C型产气荚膜梭菌β2毒素基因的克隆与表达

利用PCR技术，从C型产气荚膜梭菌染色体基因组中扩增了0．72kb的β2毒素基因，将纯化的PER产物与载体pGEM—T连接，转化至受体菌JM109中，经NcoI／Bam HI和Bam HI／Eco RI酶切鉴定及核苷酸序列测定证实，重组质粒pXCPB2中含有陡毒素基因。随后用Nco I／Bam HI酶切质粒pXCPB2,回收β2毒素基因片段，插入到事先经同样酶切处理的载体pET-28c中相应酶切位点，构建了表达质粒pETXB2，经Nco I／Bam HI和Nco I／Hind Ⅲ／Bam HI酶切鉴定及核苷酸序列测定证实，表达质粒含有陡毒素基因且基因序列和阅读框架正确。重组菌株BL21（DE3）（pETXB2）表达产物经ELISA检测和SDS—PAGE分析，重组菌株表达的β2毒素蛋白能够被如毒素抗体识别，其表达量占菌体总蛋白相对含量的13．26％。

Cloning and Expression of Beta2- toxin Gene from Clostridium Perfringens Type C

Beta2-toxin gene was amplified from chromosomal DNA of Clostridium perfringens type C by polymerase chain reaction(PCR).PCR product was inserted into vector pGEM-T directively.The cloned recombinant plasmid pXCPB2 possesses positive nucleotide sequence of beta2-toxin.A 0.72kb beta2-toxin gene fragment was cleaved with restriction endonucleases NcoI/BamHI from plasmid pXCPB2,and then inserted into an expression vector pET-28c which cleaved with NcoI/BamHI by blunt-end ligation.The recombinant expression plasmid pETXB2 was studied in detail by restriction endonucleases analysis and nucleotide sequencing.The results showed that the recombinant expression pETXB2 possessed a positive beta2-toxin gene sequence and reading frame.BL21(DE3)(pETXB2) could produce beta2-toxin and the expressed products were recognized by beta2-toxin antibodies,and the expression level of the beta2-toxin proteins were about 13.26% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.