大熊猫肠道菌抗生素抗性菌转座酶的基因克隆和表达

Cloning and expression of transposase gene of plasmid DNA from antibiotic-resistance bacteria in intestine of giant pandas

对大熊猫肠道大肠杆菌抗生素抗性质粒pAm08CD7339全序列的生物信息学分析结果表明,该质粒中存在一个编码转座酶的DNA片段,两条DNA链均可编码转座酶,其编码区长度分别为717 bp和600 bp,编码238个和199个氨基酸残基(分别命名为Transposase-238和Transposase-199).以pAm08CD7339质粒DNA为模板,利用PCR方法对2个转座酶基因进行扩增,构建相应表达质粒,转化不同大肠杆菌菌株进行表达,结果表明:Transposase-199可以在E.coli BL21(DE3)中表达,而Transposase-238则只能在E.coli BL21 Rosetta(DE3)中表达.它们表达产物几乎全是包涵体.

Bioinformatics analysis of plasmid pAm08CD7339 DNA of antibioticresistance bacteria in intestine of giant pandas from Chengdu Giant Panda Breeding Research base was analyzed. The results showed that there was DNA encoding transposase in the plasmid pAm08CD7339; the two genes were located in the same DNA fragment with reverse orientation. These two transposase were 600 bp and 717 bp in length, encoding peptides of 200 and 239 amino acids, respectively （named transposase199 and transposase238 respectively）. Utilizing the plasmid pAm08CD7339 DNA as template,the ORF DNA fragments of these two transposase genes were further amplified by PCR and inserted into pET28a to test the heterogenic expression in E.coli BL21 （DE3） and E.coli Rosatte（DE3）.The results showed thatTtransposase199 could express in E.coli BL21（DE3）, and transposase238 only expressed in Rosatte （DE3）. However, the expressed transposases were exclusively resident in inclusion body.