Gene Cloning, Expression And Characterisation Of A New Protease Inhibitor From Xenorhabdus Bovienii

Natural inhibitor (PI) is one group of small molecular proteins.They can bind with the protease active or allosteric site and inhibit the enzyme catalytic activity.There are many important proteases inside the insects,they play an important role in the process of digestion and some other metabolism.After protease inhibitors are taken,insects'digestive proteases and growth are blocked,and the insects are vulnerable to other adverse environmental factors,ultimately leads to an abnormal development of insects or even death.Therefore, the protease inhibitor has potential in controlling of the agricultural pest.We cloned a protease inhibitor gene from Xenorhabdus bovienii by PCR,and inserted it into the effective prokaryotic expression vector and attained the recombinant proteins.We purify the recombinant protein through the affinity chromatography,the bioassay results show that the recombinant proteins inhibited the growths of aphids(Myzus persicae),the plant bugs(lygus lucorum Meyer-Dur) and whiteflies(Trialeurodes vaporariorum),but no adverse effect on the normal mammalian cells and crops.The following are the results and conclusions from this study:1 We designed and synthesised two oligonucleotide primers refering to sequences of protease inhibitor gene and the ATG initiation site,TAA termination site from Genbank,then about 360bp protease inhibitor gene fragment named with Xbpi-1(Xenorhabdus bovienii protease inhibitor gene 1) was obtained using genome DNA of Xenorhabdus bovienii BJFS-526 as a template by polymerase chain reaction (PCR).2 Amplified fragment was inserted into the cloning vector pMD18-T Vector to obtain plasmid pT-PI,then transformed the plasmid into E.coli and screened using antibiotics.Positive–clone was checked by PCR and restriction enzyme digestion,and the precise results of identification was ensured. The results showed that the fragment was 360bp as we expected, with the similarity of 78% to the inhibitor gene sequence submitted to Genbank by blastn in database.3 We designed and synthesised another two primers contained restriction endonuclease sites NedI and XhoI in the 5'end separately.By PCR,these two sites were added to the 5'and 3'end of Xbpi-1 gene separately.Then the fragment digested by NedI and XhoI was inserted into the fusion expression vector pET-42a (+) digested by the same restriction endonuclease.Then T4DNA ligase was used to link the vector pET-42a (+) with the Xbpi-1 gene into pET42a-PI.PCR and restriction enzyme digestion were used to choose a positive-clone,the result showed that the Xbpi-1 gene had been successfully inserted into the expression vector.4 IPTG induced the expression and the best conditions for the Xbpi-1 gene to be highly expressed was known.The way of electrophoresis and western bloting analysis showed that the gene was highly expressed.Ni SepharoseTM6 Fast flow(GE Healthcare)column was used for purification in order to obtain the pure samples by 250mM imidazole elution buffer.After the protein was boiled for 30min, no significant changes in protein solubility showed that the recombinant protein was a heat-stable protein. 5 The bioassay results showed that the recombinant protein inhibited the growths and developments of aphids and the plant bugs.Insect mortality increased and the reproduction decreased when insects fed on diet containing the recombinant protein compared to the control.The protein also showed effects on whiteflies in the green house experiment.The results also indicated that the growth of normal mammalian cells and proteases from plants was not adversely affected by the inhibitor.