Cloning of anti-lPS factor cDNA from Tachypleus tridentatus, expression in Bombyx mori larvae and its biological activity in vitro.
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.
Wang
Dong-Ning
DN
College of Life Science and Technology, Shanghai Jiao Tong University, Shanghai, China.
Liu
Jie-Wu
JW
Yang
Guan-Zhen
GZ
Zhang
Wei-Jie
WJ
Wu
Xiang-Fu
XF
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects; D006737:Horseshoe Crabs / Q000235:genetics; D007447:Invertebrate Hormones / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D007814:Larva; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D018076:DNA, Complementary; D007447:Invertebrate Hormones; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; C034705:antilipopolysaccharide factor (Limulus)