Clone Human TRAIL CDNA Large-scale Fermentation In Pichia Pastoris And Purification

Since 1995,Wiley search the homologization conservative seqence of TNFfamily molecular in EST(expressed sequence tag),he found theTRAIL(TNF-related apoptosiinducing ligand).TRAIL is a typical IItransmembrane protein,C-terminatio extracellular domain (ECD) is highhomologization with CD95L and TNF,it can induced some kinds of tumour cellapoptosis quickly,but it can do little influence for normal cell.So,TRAIL wasconsidered a extreme potent anti-tumor factor.We found the TRAIL caninfluence 80.7% uterine cervix cancer cell ME180 apoptosis,incubationmalignancy tumor cell strain Jurkat or Ag8,after 6 hour, above 80% tumor celldead in our sreach.More surprising is 90% mortality rate or more high inincubation the TRAIL with neurospongioma cell.All these evidence suggestionthat TRAIL is quite futuer,more safely anti-tumor factor.TRAIL,one kind ofanti-tumor factor is more and more obvious .TNF (tumorne crosis factor)family had three molecular could induce cell apoptosis.They are TNF-α,Fasland TRAIL ( TNF-related apoptosis-inducing ligand).Although TNF-α andFasl could induce cell apoptosis both,but TNF-αcould active nuclear factor(NF)NF-κB and creat severe Inflammatory reaction,and Fasl could induce fatalhepatic injury. TNF-α and Fasl`s poison and adverse reaction limited theireffort in tumor therpy greatly.TRAIL was found recent years as a new memberin TNF family.Compare with Fasl and TNF-α,TRAIL's mostdifferent:①TNF-α and Fasl expressed in most actived immune cell,but TRAILexpressed most normal cell continuum.②It can induce tumor cell apoptosismost powerful,but it had no effact to normal cell.③It has more anti-tumorspectram than Fasl.④It had little active effort to nuclear factor NF-κB,even ifsystemic administration,that had no severe effort inflammation effort eventhough TNF-α.Because of this,TRAIL was considered a kind of anti-tumorfactor safty and more potent.TRAIL,also be known Apo -2L,general reside most tissue.TRAILespecially reside in spleen,parastata glandulosa and lung,but brain ,liver anddidymus didn't transcript the TRAIL factor.In heterogenesis cell,such asretrogression leukomonocyte strain K299, paristhmion T cell is especiallyabundant. Burkitt lymphadenoma Raji cell strain abundence is low.Moreoverthe fresh abstraction peripheral blood leukomonocyte's transtript number isvery low or no transtript TRAIL mRNA copy.Scientist study theTRAIL ,recombination express and physico-chemical property analysis,indenced some kinds of cell apoptosis study and research,combination aminoacids sequence analysis, scientist found :human TRAIL whole moleculeconsist of 281 amino acids, isoelectric point is 7.63. According to proteinchemisty theory deduce dissolvability molecular is 114-281 amino acids,but invivo didn't find the dissolvability molecular be enzyme cutting from icellularmembrane. it seems that amino acids sequence dosen's have metalloproteaseenzyme cut site.nodeoxidize SDS-PAGE analysis dissolvability molecular ,molecule quality is 24 ku,assembly 48 ku dimeride,trimer is assembly 66ku.TRAIL is a typical II transmembrane protein,it has not signal peptide at it's N-terminatio(intracellular region). Amino acids sequence 15-40 is hydrophobicregion that can form transmembrane construction. C-terminatio(entracellularregion)is identical with TNF and Fasl,strongly conservation,that could formtypital βplate sheet structure and βchain sandwich.Even though dissolvabilityTRAIL recombination molecular is achieve from 114-181 and 95-281 aminoacids,but eukaryon express total length TRAIL mRNA didn't achievereconstruction.Alliance use anti-tumor factor and TRAIL,not only decreasepoision and adverse reaction but also increase sensitivity on TRAILtherapy.This provide Rationale of the alliance use anti-tumor factor andTRAIL .Our research make use of gene recombination technique , thoughRT-PCR ,from human activing lympholeukocyte amplify TNF-related apoptosiinducing ligand, We created a full human TRAIL expression vectorpPICZαbased on the vector pPICZα, Then expressed TRAIL in the expressionsystem. Identify the recombinant antibody, evaluate the advantage of Pichiapastoris expression system to express full human TRAIL.1. RT-PCR amplify human TRAIL geneThis experiment make use of gene recombination technique. On the basisof NCBI announce the TRAIL mTRAIL, to design primer.Making use ofRT-PCR ample TRAIL sequence.We achieve human complete TRAIL gene.RT-PCR production purify,and recombine with pMD 18-T, Construction offull human clone expression system2. Construction of full human TRAIL expression vector pPICZαThis experiment make use of gene recombination technique., On the basisof vehicle pPICZα sequence design primer ,leading in XhoⅠandEcoRⅠenzyme cut site in sequence,amplify the cDNA ,digesting withXhoⅠand EcoRⅠand combination express pPICZα.3. Identification of the full human TRAIL expression systemCloned the human TRAIL cDNA into expression vector pPICZα.Analyzed the recombinant protein by Western blot after transformation.Analyzed the purified recombinant TRAIL by western blot, the full humanantibody of 140 KD molecular weight appeared. These results indicated thatthe functional soluble TRAIL could be induced in methylotropic yeastexpression system. The cloning and expression of full human TRAILindicated an important role for improved Pichia pastoris expression system inthe cloning, screening and expression of full human TNF-relatedapoptosis-inducing ligand.The result indicate our recombination vector is success. it also providedthe potential to prepare therapeutic tumor cell and cell apoptosis.4. Large scale fermentation and purification of TRAILTo fermentated Pichia Pastoris engineering bacteria with 80L fermentationtank.Optinizing good conditions and purication methods.It gives a foundationfor biological activity and pharmacodynamics study.5. MTT shade selected methodEstablish a set of integrity human TRAIL expressde system.It has a importantsignificance on apoptosis and tumor therapy.This investigation use PichiaPastoris expressed system.Establish a integrited human TRAIL expressedvetor.Give a theory and experiment according on TRAIL advanceddevelopment.