Abstract:

【Objective】 A gene named HarmPBP2 encoding pheromone binding protein 2 was cloned from Helicoverpa armigera and expressed in prokaryotic system, and purified HarmPBP2 was obtained. 【Method】 The HarmPBP2 gene was cloned in H.armigera using reverse transcription-polymerase chain reaction (RT-PCR). And its spatio-temporal expression pattern was studied by semi-quantitative RT-PCR analysis. HarmPBP2 was expressed using pGEX-4T-1/BL21 (DE3) prokaryotic expression system, and purified using GSTrap FF. 【Result】 The full length of HarmPBP2 (GenBank loucs: EU647241) was 457 bp, encoding 149 amino acids, including 25 aa of signal peptide. Sequencing and analysis indicated that HarmPBP2 was characterized by six conservative Cys, which shared typical feature of OBP with those of other insects. Further analysis showed that HarmPBP2 may belong to the family of PBP. HarmPBP2 was expressed in antenna, leg and wing in both male and female, and also expressed in female maxillary palpus. The expression quantity was antenna＞wing＞leg, and the female＞male. The molecular weight of recombinant protein of pGEX-4T-1/ HarmPBP2 was about 40 kD. About 14 kD HarmPBP2 was obtained by affinity chromatography and cleavage of GST-tag. 【Conclusion】 HarmPBP2 was cloned and expressed it in prokaryotic expression system ,and then the expressed protein was purified, which is helpful for further researches on molecular structure and function of HarmPBP2, and the expression pattern was studied by Semi-quantitative RT-PCR analysis.

Key words: Helicoverpa armigera')">Helicoverpa armigera, pheromone binding protein, prokaryotic expression, purification, expression pattern