摘要
为研究家蝇防御素融合蛋白对肿瘤细胞K562有抑制作用，在GenBank中检索家蝇defensin基因，设计特异引物，在家蝇幼虫组织中提取RNA，并通过PCR扩增defensin基因成熟肽部分（Maturedefensin，MD），将该基因与原核质粒表达载体pGEX．6P-1进行重组，构建家蝇抗菌肽重组蛋白pGEX-6P-1／MD。通过双酶切鉴定证明目的片段已稳定融合到载体pGEX-6P-1中并将其转化至BL21（DE3）感受态细胞中，经25℃IPTG诱导在大肠杆菌E．coliDH5u中表达。用GST—Sefinose亲和层析纯化蛋白，15％SDS—PAGE分析鉴定，可见相对分子量为约30kDa的特异性目的条带。运用琼脂孔穴平板扩散法测定表达产物的活性，得知纯化后的重组融合蛋白pGEX-6P-1／MD对大肠杆菌生长有一定的抑制作用，并首次证明家蝇防御素融合蛋白pGEX．6P．1／MD对肿瘤细胞K562有抑制作用。家蝇抗菌肽融合蛋白pGEX-6P-1／MD被成功克隆和表达，证明纯化融合蛋白pGEX-6P-1／MD对肿瘤细胞K562有抑制和损伤作用，这为以后研发蝇防御素抗菌肽药物提供了依据。
The genic sequence of defensin gene （GenBank No. AY260152） was used to design specific primers for cloning mature defensin （MD）. Total RNA was extracted from Musca domestica larva and MD was then amplified by PCR. PCR product was cut with BamHl and XhoI and put into pGEX-6P-I vector. Recombinant expression plasmid pGEX-6P-1/ MD was constructed and target fragment was put into pGEX-6P-1 vector stably through double digestion. The resultant recombinant plasmid （pGEX-6P-1/MD） was transformed into BL21 （DE3）. The fusion protein was expressed after IPTG induction at 25~C , and purified by GST-Sefinose affinity chromatography. SDS-PAGE results showed that recombinant defensin was produced after IPTG induction. Antibacterial experiments indicated that the fusion recombinant protein had certain inhibitory effect on the growth of E. coli on tumor ceils of K562 at the first time. Plasmid pGEX-6P-1/MD protein was cloned and expressed successfully, which testified the protein had inhibitory and damaging effect on K562 tumor cells. All that aforesaid provided a basis for the researches of antibacterial peptide drugs from M. domestica defensin.
出处
《寄生虫与医学昆虫学报》
CAS
2012年第4期228-234,共7页
Acta Parasitologica et Medica Entomologica Sinica
基金
山西省高校科技研究开发项目（200613011）
山西省科技攻关项目（2006031087-04）
太原市科学技术发展计划项目（081049）
关键词
DEFENSIN
抗菌肽
基因克隆
原核表达
亲和层析
肿瘤细胞
Defensin
Antibacterial peptide
Gene clone
Prokaryotic expression
Affinity chromatography
Tumour cells
