牛Cathelicidin BMAP-28基因的克隆及原核表达

根据GenBank中公布的牛BMAP-28基因mRNA序列设计引物,利用RT-PCR技术从牛骨髓总RNA中扩增出BMAP-28基因片段,将其克隆到pMD-18载体上,通过PCR、酶切和测序分析鉴定,获得重组克隆载体pMD18-T-BMAP28.以重组质粒为模板,扩增BMAP-28基因的去信号肽片段,转入原核表达载体PE...

Cloning and prokaryotic expression of Cathelicidin BMAP-28 gene in bovine

A pair of primers were designed according to the mRNA sequence of BMAP-28 gene from GenBank.Then the total RNA was extracted from bovine bone marrow and BMAP-28 gene was amplified and cloned into pMD-T vector by RT-PCR.The recombination cloning vector pMD18-T-BMAP28 was constructed after identification by PCR,enzyme digestion and sequencing.Then,the BMAP-28 gene without signal peptide fragment was amplified by using the recombinant plasmid as template and was transferred into PET28a expressive vector.The recombinant expressive plasmid was transformed into Rosetta and induced to express by IPTG,and the SDS-PAGE and Western-blot were used to detect expression products.The results showed that the sequence of the recombination cloning vector had a base sequence difference from the previously published BMAP-28 sequence,and this lead to amino acid substitutions.After induced by IPTG,E.coli containing the recombinant plasmid PET28a-BMAP28 expressed as expected fusion protein,which had bacteriostatic activity.