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Pacifastin蛋白酶抑制剂在昆虫免疫与发育中起着重要作用。为了明确其在寄生蜂中的相关功能,本研究分别克隆获得编码丽蝇蛹集金小蜂Pacifastin蛋白酶抑制剂开放阅读框的cDNA序列nvpp-1和nvpp-2,序列长度分别为723和888 bp,分别编码240和295个氨基酸残基。预测结果表明,nvpp-1和nvpp-2推导氨基酸序列N端均含一个长度为17个氨基酸残基的信号肽序列。序列分析和进化树构建结果表明,NVPP-1和NVPP-2分别含有5个和4个典型的Pacifastin保守结构域,并与疑黑瘤姬蜂Pimpla hypochondriaca毒液蛋白CVP4聚为一类。实时荧光定量RT-PCR结果表明,nvpp-1和nvpp-2于该蜂雌蜂各组织中均发生转录,且在胸、腹部残体(解剖后腹部剩余部分)和毒器官中的转录水平较高;于毒器官中,其在羽化初期(0和1 d)转录水平较高,其转录水平显著降低。Western blot结果表明,NVPP-1和NVPP-2均只在毒液中被大量检出,在其他待测组织中均未被检出,而刚羽化时(0 d)其在毒液中含量较低。利用pET-28a(+)载体分别对nvpp-1和nvpp-2进行了原核表达,并对重组表达产物进行纯化。分别测定重组NVPP-1和NVPP-2对4种不同丝氨酸蛋白酶(胰蛋白酶、糜蛋白酶、蛋白酶K和弹性蛋白酶)的抑制效果,结果表明,重组NVPP-1和NVPP-2分别能显著抑制糜蛋白酶和胰蛋白酶活性。同时还分别测定了两种重组蛋白对寄主家蝇蛹血淋巴自身的酚氧化酶活性及原酚氧化酶激活反应的影响,结果表明,重组蛋白对家蝇蛹血淋巴原酚氧化酶激活反应亦有抑制效果,但其均不能显著影响血淋巴自身的酚氧化酶活性。综上所述,丽蝇蛹集金小蜂毒液中含有Pacifastin蛋白酶抑制剂NVPP-1和NVPP-2,分别为糜蛋白酶抑制剂和胰蛋白酶抑制剂家族成员,均能显著影响寄主家蝇蛹血淋巴原酚氧化酶激活反应,从而削弱寄主体液免疫水平。本研究所获结果加深了我们对昆虫尤其是寄生蜂Pacifastin蛋白酶抑制剂作用的认识。
Pacifastin protease inhibitors play an important role in insect immunity and development. In order to clarify its function in parasitoid, the cDNA sequences nvpp-1 and nvpp-2 of Pacifastin protease inhibitor open reading frame (ORF) coding for Bombyx mandarinae were cloned and sequenced respectively. The sequences were 723 and 888 bp in length, Encoding 240 and 295 amino acid residues, respectively. The predicted results showed that the N-terminal of nvpp-1 and nvpp-2 deduced amino acid sequence contains a signal peptide sequence of 17 amino acid residues in length. Sequence analysis and phylogenetic tree construction indicated that NVPP-1 and NVPP-2 contained 5 and 4 typical Pacifastin conserved domains, respectively, and clustered with the pimpla hypochondriaca venom protein CVP4. Real-time quantitative RT-PCR results showed that nvpp-1 and nvpp-2 were transcribed in all the tissues of the female wasp and the transcript levels in the thorax and abdomen (the rest of the abdomen) In the poison organs, its transcription level was higher in the early eclosion (0 and 1 d), and its transcription level was significantly lower. Western blot results showed that both NVPP-1 and NVPP-2 were detected only in venom and were not detected in other tissues tested. However, NVPP-1 and NVPP-2 were low in venom immediately after emergence (0 d). The prokaryotic expression of nvpp-1 and nvpp-2 was carried out by using pET-28a (+) vector, and the recombinant expression product was purified. The inhibitory effects of recombinant NVPP-1 and NVPP-2 on four different serine proteases (trypsin, chymotrypsin, protease K and elastase) were determined respectively. The results showed that recombinant NVPP-1 and NVPP-2 could significantly inhibit chymotrypsin And trypsin activity. At the same time, the effects of two recombinant proteins on the activity of phenoloxidase and the activity of pro-phenoloxidase in hemolymph of housefly, respectively, were also measured. The results showed that the activation of properoxyphenol oxidase Have inhibitory effect, but none of them can significantly affect the hemolymph’s own phenoloxidase activity. In conclusion, Pacifastin protease inhibitors NVPP-1 and NVPP-2 are contained in the venom of the fruit fly pupas, which are chymotrypsin inhibitors and trypsin inhibitor family members, respectively, and can significantly affect the hemolymph Pro-phenol oxidase activates the reaction, thereby weakening the host humoral immunity. The results obtained in this study deepen our understanding of the role of Pacifastin protease inhibitors in insects, especially parasitic wasps.