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6本文以华北大黑鳃金龟Holotrichia oblita中肠为材料,依据Stratagene公司文库构建试剂盒方法,构建其中肠cDNA表达文库,该文库滴度为1.9×106pfu/mL,重组率为99.97%。依据现代免疫学原理,利用棉铃虫Helicoverpaarmigera围食膜蛋白多克隆抗体筛选文库,得到两个编码华北大黑鳃金龟围食膜蛋白的cDNA克隆Ho-Peritrophin1与Ho-Peritrophin2,其cDNA长分别为2385bp和1633bp,在polyA末端上游各有3个多聚腺苷酸信号序列AATAAA,最长开放阅读框(ORF)分别编码729个和477个氨基酸,与粉纹夜蛾Trichoplusia niCBP2(chitin bindingprotein2)的相似性最高,分别为21.9%和19.1%。结构域分析表明,Ho-Peritrophin1与Ho-Peritrophin2分别具有9个和6个几丁质结合功能域,只含有较少的O-糖基化位点,不含有类粘蛋白结构域。胰蛋白酶和胰凝乳蛋白酶对两种蛋白的作用位点主要位于几丁质结合功能域(chitin binding domain,CBD)内部,而因受几丁质结合功能域保护,这两种蛋白能够抵抗这些蛋白酶的降解。与正常CBD比较,这两种蛋白C端的CBD只含有4个Cys,只在第1与第3、第4与第5个Cys之间形成两对二硫键,缺少由第2与第6个Cys形成的二硫键。推测其N端还应包括信号肽序列和几丁质结合功能域的未知序列。
In this paper, the midgut cDNA expression library of Holotrichia oblita was constructed according to Stratagene’s library construction kit. The titer of this library was 1.9 × 106pfu / mL and the recombination rate was 99.97%. According to the principle of modern immunology, cDNA clones Ho-Peritrophin1 and Ho-Peritrophin2 were obtained by screening the library with Helicoverpa armigera polyclonal antibody against Helicoverpa armigera, and two cDNA clones encoding Ho-Peritrophin1 and Ho-Peritrophin2 were obtained. The cDNA lengths were 2385bp And 1633bp respectively. Three polyadenylation signal sequences (AATAAA) were found upstream of polyA, and the longest open reading frame (ORF) encoded 729 and 477 amino acids respectively, which was similar to Trichoplusia niCBP2 (chitin binding protein 2) Sex highest, respectively 21.9% and 19.1%. Domain analysis showed that Ho-Peritrophin1 and Ho-Peritrophin2 have 9 and 6 chitin binding domains, respectively, with fewer O-glycosylation sites and no mucin-like domains. The sites of action of trypsin and chymotrypsin on the two proteins are mainly located inside the chitin binding domain (CBD), which are protected by these chitin-binding domains Protease degradation. Compared to the normal CBD, the C-terminal CBDs of these two proteins contained only four Cys, forming two pairs of disulfide bonds only between the 1st and 3rd, 4th and 5th Cys, lacking the second and the sixth Cys disulfide bond formation. It is speculated that the N-terminal should also include the unknown sequence of signal peptide and chitin binding domains.