Cloning And Expression Of HSP And Research On Differential Proteins Of Taenia Solium Oncosphere

Taenia solium is a serious parasitic disease to human and animals. The Oncosphere growth into cysticerci can cause swine and humen cysticercosis.Finding out the key factors or differences proteins of oncosphere grow in cysticerci may explain changes of the process of oncosphere into cysticercosis and may reveal mechanism of Oncosphere break host mucosal.The heat shock protein (HSP) is closely related to organisms with growth, protein synthesis and adaptation to external stimuli. In this paper,pET28a-HSP vector has been successfully constructed and provide a theoretical basis study on relationship between the host and parasite. 1. Separation and identification of active and nonactive total proteindifferencesA method has been developed for differential display of proteins from activated and nonactivated Taenia solium oncospherein.A 2-D Liquid-phase separation(2D-LC) applied to a comparison of differential protein expression between activated and nonactivated Taenia solium oncosphere. The total of 208 and 197 specific protein bands from activated and nonactivated proteins of T. solium oncosphere were separated and 44 activated different protein and 33 nonactivated different protein included in 77 total different protein bands were detected in 0.3 pH fraction over a pH range of 7.71-5.41. According to the published characteristics of heat shock protein isoelectric point and hydrophobicity, selected five differential activated protein proteins by peptide mass fingerprint analysis.We successfully identified solium GAPDH proteins and small heat Shock protein (HSP), three other unidentified sequence. 2.Cloning and sequencing of interested gene HSPThe HSP gene was separately amplified from total RNA of activated and nonactivated Taenia solium oncosphere by RT-PCR. The PCR products were cloned into pGH vector,recombinant positive clones was sequenced after restriction enzyme digestion. The results of amplify the activation fragment oncosphere was 100% homology to HSP gene in GenBank, but not to amplified nonactivated oncosphere. Identified result accordance with peptide mass fingerprint analysis.Taenia solium oncosphere HSP gene was obtained successfully. 3.Expression of pET28a-HSP recombination antigenThe HSP gene was subcloned into pET28a expression vector,the recombinant pET28a-HSP infected into E.coliBL21.IPTG was added to induce fusion expression and the expression products was identified by SDS-PAGE and Western-blot. One fusion protein band about 35kDa was identified by SDS-PAGE after inducible expression After inducible expression, one fusion protein band about 38kDa in size was identified by SDS-PAGE. The result may lay foundations for the mechanism of invasion of between oncosphere and host, and the design of new vaccine anti-porcine cysticercosis and taeniasis.