State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China; Laboratory of Sericulture, College of Life Science, Anhui Agricultural University, Hefei 230036, China.
Insect Biochem Mol Biol. 2013 Oct;43(10):936-46. doi: 10.1016/j.ibmb.2013.07.010. Epub 2013 Aug 8.
Parasitoid wasps inject venom into the host to protect their offspring against host immune responses. In our previous study, we identified a calreticulin (CRT) in Pteromalus puparum venom. In this study, we expressed the wild-type and the coiled-coil domain deletion mutant P. puparum calreticulins (PpCRTs) in Escherichia coli and prepared polyclonal antibody in rabbit against PpCRT. Western blot analysis showed that PpCRT protein was not only present in the venom but also in all the tissues tested. Real time PCR results indicated that PpCRT mRNA was highly expressed in the venom gland. The transcript level of PpCRT in the venom gland was peaked at 2 days post-eclosion, while the PpCRT protein in the venom was maintained at a constant level. Both recombinant wild-type and mutant PpCRT proteins could bind to the surface of P. puparum eggs. Recombinant PpCRT inhibited hemocyte spreading and cellular encapsulation of the host Pieris rapae in vitro, and the coiled-coil domain is important for the inhibitory function of PpCRT. Immunocytochemistry results showed that PpCRT entered P. rapae hemocytes, and the coiled-coil domain played a role in this process. After injection of recombinant PpCRT into P. rapae pupae, real time PCR results showed that PpCRT inhibited transcript levels of host encapsulation-related genes, including calreticulin and scavenger receptor genes. In conclusion, our results suggest that P. puparum venom protects its offspring against host cellular immune responses via its functional component PpCRT to inhibit the expression of host cellular response-related genes.
寄生蜂向宿主注射毒液，以保护其后代免受宿主免疫反应的影响。在我们之前的研究中，我们在 Pteromalus puparum 毒液中鉴定出一种钙网织蛋白 (CRT)。在这项研究中，我们在大肠杆菌中表达了野生型和卷曲螺旋结构域缺失突变体 P. puparum 钙网织蛋白 (PpCRTs)，并制备了兔抗 PpCRT 多克隆抗体。Western blot 分析表明，PpCRT 蛋白不仅存在于毒液中，而且存在于所有测试的组织中。实时 PCR 结果表明，PpCRT mRNA 在毒液腺中高度表达。毒液腺中 PpCRT 的转录水平在出蛰后 2 天达到峰值，而毒液中的 PpCRT 蛋白水平保持恒定。重组野生型和突变型 PpCRT 蛋白均可与 P. puparum 卵的表面结合。重组 PpCRT 抑制了宿主 Pieris rapae 的血细胞铺展和细胞包被作用，卷曲螺旋结构域对于 PpCRT 的抑制功能很重要。免疫细胞化学结果表明，PpCRT 进入了 P. rapae 血细胞，卷曲螺旋结构域在此过程中起作用。将重组 PpCRT 注射到 P. rapae 蛹中后，实时 PCR 结果表明，PpCRT 抑制了宿主包被相关基因的转录水平，包括钙网织蛋白和清道夫受体基因。总之，我们的结果表明，P. puparum 毒液通过其功能成分 PpCRT 抑制宿主细胞反应相关基因的表达，从而保护其后代免受宿主细胞免疫反应的影响。
