPZ r (PFL11, PFL25……) from the transformants selected on SOB agar plates containing 75 μg of ampicillin per ml and 40μg of CPZ per ml. Meanwhile, the results revealed that self cycling recombinant DNA pUC19 transformants could grow on the selecting SOB agar plates. This led to low efficiency of CPZ r cloning and trouble in research work. According to pUC19 encoding resistance to CPZ and low efficiency of CPZ r cloning in which pUC19 was selected as the cloning vector, pUC19 is unsuitable for the ideal vector of CPZ r cloning.