小菜蛾PGRP基因的克隆及其在昆虫细胞中的表达

Peptidoglycan recognition proteins (PGRP) is one of the pattern recognition receptors which can recognize the peptidoglycan or a class of bacterial containing the peptidoglycan and plays an important function in the regulatory and recognition in innate immune response. In this paper, a full length cDNA sequence encoding PGRP was cloned from Plutella xylostella by homology and rapid amplification of cDNA ends (RACE) technology (GenBank Accession No. EU399240, defined as PxPGRP). The analysis of sequence showed that PxPGRP contains an open reading frame (ORF) of 588 bp, encoding 195 amino acids, with the predicted MW of 21 kDa and pI of 8.38, while the mature protein encoding with the predicted MW of 19.04 kDa and pI of 8.86. The temporal and partial expression profiles of PxPGRP was investigated by semi-quantitative PCR and real-time quantitative PCR. The analysis of semi-quantitative RT-PCR result showed that PxPGRP could be expressed at each instar stage, especially with higher expression in 4th instar and pupae of P. xylostella. The analysis of real time PCR showed that the mRNA transcription level of PxPGRP has tissue specific, such as epidermis, malpighiantubules, fat body, hemocyte and midgut from high to low. In order to obtain recombinant protein and lay a basis of structural and functional study of PxPGRP. The ORF of PxPGRP was ligated into pEGFP-C3. The converted fluorescence microscopy convinced that PGRP was expressed in Sf9 cells. The successful expression of PxPGRP in cells lays a foundation for the structural and functional study, but also provides a new target for the biological control of P. xylostella.