Study On Construction Of Expression Vector Encoding Huwentoxin-â…  Gene Quadrivalent And Bacillus Thuringiensis Engineering Strains

The promoter of insecticidal crystal proteins (ICPs) gene cryl was a sporulation-dependant and activated in the phase of sporulation. Huwentoxin-I (Hwtx- I )is a peptide neurotoxin purified from the crude vemon secreted by the Chinese bird spider Seleocosmia huwena.lt is one of an inhibitor of acetylcholine receptor and can cut the signal express between nerve and muscle.It can act as one of a N-type Ca~2+ ion channel inhibitors. Synthetic gene monomer encoding recombinant hwtx — I in fragments using Bt's preference code,based on the known protein sequence.Phosphorylated the fragments and linked them.A linker with with nucleate endonuclease Spe I and Xba I sites and termination codon (TAG) was designed. The sites sequences selected in the linker were Bt preference code and they could link hwtx-I gene monomer into multimer by head to tail linking.The vector pUA19 eliminated the ORF of instecticidal crystal proteins cry1 Ac gene after double digested by BsaB I and Mun I ,and the promoter and the terminator of crylAc gene were remained.The linker was inserted in this site,and got a new vector pUKH1. pUKH1 double digested with Sal I and Spe I ,and reclaimed the larger fragment and linked to the smaller fragment which pUKHl double digested with Sal I and Xba I ,then got pUKH2 .Repeat the digest and link step on pUKH2 as pUKH1,we would get pUKH4.Double digesting plasmid pUKH4 with Sal I and Bam H I ,reclaiming the smaller fragment and insert into the E.coli and Bt shuttle vector pHT304 between Sal I and BamH I sites, got pXH4,the hwtx-I gene quadrivalent expression vector. pXH4 was transformated into Bacillus thuringiensis acrystalliferous strain XBU001 and wild type strain Bt4.0718. The interest protein expressed successfully in recombinate strains. The production of the quadrivalent in...