抗菌肽CP10A在大肠杆菌中的融合表达

CP10A是一种由抗菌肽Indolicine经过序列改造，且对多数革兰氏阳性病源细菌具有较强抗菌活性的多肽序列。本研究根据已报道的CP10A氨基酸序列，兼顾大肠杆菌密码子偏好性，设计CP10A的核苷酸序列，利用PCR技术合成相应的DNA序列，后克隆构建重组表达载体pET32a（+）-CP10A，转入大肠杆菌AD494菌株。经IPTG诱导表达和15% SDS-PAGE电泳检测后发现产物以包涵体形式存在，且融合表达量占总蛋白的50%。在变性条件下经Ni-NTA亲合柱层析及复性，最终获得了较高纯度的可溶性重组蛋白。本研究首次实现了CP10A抗菌肽在大肠杆菌中的融合表达，为进一步研究其生物学活性及应用奠定了一定的基础,同时也为研究抗菌肽表达提供了一种方法。

Fusion Expression of Antimicrobial Peptide CP10A in E.coli

CP10A, an anionic antimicrobial peptide designed from Indolicine, has a strong resistance to Gram-positive bacterial, which usually cause clinical infections. The DNA sequence of this 13 amino acids peptide was designed according to the reported CP10A peptide sequence and synthesized by PCR amplification. The sequence was cloned into the E.coli expression vector pET32a (+) and constructed the recombinant expression vector pET32a (+)-CP10A. The recombinant plasmid was transformed into E.coli AD494 and induced with IPTG. The Expression product was detected via 15% SDS-PAGE. The fusion protein was about 50% of total cellular protein and expressed as inclusion bodies. We got the pure fusion expressed CP10A protein via degeneration, Ni-NTA affinity chromatography and refolding of the complex. The purity of the refolding protein was more than 95%. It could be of great value for providing some basis to study biological activities of CP10A. It also provides some approach to study the expression of antimicrobial peptides.