[Cloning of alpha-bungarotoxin gene and its prokaryotic expression as a non-fusion protein].
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.
Hu
Yan-Chun
YC
College of Animal Science and Technology, Sichuan Agricultural University, Ya'an 625014, China.
Zhang
Nai-Sheng
NS
Deng
Jun-Liang
JL
Zuo
Zhi-Cai
ZC
Liao
Yu-Hui
YH
Ouyang
Hong-Sheng
HS
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
D002038:Bungarotoxins; D017931:DNA Primers