Expression, Antibody Preparation, Bioactivity Tests And Application Researches Of Insect Neurotoxins

Insect neurotoxins have potential applicative value for agricultural insect pest control, but these kinds of natural neurotoxins are limited and difficult to extract. So far, there are some efficient expression systems for production of heterologous peptides and proteins, these heterologous expression systems help to obtain massive insect neurotoxins. Escherichia coli expression system is an apprehensive application heterologous genes expression system, overproduction of heterologous proteins in E. coli has emerged as a routine technique. Pichia pastoris expression system is an efficient eukaryotic expression system, many heterologous genes high-level expression in this system successfully.According to the natural mature amino acid sequence of three scorpion long-chain insect neurotoxin AaIT, BjαIT and LqhIT2 and one spider short-chain insect neurotoxin TX4(6-1), chemical synthesized the four full long mature neurotoxin genes based on the codon bias of P. pastoris. All the four synthesized insect neurotoxin genes were cloned to E. coli expression plasmid pET-30a(+) and P. pastoris expression plasmid. After transform these genes to E. coli, the transformants of these four insect neurotoxin genes were screened by PCR and all scorpion insect neurotoxin genes expressed were induced with IPTG. The three scorpion insect neurotoxin genes expressed in fusion proteins and were purified with Ni affinity columns. Antiserum of these three kinds long-chain scorpion insect neurotoxins were prepared after immunization BALB/c mice with purified fusion protein. The highest titer to AaIT come to 1:128,000, the highest titer to BjαIT come to 1:512,000 and the highest titer to to LqhIT2 was over 1:128,000. Total RNA was extracted from the highest titer mouse's spleen cells with fusion BjαIT protein immunization. The VH gene fragments and VL gene fragements were obtained by RT-PCR with total RNA respectively. The full long scFv fragments were obtained by link VH and VL with linker polypeptide DNA fragment.All four insect neurotoxin genes transformed into P. pastoris strain KM71, the transformants with different copies of neurotoxin genes were screened by PCR, and the high-level expression P. pastoris transformants of scorpion neurotoxin genes were selected by dot blotting with prepared antiserum respectively. Scorpion long-chain insect neurotoxin AaIT, BjαIT and LqhIT2 secretory expressed in P. pastoris were induced with methanol. After concentration, the bioactive test shows all these expressed scorpion long-chain insect neurotoxin have bioactivity through injection locusts or cockroaches.The signal peptide gene of BjαIT and synthesized BjαIT gene were cloned to Metarhizium anisopliae over-express plasmid successfully. After transformation the spores of M. anisopliae, the M. anisopliae transformants of BjαIT were selected with anti-herb plates, and the transfomants of BjαIT were identified by PCR template on the DNA of these M. anisopliae transformants.