Clone And Sequence Analyses Of ScFv Gene Against Cysticercus Cellulosae

Cysticercosis cellulosae is a kind of sporadic and infectious parasite disease because of the larva of taenia solium parasitising in the musle and the other organ of swine.This disease is cosmopolitan distributional and sporadic occurrence all over our country,especially popular in northeast,northchina and southwest it is a imprtant zoonosis,which not only influences swine husbandry but also seriously threats human health.At present,there are not application of biologicals for curing this disease accept chemicals.In recent years,with the developments of molecular biology,biochemistry and immunology,and constant improvment experimental method,many researchers try to study biologicals and the application of Scfv and immunotoxin provide faster and completely methods.Scfv is a kind of recombinant protain,which connect Vh and Vl by about 5～25 amino acids which is(Gly4Ser)3,compare with intact antibodies it has its advantages for exmaple a short residence time,rapid clearance speed in the the serum,strong tissues penetration force,specific screening in vitro By mmunological method and more important,it van express in bacteria and convenient for Gene operation and mass production by genetic engineering.It also can link with effector molecule and express fusion protein by genetic engineering.So it can be used as a ideal vecror of drg toxins radioactive material and the other targeting carriers.Overlap extends PCR(SOE PCR) make PCR proeucts to be overlap chain through primers have supplementary terminal,then link overlap splices in the next work through extension of overlap splices.This PCR technology gene Carry out the effective gene recombination.But it avoids avoid digestion restriction enzyme digestions and liapigated by ligase,so some products which must depend on digestion restriction enzyme digestions also be able to generate.This test is made to identify the digestion and PCR of the specification that gene Vl in monochain antibody light chain in variable region and gene Vh of scolex of Cysticercus botryoides.The electrophoresis shows that bar which appears between 330bp and 370bp is accord with the expected result.The result of sepuential analysis shows high autoploidy. The antoploidy of Vh reaches to 94%and 96%that of the light chain.Then the technology of RCR(SOE PCR) is taken.After times of tests to find out the response conditions,the denaturation temperature is defined as 94℃,40s,during the first PCR,the Tm defined as 58℃is best;During the second PCR,Tm defined as 52℃is best and the number of circle for each round is defined as 25.Connect the gene Vl in monochain antibody light chain in variable region with the gene Vh of scolex of Cysticercus botryoides to objective gene Vl-linker-Vh monochain antibody gene using the linker.The results of digestion and the PCR show that the bar which appears in 750bp is accord with the expected result and connect the joining products to the carrier of pMD-18T and transforms to permissive cell JM109.This test builds upstanding foundation for the constructions,expression and measurement of activity of monochain antibody ScFv-PE40 of recombination immunity toxin in Bacillus coli in future.