重组血管活性肠肽的制备及鉴定

为利用基因工程技术获得重组血管活性肠肽(vasoactive intestinal peptide,VIP)，根据大肠杆菌的密码偏好性，设计并人工合成编码28个氨基酸的VIP基因。克隆到表达载体PTWIN，构建重组质粒PTWIN-VIP，转化宿主菌E. coli Strain ER2566，构建表达工程菌。实现由重组VIP，内含肽与纤维素结合域（cellulose binding domain, CBD）组成的融合蛋白表达。融合蛋白经几丁质亲和层析纯化，通过改变温度和缓冲液PH值切割融合蛋白，获得目的多肽。所得的多肽经质谱测定分子量结果与理论值相符。生物活性分析表明，重组VIP能显著降低急性炎症小鼠血清中抵抗素的水平，发挥抗炎作用。重组VIP的制备及其抗炎活性的鉴定为其深入开发奠定了基础。

Preparation and Identification of Recombinant Vasoactive Intestinal Peptide

In order to prepare the recombinant vasoactive intestinal peptide ( VIP) using intein mediated rapid purification system, the cDNA encoding the recombinant VIP was designed and synthesized according to the preference of E. coli, and then was cloned into the expression vector PTWIN. The recombinant plasmid PTWIN-VIP was transformed into expression host E. coli strain ER2566.The fusion protein consisting of the recombinant VIP, intein and chitin binding domain was expressed and purified by chitin affinity chromatography. The target peptide was released from the fusion protein by changing the temperature and the pH of the cleavage buffer. The molecular weight of the recombinant VIP was determined by the mass spectrometry and the results was conformity with the theoretical value. The preliminary bioactivity assay indicated that the recombinant VIP decreased the serum resistin levels significantly in LPS-induced acute inflammation. The preparation and the characterization of anti-inflammatory effects of the recombinant VIP layed the foundation for its further application.