| Cobra neurotoxin (NT) has huge therapeutic potential in medicine as anesthetics or antidotes. However, production of natural neurotoxin is a little and it is difficult to isolate. That sharply limited the application of neurotoxin in medicine. Therefore, the objective of current study is to express, isolate, and purify the functional Guangxi cobra cobortoxin (CT) and Phospholipase A2(PLA2) in insect expression system in vitro.The technologies of segmented-PCR clone and reverse transcription (RT) PCR were employed to obtain the coding sequences of CT and PLA2. The sequences of CT and PLA2with or without His tag were inserted to the downstream of promoter PH and p10in baculovirus shuttle vector. The results showed that recombinant plasmids with and without His tag were pD-H-CT, pD-H-PLA2, pD-HCT-HPLA2and pD-CT, pD-PLA2, pD-CT-PLA2, respectively. Six shuttle vectors were transfected into DH10Bac and the antibiotic (gentamicin, kanamycin and tetracycline), blue-white screening, PCR and sequencing were used to identify the positive virus recombinant vector. The results showed that6virus recombinant vectors, BM-CT, BM-PLA2, BM-CT-PLA2, BM-HCT, BM-HPLA2, BM-HCT-HPLA2, with target genes were contructed successfully.The six virus recombinant plasmids and empty virus plasmid were transferred to Sf9cell using Cellfectin Ⅱ. The positive virosomes were identified by PCR. The SDS-PAGE was used to test the proteins expression of target genes. The results showed that6target proteins were tested in supernatant of cell lysis, indicating those proteins were soluble. The CT and PLA2proteins with and without His tag were isolated and purified using electro-elution and Ni affinity purification. Mice writhing experiment was employed to test the activity of CT and PLA2proteins. The results indicated that CT with and without His tag have significantly better analgesic effect than that PLA2(P<0.05). There are no significant differences between PLA2with and without His tag (P>0.05). |
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