Cloning And Prokaryotic Expression Of HC-pro Gene Of PVY-N Shanxi Isolate

Generally, The plant virus can't survive alone outside cell for it is a kind of molecule living creature. In the nature , thjy are carried and transmitted by vector , such as insects, nematodes ,fungi and so on. It developed many special virus transmition process between virus and ve:tor, which make the virus survive and spread naturally.Potato virus Y (PVY), a member of the family Potyviridae, is a dangerous pathogen in potato, pepper and tobacco in the vorld, leading to significant crop losses. HC gene , which is ecoded by the PVY genoire, is a protein related with aphids transmition, otherwise it takes part in many other bio-chemistry procession. In this experiment, we designed the particularity primers, and got the HC gene frament from shaanxi isolation of PVY necrotic stain with RT-PCR technology. Then, the HC gene was prokaryotic expressed. The results was those:1. We got the PVY necrotic isolae in shaanxi, then RT-PCR reverse transcript reaction was amplified, at last , we got a fragment of PVY-N HC-pro gene about 1. 4kb.2.The HC-gene was cloned into pBLueKS, then the constructed plasmid was transformated into ?coil DH5a .3. The cloned HC-pro gene was ligated into pBV221, pETSOa and pTrcHisA expression vector respectively for constructing pBVHC, pETHC and pTrcHC. They were transformated into E. coil BL21 for studying their exprression. The SDS-PAGE electrophoresis suggested that pBVHC had the highest expression.4. After a serious of experiments, we found that the diferent pre-cultivation in 37癈 lead to the difference of the pBVHC HC-pro expression level. 4 hour pre-cultivation 37 癈 and 6 hour heat-stimtlated-cultivation was the best condition for harvesting the target protein5. Purified the protein of pBVHC HC- pro and immuned the rabbit to prepare for the antiserium of the HC- pro.6. The Western blot indicated the prokaryotic expression was successful and the expression protein and the wilden-protein are homogenous.We have gained the HC-pro gene of PVY-N shaanxi isolate, and have done a primary study of expression. Based on this study, we can analyze the discrimination between different virus in Potyviridae, even the different isolate. In further research, we can separate and purify the accepted protein of the aphids with the HC-pro.