Wang Dong-Ning, Liu Jie-Wu, Yang Guan-Zhen, Zhang Wei-Jie, Wu Xiang-Fu
College of Life Science and Technology, Shanghai Jiao Tong University, Shanghai, China.
Mol Biotechnol. 2002 May;21(1):1-7. doi: 10.1385/MB:21:1:001.
In this article we report the cloning and expression of a cDNA encoding Tachypleus anti-lipopolysaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gram-negative bacterial endotoxins. First, two degenerate primers were designed based on the sequence homology of anti-LPS factors purified from different species of horseshoe crab. The total RNA was extracted from amebocytes of Tachypleus tridentatus. The cDNA was then obtained by using the RT-PCR methods. Second, the cDNA of Tachypleus anti-LPS factor (TALF) was expressed in Bombyx mori larvae using baculovirus expression system, which showed a yield of up to 600 mg/L. Last, we determined the biological activity of the recombinant proteins by LPS neutralization assay and bacteriostatic assay in vitro.
在本文中，我们报道了一种编码中国鲎抗脂多糖（LPS）因子的cDNA的克隆与表达，该因子有望作为革兰氏阴性菌内毒素共同核心亚基的潜在抑制剂。首先，根据从不同种类鲎中纯化得到的抗LPS因子的序列同源性设计了两条简并引物。从中国鲎的血细胞中提取总RNA。然后通过RT-PCR方法获得cDNA。其次，利用杆状病毒表达系统在中国家蚕幼虫中表达中国鲎抗LPS因子（TALF）的cDNA，产量高达600 mg/L。最后，我们通过体外LPS中和试验和抑菌试验测定了重组蛋白的生物活性。
