白喉毒素基因序列染色体整合质粒的构建

白喉毒素基因序列染色体整合质粒的构建
方悦群;王春晖;王涛;何星;崔萱林;谢贵林;王秉瑞
【期刊名称】《中华微生物学和免疫学杂志》
【年(卷),期】2001(021)003
【摘要】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。

%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.【总页数】4页(P312-315)
【作者】方悦群;王春晖;王涛;何星;崔萱林;谢贵林;王秉瑞
【作者单位】兰州生物制品研究所第一研究室;兰州生物制品研究所第一研究室;兰州生物制品研究所第一研究室;兰州生物制品研究所第一研究室;兰州生物制品研究所第一研究室;兰州生物制品研究所第一研究室;兰州生物制品研究所第一研究室【正文语种】中文
【中图分类】R37
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