CAJ | 학술논문

目的构建白喉杆菌染色体整合质粒，建立使外源基因置换白喉毒素基因的方法。方法 PCR方法，寡核苷酸合成方法及其他分子克隆技术。结果使用以上方法准确制备了所需特定碱基序列位置的2个与白喉毒素基因相关的染色体同源片段，插入pG+host5后又引入转录终止子和抗生素抗性基因，从而产生具有可接入外源基因并与白喉杆菌染色体进行双交换同源重组能力的质粒pLB23。用此质粒对白喉杆菌PW8进行了染色体整合，获得了其遗传变异株。结论所构建的整合质粒能够用于白喉杆菌染色体的同源重组，实验为将外源基因引入白喉杆菌染色体进行表达奠定了基础。
목적구건백후간균염색체정합질립，건립사외원기인치환백후독소기인적방법。방법 PCR방법，과핵감산합성방법급기타분자극륭기술。결과사용이상방법준학제비료소수특정감기서렬위치적2개여백후독소기인상관적염색체동원편단，삽입pG+host5후우인입전록종지자화항생소항성기인，종이산생구유가접입외원기인병여백후간균염색체진행쌍교환동원중조능력적질립pLB23。용차질립대백후간균PW8진행료염색체정합，획득료기유전변이주。결론소구건적정합질립능구용우백후간균염색체적동원중조，실험위장외원기인인입백후간균염색체진행표체전정료기출。
Objective　To construct a chromosome integrated plasmid of C.diphtheria and to establish a method by which some foreign genes could replace diphtherial toxin gene on the chromosome. Methods　Polymerase chain reaction, the synthesis of oligonucleotides and molecular cloning technique were used in the experiment. Results　Two homologous fragments, which contained the part of the toxin coding nucleic acid sequence, its promoter, SD sequence, signal peptide sequence, and the flanking sequence of 5′ end of the promoter were prepared by PCR after accurate selection of the sequence, and then the fragments were inserted into pG+host5 that was a thermosensitive plasmid with broad host range. Some restricted sites, a transcription terminator sequence and kan-resistance gene were introduced into the plasmid there by plasmid pLB23 was constructed. pLB23 could be used for the integration of some foreign genes into chromosome under control of diphthericum toxin operon by double crossover homologous recombination model. The homologous recombination occurred after pLB23s were transformed into C.diphthericum PW8 and a mutant strain was obtained. Conclusion　The constructed integration plasmids could be used for the homologous recombination of C.diphthericum chromosome and the experiment result reported provided a tool for expression of foreign gene in diphtheria.