title: [Cloning of alpha-bungarotoxin gene and its prokaryotic expression as a non-fusion protein].
abstract: On the basis of the reported amino acid sequence of alpha-bungarotoxin (alpha-BGT), DNA sequence of alpha-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized. A plasmid carrying the coding region of alpha-BGT was obtained by primer extension, PCR and ligation with pMD-18-T. The target fragment was digested with Xba I and EcoR I, recovered and ligated with pET28a(+). The resultant expression vector was transformed into BL21 (DE3), BL21 (DE3) Codon plus, and BL21 (DE3) plysS, respectively. Recombinant alpha-BGT was expressed in BL21 (DE3) and was analyzed by 15% Tris/tricine SDS-PAGE. The result showed that the recombinant protein, mostly found in inclusion bodies, accounted for 11.98% of the total bacterial lysate. The expression capacity could be increased to 16.28% by optimizing expression conditions. Western blotting results showed that the expressed protein had similar immunogenicity with the natural alpha-BGT protein purified from the venom of Krait Bungarus spp. In vivo toxicity assay of purified and renatured proteins in mice showed that LD50 was about 1.28 microg/g.
authors.affiliation: College of Animal Science and Technology, Sichuan Agricultural University, Ya'an 625014, China.
authors.forename: Yan-Chun
authors.initials: YC
authors.lastname: Hu
authors.forename: Nai-Sheng
authors.initials: NS
authors.lastname: Zhang
authors.forename: Jun-Liang
authors.initials: JL
authors.lastname: Deng
authors.forename: Zhi-Cai
authors.initials: ZC
authors.lastname: Zuo
authors.forename: Yu-Hui
authors.initials: YH
authors.lastname: Liao
authors.forename: Hong-Sheng
authors.initials: HS
authors.lastname: Ouyang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002038:Bungarotoxins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data
chemical_list: D002038:Bungarotoxins; D017931:DNA Primers