关键词: 华北大黑鳃金龟, 中肠, cDNA表达文库, 围食膜蛋白, 序列分析

Abstract: Peritrophic membrane (PM) proteins of a coleopteran insect Holotrichia oblita were studied by constructing a H. oblita larval midgut cDNA expression library with the cDNA Synthesis Kit of Stratagene Company and screening with a PM protein polyclonal antiserum from Helicoverpa armigera according to the theory of modern immunology. The titer of the primary cDNA expression library was 1.9×106 pfu/mL and the recombination rate was 99.97%. Two positive cDNA clones named Ho-Peritrophin1 and Ho-Peritrophin2, with the sizes of 2 385 and 1 633 bp, were screened and sequenced from the library, respectively. The longest open reading frame of Ho-Peritrophin1 and Ho-Peritrophin2 coded for 729 and 477 amino acids respectively, both of which showed the highest similarity to CBP2 of Trichoplusia ni, though their similarities to the latter were only 21.9% and 19.1%, respectively. Ho-Peritrophin1 and Ho-Peritrophin2 contained nine and six chitin binding domains (CBDs) respectively with a little O-linked glycosylation sites and no mucinlike domain was found in the two protein sequences. The cleavage sites of trypsin and chymotrypsin are mainly located inside CBDs of the Ho-Peritrophin1 and Ho-Peritrophin2, but the two proteins are protected by the intradomain disulfide bonds, so they can resist the enzymes and exert physiological function in the midgut. Compared with the normal CBD, the CBD of carboxylic terminal of the two proteins contains only four cysteines, which form two pairs of disulfide bonds between the 1st and 3rd cysteines and the 4th and 5th cysteines, lacking the one between the 2nd and 6th cysteines. It is inferred that there are one signal peptide and unknown CBDs in the N end of Ho-Peritrophin1 and Ho-Peritrophin2 respectively and these require further study.

Key words: Holotrichia oblita, midgut, cDNA expression library, peritrophic membrane (PM) proteins, sequence analysis