Study On Fusion Cloning, Expression Of Cry1Ac Gene From Bacillus Thuringiensis With A Neurotoxin Gene Hwtx-Ⅰ And Bioassay Analysis Of Fusion Protein

At present, researches on appliance of Bacillus thuringiensis are mainly about constructing more toxic recombinant protein and recombinant strain by the technology of genetic engineering such as site-directed mutation, constructing fusion gene, constructing transgenic plants of Bt toxin gene and so on. In order to construct the recombinant strain with higher toxicity and broader activity spectrum, this work construct the fusion gene of crylAc gene and neuron toxin gene to enhance the toxicity of crystal proteins.Appling the way of Biology informatic to analyze the sequence homeology of crylAc genes, and amplifying the crylAc gene with the upstream promoter region from B. thuringiensis strain 4.0718 with primers Ac-F and Ac-R. Then designing and synthesizing the sequence of hwtx- I gene and Enterokinase site with partial codon of B. thuringiensis, and ligating the sequences synthesized with phosphorylation and annealing ligation. Amplifying the hwtx- I gene and Enterokinase site fragment with primers Ltx-F and Ltx-R, then ligating the two PCR fragments above and amplifying the fusion gene fragment with primers Ac-F and Ltx-R. Through a series of digestions and subclonings, the fusion gene with the upstream promoter region and downstream terminator region was cloned in the shuttle vector pHT304, the resulting plasmid was named pXL43, then the expression vector pXL43 was electro-transformed in the B. thuringiensis acrystalliferous strain XBU001 and B. thuringiensis strain 4.0718 and the recombinant strains were named XL002 and XL004 respectively.SDS-PAGE analysis showed that the 138kD fusion proteins were expressed in the B. thuringiensis acrystalliferous strain XBU001 and the qualified analysis indicated that the expressed proteins were accounted up...