Construction Of Cysticercosis Cellulosae ScFv And PE40 Recombinant Vector

Cysticercosis cellulosae is a kind of global parasitic helminthic disease,which is more frequently in the developing countrys especially in our country.This disease did serious harms to our living and productions even to human's health.At least 31 provinces and autonomous regions had been reported the disease,not only ten millions of infected patients but also twelve millions of infected pigs had to abatage every year,which costed about two billions.Cerebral cysticercosis is one of the main reasons,which threaten human health induced them death in the developing countrys.When drug fast and medicine residual phenomenon came out unceasingly,immunoprophylaxis instead drugs became the necessity.Immunotoxin is a kind of targeted drug,which make gene fusion obout toxin gene and target gene,then express the fusion protein,which contain targeting and toxicity sections.It has the fuction of discrimination specificity acceptor's target cell selectivily. Pseudomonas aeruginosa's ectotoxin usually used as the noxious property component of recombine tox which catalyzes the elongation factor in eucaryotic cell among the ADP ribosylation so as to exercise the function of arresting chain elongation and abscising protein synthesis.The targeting parts of recombinate toxic,which not only can combine with target cell surface receptor,but also can fuse toxin gene and express fusion protein,the two parties do not affect each independence and functions.Connecting the Cysticercus botryoides scolex's single-chain antibody gene with Pseudomonas aeruginosa exotoxin PE40 gene,and then building in the expression vector PET-28(a) to get recombinated toxin,in which we can study the killing effect to Cysticercus botryoides.The genome was extracted from Pseduomonas-aeruginosa ATCC27853 strain.According to Pseduomonas-aeruginosa's exogenous toxin gene complete sequence,we should design primers and draw into two enzyme-situs.Amplificating the PE40 by PCR,adding glycerine into it by 10%of the bulk volume,detecting by Electrophoresis,purificating of the PCR product and conjugating carrier pMD18-T to construct pMD18-T-PE40,which is used to put into E.coli's competence:JM109,select the masculine to clone by the method of blue-white selection,Purifying its plasmid to identify by PCR,and then determine the sequence.Duplicate bands will turn out after XbaⅠand HindⅢthrough double-enzyme-cutting pMD18-T-PE40,that is 2692bp and 1083bp.Sequencing result shows that there is 10 basic radical,which has caused 7 amino acids changed,but there is no change about the active site of PE40 and PA103.There is pMD18-T-ScFv containing ScFv which has enzyme cutting site that EcoRⅠand XbaⅠ.Using XbaⅠand HindⅢto double-enzyme-cut pMD18-T-PE40 and pMD18-T-ScFv,Using the double-enzyme-cut site connect PE40 and pMD18-T-ScFv,then put it into E.coli's competence:JM109.picking masculine bacterium to be identified.Through the electrophoretic analysis,we got the expecting fragments,about 1800bp,which proved that the connection of ScFv and PE40 had been succeed.Enzyme-cutting pMD18-T-ScFv-PE40 and pET-28 (a) by EcoRⅠ和HindⅢ,putting ScFv-PE40 into pET-28(a) to construct fused toxin:pET28-ScFv-PE40 and put it into E.coli's competence cell-DH5α,picking masculine bacterium to abstract plasmid.Through the electrophoretic analysis,we got the expecting fragments,about 1800bp.Preserving the strain,settling foundation for the next step of researching the killing effect to Cysticercus botryoides by recombinant immunotoxin.