Cloning And Expression Of Venom Allergen Soli1 And Soli4 Genes In The Solenopsis Invicta

The imported red fire ant (Solenopsis invicta) is a new baneful species that appear for the first time in China.It is an aggressive, territorial ant that results in serious health, economic, and environmental impacts to the communities. For these reasons this insect was listed as one of the most aggressive and destructive imported pest worldwide. Anaphylaxis,even deaths,maybe induced by red imported fire ant stings because it's venom allergen is an extremely potent allergy-inducing agent. Up to now, the molecular mechanism of such kind of extremely potent allergy-inducing is unclear, which affects the effective therapies of the fearful anaphylaxis induced by red imported fire ant stings.Therefor, the work in this thesis include:The Solenopsis invicta venom allergens Sol i 1 gene and its fragment Sol i la encoding the truncated peptide without transmembrane region and Sol i 4 were amplified by RT-PCR and nPCR, and then were cloned into pET43.1a vector and got high-level expression. The purified recombinant proteins had good allergenic activity and immunotherapy potential.1 Amplification and sequence analysis of Sol i 1 and Sol i 4 gene cDNA fragmentsTotal RNA was extracted from mature red fire ant ergate, and the cDNA fragments with the expected size of 1050bp and 354bp was amplified by RT-PCR. The sequencing result and the reported cDNA sequences were compared and analyzed, indicating that Sol i 1 shared a high identity (99%) in nucleotides with the reported sequences, and Sol i 4 shared a high identity of 97% in nucleotides with the reported sequences.2 Bioinformatics analysis of Sol i 1 and Sol i 4Amino acid sequence of Allergen Sol i 1shared an identity of 99% with the reported ones, and an identity of 40% with allergen Vesg1 and venom phosphate enzyme Al respectively. Apart from sharing the same amino acid sequence with the reported allergen Sol i 4.01, Allergen Sol i 4 shared an identity of 97%, 96%, 88%,36% in amino acid sequence with the reported allergen Sol i 4.02, sol-i-4, Sol g 4 and Sol i 2 respectively.Mechanism of three proteins were studied using online server and biosoft, and the results indicated: Sol i 1 protein contained 346 aa with calculated molecular weight of 38344.7Da, theoretical pI of 8.67, atomic composition of C₁₇₂₃H₂₆₃₁N₄₆₃O₄₉₄S₁₂, and instability index of 32.88, which showed that Sol i 1 might be a stable protein. Sol i 4 protein contained 118 aa with calculated molecular weight of 13556.2Da, theoretical pI of 9.83, atomic composition of C₆₁₀H₁₀₂₀N₁₆₈O₁₆₄S₇. Instability index of 29.83, which showed that Sol i 4 might be a stable protein.Sol i 1 protein had three hydrophilicities, five a-helix and fourβ-sheet. Sol i 4 protein had two hydrophilicitis, three a-helix and sixβ-sheet. Sol i 1 protein had a Pancreatlipase like (Pancreatic lipase-like enzymes) domain, and a Lipases-serine active site. Sol i 1 is a lipase of the lipoprotein lipase superfamily, a member of pancreatic lipase homologous family. 3 Expression, purification and activity analysis of Sol i 1 and Sol i 4Sol i 1's fragment sol i la encoding the active peptide was amplified by nPCR. Sol i la protein contained 125 aa with calculated molecular weight of 13408.2Da, theoretical pI of 7.85, atomic composition of C₆₀₀H₉₂₃N₁₅₇O₁₇₈S₇ and instability index of 29.96, which showed that Sol i la might be a stable protein.Sol i 1, Sol i la and Sol i 4 were cloned into pET43.1a vecter to constructed the recombined plasmid pET43.1a- Sol i 1,pET43.1a- Sol i 1a andpET43.1a- Sol i 4. After being verified by RE digestion and sequencing, the recombinant plasmids were transformed into E.coli BL21(DE3) and induced by IPTG successfully. The results of SDS-PAGE and Western blot analysis showed that the recombinant fusion proteins were high-level expressed which contained an molecular weight of 99kD, 74kD and 74kD respectively. After being purified by affinity chromatograph, the purified recombinant proteins were used to inoculate rabbits for furtherly analyzing their allergenic activity and the potential in therapy. The rabbits' scratch test indicated that all of the three recombinant proteins had allergenic activities. The rabbits' protein skin test and the immunotherapy test indicated that these recombinant proteins could be used for both irritability diagnosis and immunotherapy.This work might be used for further study of molecular mechanism of the red fire ant venom allergen, affording a great of credible protein preparation for the diagnosis, therapy and prevention of the allergic by the red fire ant sting, and manufacturing special diagnosis and effective therapy productions.