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Biochemical Characterization of Insect Intestinal Mucin HaIIM72from Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae)

Author: ZhangXiaoGang
Tutor: GuoWei
School: Agricultural University of Hebei
Course: Biochemistry and Molecular Biology
Keywords: Helicoverpa armigera Peritrophic Membrane HaIIM72 Expressed in insectcell lines Biochemical characterization analysis
CLC: S435.622
Type: Master's thesis
Year: 2013
Downloads: 8
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Abstract


The Helicoverpa armigera (Hübner) is an important Lepidopteran pest in the world andcan damage more than200different sorts of plants including cotton, wheat, broomcorn,tomato and so on. Insects midgut cells secrete an unique noncellular structure that lines thedigestive tract called peritrophic membrane(PM).It has muitiple physiological functionsand can protect the digestive tract from physical damage or microbial infections, eveninactivate ingested toxins.Insect intestinal mucin(IIM) is an important protein constituentof the peritrophic membrane.To characterize the HaIIM72in vitro, HaIIM72was designed to express in insect celllines, the haiim72was obtained by PCR method and cloned into pFastBac.Therecombinant plasmid was transformed into E. coli DH10Bac for getting recombinantbacmid-haiim72.Using the liposome method,the recombinant bacmid was transfectedinsect cell lines sf9.After72h, the virus was harvested in the media of cell.For amplifyingthe virus,P1infected Sf9for one time and P2was got. HaIIM72protein was expressed inBTI-Tn-5B1-4(HighFive).HaIIM72was detected by Western blot analysis with antibodiesto His-Tag.The results showed the protein had expressed successfully about260kDa and itwas predicted84.2kDa.After binded with regenerated chitin，HaIIM72was solubilized from the bound chitinby1%Calcoflour or by2%SDS in the presence of5%β-mercaptoethanol.It indicatesHaIIM72can strongly bind chitin in the network of peritrophic membrane. TheO-glycosylation analysis found the threonine as a potential O-glycosylation sit possessamino acid residues14.98﹪from76to251in mucin domain of HaIIM72.Using theO-glycosidase to treat HaIIM72,a reduction band from HaIIM72was shown apparentlyabout180kDa.However,in the presence of O-glycosidase and Trypsin, HaIIM72wasdegraded.The result has been proved that O-glycosylation is helpful to resist digestion ofprotease for HaIIM72.Cysteine-rich domains are common in mucins and have beensuggested to cause oligomerization of mucins by disulfide bonding.In our test, using theDTT to treat HaIIM72,has demonstrated that disulfide cross-linking bonds of cysteine-richpossessing6.19﹪were involved in maintaining a digestive protease-resistant structure.It isremarkable that the HaIIM72can be degraded by enhancin.So this study may contribute tocontrol H.arnigera with the HaIIM72as a target in future.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Crop pests and diseases and their prevention > Economic crop pests and diseases > Fiber crop pests and diseases > Cotton pests and diseases > Insect pest
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