The Clone Of Human Eotaxin Gene And Its NH₂-terminal Mutations And The Expression Their Prokaryotic Expression Vectors

BackgroundThe recruitment and activation of eosinophils to the sites of allergic inflammation,and hereafter mediators released from eosinophils play a crucial role in the pathogenesis of allergic disease.The recruitment of eosinophils to the inflammation site is mediated by chemokines,in which Eotaxin has the strongest eosinophil chemotaxis by binding with its sole receptor-CCR3 expressed in eosinophils.It has been proven that blocking of CCR3-Eotaxin axis by anti-CCR3 antibody,etc.could alleviate the allergic disease.The study for relationship of structure and function for wild type Eotaxin showed that the NH2-terminal region is critical for the biological activity,and modifications of this region of Eotaxin can profoundly and specifically alter the activity of the Eotaxin,which results in a possible CCR3 antagonism that can bind with CCR3 but can't induce cellular signaling. Theoretically,CCR3 antagonism obtained from Eotaxin mutant has higher specificity and stronger activity.ObjectivesTo obtain the gene of humen Eotaxin and a series of N-terminal mutations of humen Eotaxin(Met-Eotaxin),and to construct prokaryotic cell expression vectors of human Eotaxin mutations by genic technique for the farther expression,and detecting their activity of CCR3 antagonism.Methods1.Extract the total RNA from spleen.The cDNA sequence of Eotaxin and the fragment of humen Eotaxin gene was obtained by RT-PCR.The fragment was sequenced and analysed by PCR,DNA restrictive enzyme Kpnâ… and Xhoâ… after cloning into T vector(pTZ57R).The recombinate plasmid was named PT-Eotaxin.2.According to the sequence of PT-Eotaxin we designed eight Eotaxin mutations primers by special software.By site direct mutating,eight amino acids residues in the NH₂-terminal of Eotaxin have been individually mutated to methionine(Met-Eotaxins) and then were transformed into e.coli XL1-Blue and were filtered by ampicillin.The mutations were sequenced and analysed,and were named Met-Eotaxins for their mutative site.3.According to the mature chain of Met-Eotaxins,we designed 5 pairs primers. Eotaxin and Met-Eotaxins' mature chain fragments were extend and direct cloned to prokaryotic cell expression vector- pET30a(+) and were filtered by kanamycin and then were sequenced and analysed by PCR with T7 primer and by DNA restrictive enzyme Kpnâ… and Xho.The prokaryotic expression vectors recombinants were named pET30a(+)-Eotaxin and pET30a(+)-Met-Eotaxins.4.To transformation 9 recombinants which were identified into an expression host (DE3 lysogen),obtain appropriate expression hosts after filtering which can express the mature chain of Eotaxin or Met-Eotaxins.Pick a single colony from a freshly streaked plate and inoculate 50 ml LB containing kanamycin in a flask.Incubate with shaking at 37℃until the OD600 reaches 0.6-1.0.Add IPTG and continued the incubation for 1-6 h. Harvest the cells.And then got the induced proteins for SDS-PAGE analysis and Western-Blot.Results1.The plasmid PT-Eotaxin was analysed by PCR with Eotaxin special primer and cut by DNA restrictive enzyme Kpnâ… and Xhoâ… .The target fragments were approved to the same size as in theory.And the result of target fragments sequence were the same as which in genbank.2.The mutations(Met-Eotaxins) were sequenced and analysed and were identical with our design as expected.3.The recombinant prokaryotic expression plasmids were sequenced and analysed by PCR with T7 primer and cut by DNA restrictive enzyme Kpnâ… and Xho.The target fragments were approved to the same size as in theory.The prokaryotic expression vectors recombinants were named pET30a(+)-Met-Eotaxin.And the result of target fragments sequence are right-on.4.The target induced proteins analysed by SDS-PAGE.The result indicate that each recombinant can express the target fusion protein.Further more,the level of expression were high.The quantity of the expressed target protein was 32～35.5%of the total cell protein.More result of analysis of Western-Blot show that the expressed proteins were the target protein.ConclusionIn this study,the human eotaxin gene was cloned correctly.Prokaryotic cell expression vectors of humen eotaxin,eight NH₂-terminal mutations of human Eotaxin(pET30a(+)) were obtained by genic technique,which could be the foundation for screening out the CCR3 antagonism by large scale expression and identifying the activity of the corresponding proteins.