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Cloning, Prokaryotic Expression and Construction of Eukaryotic Expression Vetor of Elastase 2B Gene in Human

Author: GuXinXi
Tutor: TianHongTao；XuWenTao
School: Agricultural University of Hebei
Course: Microbiology
Keywords: Human elastase2B Vectors construction Pichia expression system Protein expression
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 103
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Abstract


Elastase IS characterized by their ability to hydrolyze elastin which a fibrous insolubleprotein for a class of broad—spectrum proteolytic enzyme.It has important prospects for thedevelopment in food,medicine,daily chemical,and environmental protection.Elastase isusually extracted from the pancreas of animals,which is restricted on raw materials andproduction techniques.As for such pattern,it will cost higher but produce lower,lag farbehind the market demands,and finally will limit the application as well as the promotionof elastase in future . In China and abroad , there are extensive microbial fermentationproductions of elastase,which,to a certain extent,have alleviated the demands ofindustrial enzymes,such as meat tenderizing agents,cosmetic additives . However,as forthe biochemical medical purpose , it is still restricted.This was mainly due to theinsufficiency of microbe fermentation pattern in the tests of bio—security and toxicity.Besides,microbial elastases from different sources are distinguished in nature,whichmakes the commodity production even in the biochemical medicine inaccessible to itsscheduled purposes.In regard to the consideration above,the research approached to the gene engineering techniques get elastase 2B gene by reverse transcription.Through use of E.coli and Pichiapastoris expression system to express the human elastase 2B gene.It is laid the foundationfor the security and large-scale production the enzyme,and provide the material conditionsfor researching the structure and characters ofhuman ELA2B.In this study,the human ELA2B gene was cloned from total RNA which was extracted from kidney of human by reverse transcription polymerrase chain reaction(RT-PCR),thena recombinant plasmid pGEM—T easy/ELA2B was obtained.After identification andsequencing,the sequence analysis revealed that the human ELA2B gene contained 809nucleotides which encoding 269 amino acids,the signal peptide contained 1 6 amino acids,a predicted activation peptide of 1 2 amino acids and the mature ELA2B gene composed of 241 amino acids.Then the successful constructed recombinant prokaryotic expression plasmidpET30a / ELA2B and eukaryotic recombinant expression plasmid pPIC9K/ ELA2B,were transformed into E.coli host BL2 1(DE3)and Pichia pastoris GS 1 1 5 respectively to the performance of expression researches. SDS-PAGE analysis of the IPTG induced expression of pET30NELA2B showed that the weight of target protein was correct,and itwas approximately 28kDa.However,after refolding of inclusion body,transparent circlewas not found in activity monitor flat panel which suggested that the ELA2B wascompletely devoid of proteolytic activity.pPIC9K/ ELA2B was linearized with SacI,and electroporated into P.pastoris GS 1 1 5. 1 0 1 transformants were selected on the MD agar plates, and 6 highly expressiontransformant were screened by YPD plates containing G41 8. After expression ofrecombinant elastase in shaking flask by methanol induction, we found the size ofrecombinant elastase was approximately 28kDa by SDS—PAGE analysis.The casein protein can be hydrolyzed by fermentation supernatant that initially indicated theexpression product has biological activity.The experiment cloned a human ELA2B gene , constructed prokaryotic and eukaryoticexpression plasmid and expressed the protein successfully. So that, the fermentation humanELA2B is possible, and it is laid the foundaion for further study of the structure andbiological activity of ELA2B.

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